Compatibility behaviour of some newly isolated F-like R factor. The incompatibility reactions of four new R factors have been determined against reference plasmids of the compatibility groups FI, FII, FIII, FIV, FV, FVI and various not F-like groups. Two R factors have been found to belong to group FII, while one of the two other plasmids was incompatible with representatives of the FII and FI groups. The last R factor was incompatible with plasmids of FII and P groups. It seems, therefore, that R factors with incompatibility for more than one group of plasmids occur frequently in nature.


Brochial stump closure techniques following pneumonectomy: A serial comparative study. Three methods of closing bronchial stumps were evaluated in 90 dogs undergoing left pneumonectomy:interrupted 3-0 silk sutures, interrupted 3-0 nylon sutures, and stapling with an automatic device. The dogs were sacrificed at 24 hours, 3, 5, 7, 10, and 14 days after pneumonectomy. Brochial stump leakage pressures and histological studies were performed on all specimens. Differences in the exudative reaction between silk and staple closures were significant on day 5(238.19+/- 23.28 SD PMN's/HPF in silk closures, 34.95+/- 20.37 SD PMN's/HPF in staple closures) and therafter(P less than 0.001). The reaction to nylon was intermediate between silk and staple. Collagen levels were highest at all periods for staple closures. Staple leakage pressures were greater than silk or nylon(day 5: 145.3 mm Hg +/- 64.9 SD in staple closures, 40.2 mm Hg +/- 15.4 SD in silk closures). These results have clinical relevance to the selection of suture materials for stump closures.


[Pharmacokinetics of DL-norephedrine 14C in the rat]. After i.v. administration of DL-norephedrine 14C to the rat (535 mug/kg), approximately 75% of the radioactivity was eliminated during the first 18 hr in the urine. Beside the unchanged drug (71.5% of the total urinary radioactivity), the main metabolite was alpha-methyloctopamine (18%), a parahydroxylated derivative of which 68% were excreted as glucuronide conjugates. Kinetic studies of DL-norephedrine in different organs showed that the affinity of tissues for the amine did not change with time. Captation was important and rapid in lungs, adrenals, spleen and heart. The brain was marked by a slow increase of drug level during the first 2 hr. However, the terminal semi-exponential kinetic slopes of DL-norephedrine concentration showed that the elimination processes of the drug were the same in each tissue. The affinity of tissues for alpha-methyloctopamine was shown to be much more specific; its uptake was higher in organs which are rich in adrenergic nerve endings (heart, adrenals and spleen). These results confirm the role of this hydroxylated amine as a false neurotransmitter. A whole body autoradiography in the mouse showed an important uptake of DL-norephedrine 14C in salivary glands and periocular structures.


Intraventricular antipyretics and bacterial pyrogen fever. In adult fowls with cannulae chronically implanted into the IIIrd cerebral ventricle or into the hypothalamus, the effects of various hydrosoluble antipyretics, given intraventricularly, on bacterial pyrogen fever were studied. It has been shown that fever evoked by intrahypothalamic or intraventricular infusion of O somatic antigen of Shigella Dysenteriae was reduced by intraventricular administration of acetylsalicylate-lysine, indomethacin-methylglucamine or ibuprofen-lysine given during the febrile plateau. However, a 3-day intraventricular pretreatment with acetylsalicylate or indomethacin, or a single administration 30 min before, did not prevent fever by subsequent intraventricular or intrahypothalamic injection of pyrogen. On the contrary, intraventricular infusion of indomethacin or acetylsalicylate substantially reduced pyrogen fever when given after pyrogen latency period, e.g. just at the beginning of the febrile response. A possible involvement of E prostaglandins as mediator to pyrogen fever is discussed.


Tracheobronchial epithelial multinucleation in preeclampsia. Smears were made from the tracheobronchial washings from 40 parturient women undergoing operative delivery while under general anesthesia administered via an endotracheal tube. There was a significantly greater incidence of epithelial multinucleation in the smears from 12 patients who had had preeclampsia for more than 24 hours than in those from 13 healthy pregnant women, from eight women with preeclampsia of less than 24 hours' duration, and from seven women with associated diseases other than preeclampsia. Although we are unable to explain this phenomenon, we have reviewed its possible causation in the face of existing theories on the cause of preeclampsia.


Abnormal cilia in human bronchial epithelium. Abnormal cilia are a common occurrence in human bronchial epithelium. Bronchial specimens were obtained at surgery or at "immediate autopsy" from patients with neoplastic and nonneoplastic diseases; these patients included smokers and one known nonsmoker. Areas of bronchial epithelium far removed from the tumor mass were studied. Ciliary abnormalities fell into four major categories: (1) cilia with a single axoneme and excess cytoplasmic matrix; (2) compound cilia; (3) intracytoplasmic microtubular doublets; and (4) cilia within periciliary sheaths.


A rapid method for processing liver biopsy specimens for 2 micron sectioning. We will describe a procedure for the preparation of thin sections (2 micron thick) of percutaneous liver biopsy specimens for routine histological analysis. The process results in better preservation of both cellular and structural detail. The thinner sections provide clearer definition of inflammatory infiltrates, cellular changes, and intracellular deposits. Re-examination of the pathological features of the liver with the use of this technique will provide stronger correlation of the structural and functional aspects of hepatic disease processes.


Procainamide-induced systemic lupus erythematosus. Renal involvement with deposition of immune complexes. A 54-year-old man sustained an acute anterior myocardial infarction and was subsequently treated with procainamide hydrochloride for 5 1/2 months, resulting in development of a drug-induced systemic lupus erythematosus (SLE) syndrome with renal involvement. In over 60 reported cases of procainamide-induced SLE, clinical renal evaluation has led to the concept that renal involvement is a rarity in this disease. Direct immunofluorescence examination of necropsy kidney tissue revealed mesangial deposition of immunoproteins and C3 in a granular pattern characteristically seen in idiopathic SLE. Ultrastructural studies confirm mesangial deposition of immune complexes. The immunological and ultrastructural pattern of immune complex deposition in glomeruli suggests similar pathological mechanisms for glomerular injury in procainamide as seen in idiopathic SLE.


Alternate-day prednisone therapy in recipients of renal allografts. Risk and benefits. Forty-five patients receiving renal allografts were gradually converted from daily to alternate-day prednisone therapy. Indications for conversion included aseptic necrosis, growth retardation in children and adolescents, obesity, diabetes, cataract formation, and cosmetic appearance. Eight of the 45 patients developed acute or chronic rejection during or just after completion of alternate-day steroid therapy. The remaining 37 patients had a notable decrease in the degree of hypercorticism, with return of growth in children and adolescents. However, there was no improvement once aseptic necrosis or cataract formation had occurred. Although the use of alternate-day prednisone therapy is of benefit in reducing the untoward side effects of corticosteroids, the risk of precipitating allograft rejection is a significant threat and must be carefully considered whenever this form of treatment is undertaken.


Organized kidney retrieval on a regional basis. A new approach. A network of small to medium-sized communities has been developed to increase the supply of cadaveric kidneys for renal allotransplantation. After a training period, community physicians and the community hospitals were designated as members of the transplant team to retrieve kidneys. A threefold increase in the number of cadaveric kidneys has resulted from this project. This mechanism for organized kidney retrieval on a regional basis could be used to augment the supply of cadaveric organs in other areas of the United States.


Aneurysm of the splenic artery.

Aneurysm of the splenic artery. Aneurysm of the splenic artery is found in up to 10% of the elderly. Usually, the lesion is discovered by change and does not produce symptoms unless rupture occurs. Although there is general agreement that surgery is required in symptomatic patients and in women of childbearing age, controversy persists as to management for the asymptomatic patient. Since rupture is uncommon, and surgical mortality low, those patients over the age of 60 can be followed up, and not operated on because of the diagnosis alone.


Knee dislocation. An illusive cause of critical arterial occlusion. Popliteal artery injury is a hazardous and often subtle complication of traumatic knee dislocation. Review of the literature in which series of knee dislocations were reported has revealed a 28% incidence of thrombosis or rupture of this critical vessel. Fifty-seven percent of these patients required leg amputation. The most frequent cause of limb loss has been absence or delay of direct surgical repair of the associated popliteal artery injury. This report describes a management plan that is intended to minimize the likleihood of future disabling leg amputations following knee dislocation.


[Clinical importance of the immunoserological data on the bone allotransplantation (author's transl)]. The clinical and immunoserological data in 66 bone grafted patients are analysed. The importance of the immunological histocompatibility in bone allotransplantation is discussed in a short introduction. The method of transplantation is given and the clinical material is analysed in 3 tables. It is clear from the discussion, that in 6 out 66 patients there are data for an isoimmunization in result of the transplantation. Cytotoxins in the patients' sera are demonstrated after transplantation of frozen grafts. No HL-A antibodies are demonstrated following transplantation of bone grafts, treated after the method of Maatz and Bauermeister. The humoral antibodies are of a greater importance in transplantation of larger bone grafts, as well as of bone-joints complexes. It is acceptable that the immune conflict is of importance as regards to the pathological resorption of the graft. Often, the histocompatibility is a basical reason for the suppuration and sequestration of the graft. The antigenicity of the graft possesses an inductive action concerning the new bone formation. The importance of the osteogenic possibility of the bone-bed-recipient is underlined. It is desirable for a modern tissue bank to be able to offer bone grafts with determined specificity concerning ABO, Rhesus-D and HL-A systems. This will be a new perspective in the bone allotransplantation.


[Morphological results after an experimental transplantation of autogenous cancellous bone in dogs (author's transl)]. Cell activity during the period of healing of an autogenous cancellous graft inserted into an artificial continuity defect of the ulna was studied in 20 adult dogs by light microscopy using the semithin sectioning technique. The defects were stabilised by osteosynthesis using metal plates and screws. The animals were divided into 4 groups and the autografts were investigated 1, 2, 4 and 8 weeks after transplantation. Histologic analysis showed, that ... 1. one week after transplantation, undifferentiated cells resembling reticular cells, haemocytoblasts and many closely packed osteoblasts were scattered among the trabecular network of the autogenous cancellous graft. The survival of transplanted cells in our opinion is evident, by the absence of any sign of cell death or macrophage activity. 2. two to 4 weeks after transplantation a remarkable increase of the osteoblast activity was noted. This osteogenic cell activity was mainly directed toward bone formation along the surface of contact of the autograft with the adjoining bone and there was more than one layer of osteoblasts and osteoid. Osteoblasts were also found lying along the trabecular surface of transplanted cancellous bone, but here they were arranged in a layer, which was usually only one cell deep. 3. eight weeks after transplantation there was a remarkable change in the appearance of the autogenous cancellous graft. The activity of the multinucleated osteoclasts was significantly increased in the direction of the surface of contact of the autograft with the compact receptor bone. Evidently the osteoclasts produce the prior conditions for a closer contact of the autogenous cancellous graft with the adjoining compact bone.


Modulating effects of interferon preparations on an antibody response in vitro. The effect of mouse interferon preparations on the primary in vitro antibody response of mouse spleen cells was studied. Concentrations of interferon greater than 8 units per ml significantly inhibited the antibody response while low concentrations of 0-08-0-8 units per ml could be shown to be mildly enhancing. Various treatments which affected the antiviral activity of the interferon preparations reduced the immunosuppressive activity to a similar extent. Interferon acts during the first few hours of a response but the effect is not apparent for at least 50 h. Interferon had no effect when added after 48 h. The kinetic data has been interpreted as demonstrating interferon-sensitive and interferon-resistant components of the in vitro response. The results from investigations of the polyclonal response to lipopolysaccharide support the view that interferon acts mainly on B cells alone, although effects on T-B interactions cannot be excluded.


The identification and ultrastructure of macrophages from the mammary gland of the ewe. Cells in the secretion of involuting and non-lactating mammary glands of the ewe were studied. Light microscopy studies revealed that the majority of the cells were mononucleated, but it was impossible to distinguish macrophages from sloughed epithelial cells. Electron microscopy studies showed that the majority of the cells were macrophages and that they phagocytosed polystyrene latex spheres. Very few epithelial cells were found and they were distinguished from macrophages by numerous microfilaments and blunt microvilli. Epithelial cells could be further distinguished from macrophages by the presence of tight junctions. Tissue cultures of cells in secretion of mammary glands revealed the predominant cell type to be the macrophage. After 3 days in culture these macrophages formed multinucleated giant cells which contained acid-phosphatase granules. Fluorescein-labelled antiserum prepared against the cell cultures, stained cells in the lumens of alveoli and ducts, but not epithelial cells, in frozen sections of mammary tissue. The study confirms previous findings that the large majority of cells from mammary glands in late involution are macrophages. It also points to the usefulness of non-lactating mammary glands as a source of sheep and cattle macrophages.


Simplified body-composition analysis using deuterium dilution and deuteron photodisintegration. A number of approaches to estimating human body composition (water, lean mass, and fat) are available, and each approach has its own limitations. Deuterium dilution is a comparatively safe and valid procedure for assessing body composition; however, its use has been restricted by the relative complexity of measuring deuterium at low concentrations. A simplified scheme for quantifying low levels of deuterium in body fluid samples promises to expand the use of D2O in body composition studies. The deuterons are quantified by measuring the neutron emission induced by gamma-ray irradiation from 228Th(2.61 MeV). The in vitro aspects of the procedure exhibit coefficients of variation of under 1% throughout the range of assay and near 100% recovery of added D2O. In vivo comparison studies with a reference tritium dilution procedure indicate close agreement in a group of 38 subjects. The chief advantages of the procedure are a small oral dose of D2O and the use of either serum or urine samples. The physical condition of the sample does not affect the measurement so distillation is not required. Thus, small medical facilities can enjoy the advantages of low cost, minimum patient inconvenience, and technical simplicity by submitting subject samples to larger agencies for analysis.


Perspectives and priorities in food and nutrition planning. I have taken this opportunity to challenge a trend that I have seen in recent meetings toward the promotion of a holistic, but perhaps impractical, approach to food and nutrition policy planning and implementation. While I support the development of modeling as an approach to the conceptualization of the problems and interactions, it would appear that the data requirements of the integrated nutrition policy approach exceed our present capabilities. I fear that this can result in inactivity at a time when we cannot afford inactivity--inactivity consequent to delay for data collection, or inactivity consequent to a belief that because comprehensive planning is not yet feasible, there is no point in doing any systematic planning. The precision of the definition of the nutrition problem is a function of the stage of development of the operational planning process. Let us not advocate the collection of data we are not equipped or prepared to interpret and use. In many areas of the world, relatively simple information, often already available, can define the nutrition problem with sufficient precision to permit program development within the individual sectors of government. Surely that must be our immediate goal.


The environment of the malnourished child. The study of the relation of man to his environment in developing countries emphasizes the inevitable need for societies to recognize the true causes of infection, malnutrition, and poverty. The need is for improvement in the quality of human life in less developed nations, a recommendation easy to prescribe but difficult to accomplish. Although our pool of knowledge is incomplete, it is adequate to suggest ways to diminish infection, increase food production, utilize food more efficiently, improve education, and provide systems of justice to protect the classes most in need. The physical environment in tropical and subtropical regions, and the socioeconomic characteristics of the population inhabiting such regions, favor maintenance and transmission of a variety of viruses, bacteria, and parasites that make agricultural progress and social development difficult, and that contribute to poor fetal growth, nutrient wastage, and deficient postnatal physical growth, accounting for most of the childhood morbidity and mortality. In this regard, infections contribute indirectly to the overall food problem in a similar fashion as pests do in terms of food losses and spoilage. The overall effect could be comparable or greater than that resulting from an inadequate capacity to produce or to purchase the food needed. Thus, my objective has been to stress, within the whole environment, the importance of infection and the need to diminish it. Ways to control and prevent infection are readily known. They have to do with education of the population to improve personal and environmental hygiene. Economic investment is necessary to improve housing and water supply systems, waste disposal, and such preventive measures as immunization programs. Although such measures may appear expensive when first implemented, they have long-lasting effects and many require minimal expenditure once they are established. Large segments of the population stand to benefit, and other development interventions can then be introduced. However, these measures should not be implemented singly. They should be accompanied by community development, family planning, social legislation--in other words, the holistic approach to health and welfare. To do otherwise may aggravate the problem by stimulating demographic growth, perpetuating malnutrition and infection, and maintaining underdevelopment.


Purification and some properties of nitrate reductase (EC 1.7.99.4) from Escherichia coli K12. 1. Nitrate reductase was purified 134-fold from Escherichia coli K12. The purification procedure involves the release by Triton X-100 of the enzyme from the cell envelope. i. The purified enzyme exists in aqueous solution either as a monomer (mol. wt. about 220 000) or as an associated form (probably a tetramer; mol.wt. about 880 000). 3. The purified enzyme has three subunits with apparent mol.wts. of 150 000, 67000 and 65000. An additional subunit of apparent mol.wt. 20000 is present in a haem-containing fraction that is also produced by the preparative procedure described. 4. None of the enzyme subunits is present in the cell envelope of cells grown in the absence of nitrate. 5. Reversible changes in the activity of nitrate reductase in vitro with FMNH2 as reductant can be induced under circumstances which are without effect on the reduced Benzyl Viologen-NO3-activity.


Acyl and amino intermediates in reactions catalysed by pig pepsin. Analysis of transpeptidation products. The action of pig pepsin on a variety of small peptides including Leu-Trp-Met-Arg, Leu-Trp-Met, Leu-Leu-NH2, benzyloxycarbonyl-Phe-Leu and Gly-Leu-Tyr was studied. Leu-Leu-Leu was found to be the major product from the substrates Leu-Trp-Met-Arg and Leu-Trp-Met, indicating that the predominant reaction at pH 3.4 was a transpeptidation of the acyl-transfer type. Leu-Leu-Leu was also formed in high yield by amino transfer from benzyloxycarbonyl-Phe-Leu. Like the amino-transfer reactions the acyl transfer proceeded via a covalent intermediate, since [14C]leucine was not incorporated into transpeptidation products and did not exchange with enzyme-bound leucine in the presence of acceptors. With Leu-Trp-Met both acyl and amino transpeptidation products, namely Leu-Leu, Leu-Leu-Leu, Met-Met and Met-Met-Met, were formed in addition to methionine and leucine. With Leu-Trp-Met-Arg (1 mM) the pH optimum for the rates of hydrolysis and acyl transfer is about pH 3.4. At this pH the rate of acyl transfer exceeds that of hydrolysis; at pH 2, however, hydrolysis was faster than transfer. A comparison of the effect of the length of substrates and products on the reaction rates allows the conclusion that the binding site can extend over eight to nine amino acid residues. Although the experiments provide no conclusive evidence for or against the involvement of amino and/or acyl intermediates in the hydrolysis of long peptides and proteins, the high yield of transpeptidation reactions of both types observed with some substrates suggests a major role for the intermediates in pepsin-catalysed reactions. The results also show that when pig pepsin is used for the digestion of proteins for sequence work, the likelihood of the formation of transpeptidation products is considerable. In this way peptides not present in the original sequence could easily form in a reasonably good yield.


Effects of secondary binding by activator and inhibitor peptides on covalent intermediates of pig pepsin. A number of peptides were found to increase the activity of pig pepsin towards small synthetic substrates. The activators increase transpeptidation of both the acyl-transfer and the amino-transfer types by as much as 45-fold. The effect on hydrolysis varies from inhibition to modest activation, but is always less than the effect on transpeptidation. The kinetics of substrate cleavage are the converse of non-competitive inhibition and show an increase in kcat. and no effect on Km values. Lineweaver-Burk plots of results obtained in the presence of the activators indicate a substrate activation at high substrate concentration. This appears to be a co-operative effect, since it is not observed in the absence of the activators. The activation is greatest at pH 4.7, less at pH 3.4, and at pH 2.0 is observable only with some of the activator peptides. The results show directly the effect of secondary binding on the catalytic efficiency of pepsin. The most effective activators are those that are most hydrophobic. The results suggest that binding in the secondary binding sites causes an increase in hydrophobicity in the catalytic site which results in increased stability of the acyl and amino intermediates, and preferential reaction with acceptors other than water. The implication that the present results strengthen the case for a role of covalent intermediates in the hydrolysis of good substrates (high kcat. values) is discussed.


The effect of N-acylglucosamines on the biosynthesis and secretion of insulin in the rat. The effects of N-acylglucosamines on insulin release have been studied. N-Acylglucosamines stimulated insulin release from rat islets in vitro only if a sub-stimulatory concentration of glucose was also present, and this secretory response was abolished by mannoheptulose. In perifused islets the rapidity of the secreotry response to N-acetyl-D-glucosamine was similar to that observed with D-glucose. Increasing acyl-chain length from N-acetyl- to N-hexanoyl-D-glucosamine impaired the secretory response; however, N-dichloroacetyl-D-glucosamine was a more potent stimulator of release than was N-acetyl-D-glucosamine. Polymers of N-acetyl-D-glucosamine containing two to six monomers linked alpha1-4 did not stimulate insulin release; glucosamine linked to dextran via a propionyl or hexanoyl spacer group was also without insulin-releasing ability. N-Acylglucosamines were also effective in eliciting insulin release in vivo when injected into conscious rats. At the dose used (86 mumol), N-acetylgucosamine elicited a rapid rise in plasma-insulin concentration; N-butyrylglucosamine was less effective, and there was little or no response to N-hexanoylglucosamine. The response to N-dichloroacetyl-glucosamine was greater than that to N-acetylglucosamine; an increase in plasma insulin concentration could be elicited by N-dichloroacetylglucosamine at a dose (17 mumol) at which neither glucose nor N-acetylglucosamine was effective. The secretory response to acetylglucosamine is not mediated by conversion into glucose. Rates of (pro)-insulin biosynthesis by rat islets have been measured (Pro)-insulin biosynthesis was stimulated by glucose, and this response was abolished by mannoheptulose. N-Acetylglucosamine also stimulated (pro)-insulin biosynthesis; this effect of N-acetylglucosamine did not require the presence of glucose, and was not abolished by mannoheptulose. It is concluded that there are differences in signal reception and/or transduction for the processes of insulin biosynthesis and release.


Dissociation and catalysis in yeast hexokinase A. 1. The specific activity of yeast hexokinase A depends on the concentration of the protein in the solution being assayed. When a solution containing 13.5 mg of hexokinase A/ml is diluted 10--100-fold at various values of pH and temperature, there is a gradual decline in the specific activity of the enzyme until an equilibrium value is reached, which varies with the chosen experimental conditions. 2. The catalytic activity lost when hexokinase A (1 mg/ml) is incubated at 30degreesC is recovered by lowering the temperature to 25degreesC. 3. These concentration- and temperature-dependent phenomena are consistent with the existence of a monomer-dimer equilibrium in which the dimer alone is the catalytic form of the enzyme. 4. Glucose alone prevents the decline in specific activity of hexokinase A after dilution, but it does not re-activate dilute solutions solutions of the enzyme. It is concluded that glucose binds to both the dimer and the monomer and prevents both association and dissociation. 5. The progress curve describing the phosphorylation of glucose catalysed by hexokinase A does not attain a steady state. It is possible that dissociation of catalytically active dimers in a ternary complex with glucose and ATP (or glucose 6-phosphate and ADP) could explain the non-linearity of this progress curve.


The dissociation of exocytosis and respiratory stimulation in leucocytes by ionophores. By exploiting the unique characteristics of three ionophores, experimental conditions were found which permit the dissociation of respiratory stimulation from secretion in polymorphonuclear leucocytes. A marked stimulation of respiration was produced by ionophore X537A, which binds and transports both alkali-earth and alkali cations. The stimulatory activity of this ionophore was the same at either high or low Na+/K+ ratios in the medium and was virtually unaffected by extracellular Ca2+. A slight stimulation of oxygen consumption was also caused by the K+-selective ionophore valinomycin and by ionophore A23187, which complexes and transfers bivalent cations. Ionophore X537A and valinomycin were unable to stimulate selective release of granuleassociated beta-glucuronidase and gradually increased cell fragility, as monitored by increased leakage of lactate dehydrogenase. Ionophore A23187 slightly increased exocytosis of beta-glucuronidase. In a Mg2+-free medium, Ca2+, added simultaneously with ionophore A23187, greatly enhanced respiration and secretion of the granule enzyme. If Ca2+ was added a few minutes after the ionophore, exocytosis occurred, but no respiratory burst was observed. If the latter experiment was repeated in the presence of extracellular Mg2+, both secretion and respiration were stimulated. This effect was not produced by Mn2+ or Ba2+. It is proposed that Ca2+ is required for triggering selective secretion of granule enzymes from leucocytes is caused by an intracellular redistribution of cations, which may invovle Mg2+-dependent mechanisms.


Studies on the biosynthesis of pancreatic glucagon in the pigeon (Columba livia). The biosynthesis of glucagon was studied in microdissected pigeon pancreatic, islets. [3H]-Tryotophan and [3H]leucine were incorporated into big and little glucagon. No precursor-product relationship was evident between big and little glucagon after radioactive pulsechase and immunoreactive chase incubations. Radioactive and immunoreactive little glucagon and immunoreactive big glucagon were actively secreted and the synthesis of both glucagons was inhibited by high concentrations of glucose. [3H]Tryptophan and [3H]leucine were incorporated into an islet protein of about 20000mol.wt. Gel filtration of extracts of turkey pancreas revealed the presence of an immunoreactive peak of mol.wt. approx. 20000. This glucagon-immunoreactive component was also present in dog and ox pancreas and was stable to chaotropic agents and elution at various pH values. A similar-sized glucagon-immunoreactive species was present in the dog circulation. These results are discussed in the light of the presently accepted mechanisms of glucagon biosynthesis.


Glucose transport as rate-limiting step in the growth of Escherichia coli on glucose. Over a wide range of growth rates, two strains of Escherichia coli growing aerobically in continuous culture under glucose limitation utilized glucose at rates identical with those at which cells harvested from the chemostats transported [14C]glucose.


[Activity in vitro and pharmacokinetics of minocycline (author's transl)]. The in vitro activity of doxycycline and minocycline (Klinomycin) was determined by serial dilution test in 100 strains of E. coli, 101 strains of enterobacter, 91 tetracycline-sensitive and 52 tetracycline-resistant strains of staphylococci. Only staphylococci were more sensitive against minocycline than against doxycycline whereas other species showed nearly the same sensitivity against both antibiotics. After i.v. infusion of 200 mg minocycline (during 1 h) mean serum levels fell from 3.5 mug/ml to 0.6 mug/ml (after 24 h). Half-life was calculated as 15.7 h, urine recovery as 5.9%. After oral application of 200 mg minocycline serum level peaks were 2.7 mug/ml, serum levels after 24 h 0.7 mug/ml. At repeated administrations daily dosage of 100 mg was too low of 200 mg sufficient to obtain the same serum levels as after the initial dose of 200 mg. CSF levels after oral administration of 0.4 g minocycline were 0.74 +/- 0.09 mug/ml (in serum at the same time 2.2 +/- 0.2 mug/ml). Half-life of minocycline in chronic renal failure (7 adult patients) was not prolonged (15--20 h). Minocycline is especially suitable for treatment of infections of unknown bacterial origin (including such caused by staphylococci). I.v. infusion is indicated only in unconscious or vomiting patients.


Metabolism of phenylbutazone in man.

Metabolism of phenylbutazone in man. One male volunteer received a single oral dose of 400 mg of 14C-labelled phenylbutazone (Butazolidin) and a second volunteer a repeated oral dose of 3 X 400 mg. The absorption from the gastro-intestinal tract was found to be rapid and complete. The integrated concentration of unchanged phenylbutazone in plasma, as estimated from the area under the concentration curve (AUC) between 0 and 336 h, was 63% of that of total 14C-substances. The corresponding AUC of three specifically determined metabolites, i.e. oxyphenbutazone, gamma-hydroxyphenylbutazone and p,gamma-dihydroxyphenylbutazone were 23%, 2% and 0.5%, respectively. A single oral dose was slowly excreted from the organism, since within 21 days only 88% was recovered, 61% from urine and 27% from faeces. About 1% of total urinary radioactivity was excreted as unchanged drug. The sum of specifically measured metabolites (oxyphenbutazone, gamma-hydroxyphenylbutazone, p,gamma-dihydroxyphenylbutazone) and phenylbutazone in urine did not cover more than about 10%. Solely oxyphenbutazone was present as an O-glucuronide, but only in small amounts. About 40% and 12% of total urinary radioactivity was due to C(4)-glucuronides of phenylbutazone and gamma-hydroxyphenylbutazone, respectively. Their structures were established by spectroscopic means. These metabolites contain pyrazolidine rings directly attached to glucuronic acid via a C-C bond, thus representing a novel class of drug metabolites.


Combination therapy with naproxen and aspirin in rheumatoid arthritis. Thirty-six patients with rheumatoid arthritis were studied to determine the effectiveness and safety of combined therapy with naproxen and aspirin. An 8-week double-blind crossover trial was performed in which naproxen and placebo were administered on a background of constant-dose aspirin. Combination therapy was demonstrated to be more effective than aspirin alone. Tolerance of the two regimens was comparable.


[Design and evaluation of the controlled clinical trial/demonstrated by an efficacy test of the combination pentosan polysulphate/nicotinic acid in disturbed cerebral circulation (author's transl)]. Design and evaluation of the controlled clinical trial are thoroughly discussed by giving an example for testing a sodium pentosan polysulphate/nicotinic acid combination (Compuron). 60 patients with cerebral vascular disorders were randomly allocated to the two treatments and received medication over a period of eight weeks. A detailed biostatistical analysis of the data led to the following conclusions: 1. Regarding the target symptoms headache, nausea, sleep disturbance, reduced alertness, reduced ability for contacts and moods significant differences in favor of the active medication beginning with the sixth treatment week. 2. Regarding the psychological tests substantial and statistically highly significant (P less than 0.001) therapeutic effects. 3. Statistically significant decrease of the cholesterol and triglycerides level, absolutely as well as relative to placeo medication. 4. No treatment related side effects during the entire trial period of eight weeks.


Prednisone and azathioprine compared to prednisone plus low-dose azathioprine and cyclophosphamide in the treatment of diffuse lupus nephritis. A 1-year double-blind crossover study comparing prednisone and azathioprine to prednisone plus low-dose azathioprine and cyclophosphamide was carried out in 14 patients with diffuse lupus nephritis. Low-dose triple therapy had no apparent therapeutic advantage over prednisone plus azathioprine. Cyclophosphamide-induced ovarian failure and hematuria were not avoided by its use in low dose.


[Profile comment on clinical and experimental studies of pramiverine and pramiverine/metamizole (author's transl)]. The profile of the spasmolytic 4,4-diphenyl-N-isopropyl-cyclohexylamine-hydrochloride (pramiverine, Sistalgin) is outlined on the basis of fundamental research. Evidence is provided that a combination with metamizole is useful in the case of Sistalgin comp. Description of the clinical and clinico-experimental examinations confirmed the margin of indications for Sistalgin and Sistalgin comp. Reference is made to the applications with a view to facilitate endoscopic and radiological procedures. The tabulated list of 2368 patients demonstrates the positive ratio between therapeutic effect and undesirable cholinolytic concomitant effect of Sistalgin comp.


[Clinical trial of a novel spasmolytic, pramiverine, and its influence in colitis (author's transl]. In a double blind comparison of 4,4-diphenyl-N-isopropyl-cyclohexylamin-hydrochloride (pramiverine, Sistalgin) with placebo 51 patients with parasitis and bacterial colitis and enterocolitis as well as mixed forms of both were evaluated. The antiparasitic and antibacterial basic therapy was standardised in both groups. In the 27 patients treated with pramiverine the regression of the colitis syndrome starting within a few days was evaluated as excellent to good in 21 patients. In the placebo group similar improvements were observed only in 9 patients out of a total of 24. The good tolerance as compared to other spasmolytics should be equally emphasised as well as the slight inhibition of gastric secretion under prolonged therapy.


[Pramiverine/metamizole - a spasmoanalgesic for routine therapy of symptoms in emergency/a clinical trial in patients with biliary colics (author's transl)]. The authors refer to a previous trial with the combination 4,4-diphenyl-N-isopropyl-cyclohexylamine-hydrochloride (pramiverine Sistalgin) and N-methyl-N-(2,3-dimethyl-5-oxo-1-phenyl-3-pyrazolin-4-yl)-aminomethanesulfonate (metamizole) (Sistalgin comp.) in patients with renoureteral lithiasis and report results obtained with the same spasmoanalgesic and a comparative preparation in patients with biliary colics. On the basis of parameters selected for the trial, the success of pramiverine/metamizole therapy in subjective and objective complaints induced by biliary stone diseases is considered positive.


[Therapeutic results with the combination pramiverine/metamizole in a multicentre trial (author's transl)]. 279 physicians took part in a multicentre trial to assess the efficacy and acceptability of the combination of 4,4-diphenyl-N-isopropyl-cyclohexylamine-hydrochloride (pramiverine, Sistalgin) and N-methyl-N-(2,3-dimethyl-5-oxo-1-phenyl-3-pyrazolin-4-yl)-aminomethanesulfonate (metamizole) (Sistalgin comp) in the forms of ampoules, coated tablets and suppositories. 1301 out of 1329 case reports could be evaluated; in 28 cases treatment was discontinued. The trial included 59% acute colics and 41% subacute recurrent painful spasms of the caval organs. A single presentation form was used in 44%; all other patients were treated with the combined regimen of ampoules/coated tablets/suppositories. A good or very good result was obtained in 91% of cases, with a low rate of undesired side-effects. In 3%, the result was unsatisfactory. The patients' judgment was in agreement with that of the physician. Both judgments confirmed the rapid onset, long duration and reliable effect of pramiverine/metamizole in treating colics of the intestinal, biliary and urinary tracts as well as recurrent spastic pain. Similarly favourable results were obtained with all presentation forms.


[Antimicrobial action of the combination sulfamoxole/trimethoprim in vivo (author's transl)]. The experimental infections of the mouse by gram-positive and gram-negative germs are effectively treated with the combination of N1-(4,5-dimethyl-2-oxazolyl)-sulfanilamide (sulfamoxole) and 2,4-diamino-5-(3,4,5-trimethoxy-benzyl)-pyrimidine (trimethoprim) (CN 3123, Nevin, Supristol). The potentiating effect of both substances is demonstrated by the calculated index of the synergistic effect. The therapeutic efficacy of the combination sulfamoxole/trimethoprim is proved by two models of experimental urinary infections in the rat. In the malaria of chicken and mice a notable therapeutic success is observed.


[Clinical trial of the antibacterial combination sulfamoxole/trimethoprim (CN 3123). 1st communication. Efficacy activity spectrum--resistance in therapy (author's transl)]. The clinical trial of the combination N1-(4,5-dimethyl-2-oxazolyl)-sulfanilamide (sulfamoxole) and 2,4-diamino-5-(3,4,5-trimethoxy-benzyl)-pyrimidine (trimethoprim) (CN 3123, Nevin, Supristol) showed that, when given in the ratio of 5:1 and in a suitable dosage computed from pharmacokinetic information, it exhibited antibacterial activity and had a therapeutic effect in about 90% of the patients. In view of the numerous types of pathogen identified in the clinical trial, CN 3123 can be described as a broad-spectrum chemotherapeutic agent. CN 3123 was also effective against infections caused by sulfonamide-resistant or antibiotic-resistant pathogens.


[Clinical trial of the antibacterial combination sulfamoxole/trimethoprim (CN 3123)/Third communication: Clinical results of a field trial with CN 3123 in bronchopulmonary and ENT (ear/nose/throat) infections (author's transl)]. The following conclusions emerged from an analysis of the therapeutic results documented during the clinical trial of N1-(4,5-dimethyl-2-oxazolyl)-sulfanilamide (sulfamoxole) and 2,4-diamino)-5-(3,4,5-trimethoxy-benzyl)-pyrimidine (trimethoprim) (CN 3123, Nevin, Supristol) in bacterial infections of the respiratory tract and ENT organs: 1. In bacterial infections of the bronchopulmonary system, the antibacterial activity of CN 3123 was demonstrated in 94% of the patients, in that 75% of patients were cured and there was a definite improvement in 19%. 2. In bacterial infections of the ENT organs, the antibacterial activity of CN 3123 was demonstrated clinically in 95% of the patients, in that 89% of the patients were cured and there was a definite improvement in 6%. 3. The clinical assessment of the activity of CN 3123 correlates closely with the results of bacteriological checks.


[Clinical trial of the antibacterial combination sulfamoxole/trimethoprim (CN 3123)/Fourth communication: Clinical results with CN 3123 in infections of the gastrointestinal tract, the skin and gynecological adnexes (author's transl)]. The antibacterial activity of N1-(4,5-dimethyl-2-oxazolyl)-sulfanilamide (sulfamoxole) and 2,4-diamino-5-(3,4,5-trimethoxy-benzyl)-pyrimidine (trimethoprim) (CN 3123, Nevin, Supristol) was studied in various bacterial infections in the fields of internal medicine, dermatology and gynecology. It was found that: 1. CN 3123 exhibits antibacterial activity in bacterial infections of the intestinal tract, particularly in salmonellosis and shigellosis. 2. CN 3123 is effective against bacterial infections of the female genital organs. 3. CN 3123 is very effective in patients with bacterial infections of the skin. 4. The antibacterial activity of CN 3123 in these indications is comparable to its action on bacterial infections of the urinary organs and respiratory tract and in ENT infections.


[Clinical trial of the antibacterial combination sulfamoxole/trimethoprin (CN 3123). Fifth communication: Comparison of two double-blind studies with CN 3123 undertaken for demonstration of its efficacy (author's transl)]. Two double-blind trials which, independently, revealed the same conclusion that there is no significant difference between the therapeutic effect of N1-(4,5-dimethyl-2-oxazolyl)-sulfanilamide (sulfamoxole) and 2,4-diamino-5-(3,4,5-trimethoxy-benzyl)-pyrimidine (trimethoprim) (CN 3123, Neven, Supristol) and a reference preparation, were compared with each other in respect of rating the therapeutic result. The authors discuss the greater occurrence of Proteus pathogens at one trial centre, and its bearing on the rating of the therapeutic effect.


[Clinical trial of the antibacterial combination sulfamoxole/trimethoprim (CN 3123)/Sixth communication: Results of compatibility of CN 3123 (author's transl)]. 1. During the clinical trial of N1-(4,5-dimethyl-2-oxazolyl)-sulfanilamide (sulfamoxole) and 2,4-diamino-5-(3,4,5-trimethoxy-benzyl)-pyrimidine (trimethoprim) (CN 3123, Nevin, Supristol) side-effects were reported in only 56 (=4.1%) of the 1371 patients who took part. 2. The numerous laboratory parameters studied in the whole group and in the group which received long-term treatment did not show any significant changes due to CN 3123. 3. The frequency and the type of side-effects which occurred under the combined preparation CN 3123 were the same as those known to be caused by sulfonamide treatment. 4. The good tolerance, which is a familial feature of sulfonamide treatment, is not impaired when sulfamoxole is combined with trimethoprim.


Radioactive colloidal solutions and suspensions for medical use. Radioactive colloidal solutions and suspensions are being widely used in the diagnosis and treatment of various diseases. The author considers the behaviour or radioactive disperse preparations in the body and devotes particular attention to the reasons for isotope transition from the colloidal to the ionic state. Questions concerned with producing and investigating radioactive colloidal solutions are discussed. In the light of the physical and chemical properties of the radioisotopes and of the disperse systems themselves, the author considers methods of producing the more important radioactive disperse preparations: colloidal solutions of noble metals; colloidal solutions containing phosphorus-32, yttrium-90 and isotopes of the rare-earth elements; colloidal solutions with with indium-113m, gallium-68, technetium-99m and rhenium-186; suspensions (macroaggregates and microspheres) for the diagnosis of lung diseases; and radioactive colloidal solutions based on quaternary ammonium base compounds.


Oesophageal pressure measurement in rabbits and human infants using an air-filled balloon system. In ventilated anaesthetized rabbits the relationship between the oesophageal pressure and the intrapleural pressure, measured with an air-filled balloon system, was studied. A balloon 15 mm long and 3 mm diameter detected 95% of the measured intrapleural pressure, provided the balloon was positioned accurately. In babies undergoing IPPV, the best balloon position was in the lower third of the oesophagus. When synchronous spontaneous respiration occurred in the course of IPPV the mean of intrapleural pressure remained negative in spite of positive ventilating pressures in the range 30-45 cm H2O.


Clinical evaluation of meptazinol. a new analgesic, in postoperative pain. In a single-blind study of several doses of a new hexahydroazepine analgesic, meptazinol, in 45 patients recovering from major abdominal surgery, there was a dose-related significant analgesic effect from i.m. doses exceeding 50 mg. Pain relief was maximal at the end of 1 h and the duration of effect of the 100-mg dose was approximately 5 h. There was a good correlation between pain relief assessed by the patient, by the physician, and indirectly by pulmonary function tests. Heart rate and arterial pressure were not affected by meptazionol. There were no abnormal biochemical or haematological findings and the only side-effect was vomiting in a few patients receiving multiple doses of the drug.


A soft agar colony assay for Lewis lung tumour and B16 melanoma taken directly from the mouse. A soft agar colony assay has been developed for the B16 mouse melanoma and the Lewis lung tumour. The special features of the technique are the use of a gas phase with 5% O2 instead of air and the addition of rat red blood cells. Single cell suspensions are prepared by trypsinization from the solid tumour and the cells are plated out in 0-3% agar over a layer of 0-5% agar in 30-mm Petri dishes. After 8 to 15 days' incubation in 5% O2, colonies of more than 50 cells are produced. Plating efficiencies of between 30 and 50% are usually obtained. The addition of up to 10(4) heavily irradiated tumour cells gives some further improvement in plating efficiency for the B16 melanoma but not for the Lewis lung tumour. Applications of the technique to measure cell survival in the two tumours after treatment with cytotoxic drugs and radiation are reported. The scatter of experimental points is relatively small, and in comparative experiments good agreement has been obtained with results using in vivo assay techniques.


A controlled trial of inhaled corticosteroids in patients receiving Prednisone tablets for asthma. The theraprutic efficacy of inhaled beclomethasone dipropionate and inhaled betamethasone valerate in chronic asthma has been studied in 14 treatment centres in 158 patients who had previously been taking prednisone tablets regularly. Doses of 400 mug daily of beclomethasone dipropionate and a dose of 800 mug daily of betamethasone valerate allowed approximately 80% of patients to discontinue prednisone initially and 60% to remain off daily prednisone for 24  weeks. A mean reduction in daily prednisone dose of 8 mg was achieved by patients inhaling corticosteroids whilst placebo inhaler permitted a 5 mg reduction. The three inhaled corticosteroid preparations were equally effective in facilitating prednisone reduction and provided equally good control of asthma, alone or as an ancillary to prednisone. The higher dose of beclomethasone dipropionate was superior to the lower in permitting more patients to remain off daily prednisone for the period of the trial. Although 82% of patients recovered a normal adrenal response to tetracosactrin 24 weeks after prednisone was discontinued and inhaled corticosteroids subsituted, 18% still showed some suppression of adrenal function. There was no significant difference between the treatment groups in this.


Depression of bursal follicle formation by Candida albicans infections. Sub-lethal i.v. inoculations of Candida albicans into 12-day-old chick embryos have been shown to cause a depression of follicle formation in the bursa of Fabricius. This depression occurred in the absence of any infection in the bursa, although candidal encephalitis was a frequent manifestation of the disseminated infection. A similar depression of bursal follicle formation was caused by the administration of viable C. albicans on to the embryonic chorio-allantoic membrane, in the absence of any disseminated infection. Non-viable extracts of disrupted C. albicans administered on to the chorioallantoic membrane also caused a depression of bursal follicle formation. This immunodeficiency produced by an endotoxin-like principle at a susceptible stage in the development of the chick has been considered in terms of human immunological development and the neonatal occurrence of C. albicans.


Insulin secretion after injuries of differing severity in the rat. The effects on insulin secretion of injuries of differing severity have been studied in the rat. The injuries used were dorsal scalds to 20% and 40% of the body surface area, and a 4-h period of bilateral hind-limb ischaemia. These injuries resulted in 48 h mortality rates of 0/10, 7/10 and 5/10 respectively. Rats were studied 1-5-2 h after scalding or removal of tourniquets. The blood glucose concentration was markedly raised after all these injuries, and the plasma insulin concentration was also raised, so that the insulin to glucose ratio in any group did not differ significantly from that in non-injured controls. Injection of glucose (0-5 g/kg i.v.) induced a rise in insulin concentration in all groups, although the insulin to glucose ratio after the lethal 40% scald was lower than in control rats. It was concluded that in the rat normal insulin secretion is maintained even after lethal injuries, although some suppression of the insulin response to exogenous glucose may occur. Insulin resistance is more important in the rat than impairment of insulin secretion even at an early stage after injury.


Pathological changes in the pancreas of mice following infection with Coxsackie B viruses. Infection of pregnant or non-pregnant mice with Coxsackieviruses B1, B3, B4 or B5 produced a severe pancreatitis consisting of a degeneration of the acinar cells, loss of zymogen granules, infiltration of mononuclear and plasma cells and a replacement of the exocrine tissue with fatty tissue. Coxsackieviruses B2 and B6 did not cause these changes in the period up to 6 weeks following virus injection. Suckling mice did not appear to be more susceptible to pancreatic damage due to these two viruses. Sequential studies on the development of Coxsackievirus B-induced pancreatic lesions indicated that although the changes due to B1, B3, B4 or B5 were similar in type, B3 and B4 exhibited a more rapid action in the tissue and more severe lesions than either B1 or B5. In this work, none of the Coxsackie B viruses examined elicited pathological changes in the islets of Langerhans detectable with the light microscope.


The effect of T and B lymphocyte depletion on the protection of mice vaccinated with a Gal E mutant of Salmonella typhimurium. Immunosuppressive agents were used to determine the relative importance of T and B lymphocytes in conferring protection to mice vaccinated with a live gal E mutant of Salmonella typhimurium, strain G30D. Lymphocyte transformation and serum agglutination tests showed that while cyclophosphamide (CPA) suppressed B lymphocytes, antilymphocyte sea (ALS) suppressed both T and B cells. The humoral response of vaccinated animals treated with ALS was therefore supplemented by the i.v. injection of serum from untreated vaccinated mice. CPA-treated mice could not control multiplication of the vaccinal strain which eventually killed them. There was little multiplication of the vaccinal strain in the controls and ALS-treated mice, all of which survived to challenge. The vaccinated controls and vaccinated ALS treated groups each survived infection with the challenge strain which was gradually eliminated. It was concluded that humoral immunity was of greater importance than cellular immunity in mice vaccinated i.p. with strain G30D.


Leucosis and skin: acute lymphoid immunoblastic leukaemia. The clinical course of a patient with acute lymphoid immunoblastic leukaemia and prominent nodular haemorrhagic skin lesions is described. Cytological, cytochemical and electronmicroscopic studies were performed on bone marrow and skin blast cells. The absence of surface immunoglobulins and of the other markers for B lymphocytes (EA and EAC rosettes) and the presence of 30% of spontaneous sheep erythrocyte rosettes excluding an acute leukaemia with Burkitt's tumour cells, suggest that T cells are involved. Complete haematological and cutaneous remission was obtained with prednisone therapy.


Ciclopirox: a new topical pyrodonium antimycotic agent. A double-blind study in superficial dermatomycoses. One percent Ciclopirox solution, a new topical antimycotic agent, was used in a double-blind controlled trial in 105 dermatomycosis patients. Twelve of the patients were lost to follow-up. Of the remaining ninety-three patients, forty-four were treated with the active drug, forty-nine with a placebo. The cure rate amongst those using ciclopirox was significantly superior to that of the placebo. The drug was effective against Trichophyton rubrum, T. violaceum, T. mentagrophytes and Malassezia furfur.


Clinical and bacteriological evaluation of a twice daily regimen of epicillin capsules for the treatment of skin infections. Fifty-five patients with impetigo, ecthyma, or folliculitis caused by epicillin-sensitive strains of Streptococcus pyogenes or Staphylococcus aureus were treated with 500 mg epicillin capsules twice daily for an average duration of 9 days. Bacteriological and clinical examinations were performed at 2-day intervals. Favourable clinical responses (excellent or good) were obtained in 95% of the patients. No side effects were observed.


Dithranol (anthralin) versus triacetoxyanthracene. Investigations in vitro. In two in vitro models for evaluating antipsoriatic activity, dithranol (anthralin) and triacetoxyanthracene were compared. Both compounds exhibited an about equal effect on oxygen consumption of surviving human skin. Dithranol, however, exerted a stronger inhibiting effect on glucose-6-phosphate dehydrogenase (-G-6-PDH) activity of human skin homogenates.


Identification of Haemoglobin S in red cells and normoblasts, using fluorescent anti-Hb S antibodies. Anti-haemoglobin S antibodies were raised in horses and purified by affinity chromatography. These antibodies recognize beta6 valine, while they fail to bind to haemoglobins with a glutamy1 (Hb A) or a lysy1 (Hb C) residue in this position. The purified anti-Hb S antibodies were composed of equine IgG(a, b) and IgG(T) subclasses and failed to cause precipitation with Hb S, evidently because of the bivalencey of both antibodies and antigen. Identification of Hb S in single erythrocytes was achieved by reacting the fluorescein isothiocyanate-conjungated anti-Hb S antibodies with the haemoglobin antigen in fixed blood smears. All the red cells from persons with sickle cell trait and sickle cell anaemia are labelled with the anti-Hb S-FITC, while red cells from persons with normal haemoglobin or other mutant haemoglobin genotypes are not. Erythrocytes from newborns with sickle cell trait bind the anti-Hb S antibody, although the intensity of labelling varies from cell to cell. This method of cellular identification of Hb S preserves the morphological characteristics of the reactive cells and permits observations of the pattern of haemoglobin synthesis among cells of various degrees of maturity. Fluorescent identification of Hb S in fixed blood smears may be applicable to prenatal diagnosis of haemglobinopathies.


Cadaver bone as orbital implant.

Cadaver bone as orbital implant. Fifty-four cases of cadaver bone orbital implant are described. The results were encouraging. Cadaver bone implant is easily available and can be prepared in various sizes to fit the individual patient.


Vicryl (polyglactin 910) in cataract surgery. A controlled trial. In a controlled, randomized trial in 45 cataract operations a new synthetic absorbable suture (8-0 monofilament Vicryl, made by Ethicon), was compared with monofilament nylon or virgin silk normally used. There was a higher incidence of early complications-hyphaema, shallow anterior chamber, and choroidal detachment (15-4 per cent v. 0 per cent)-with the absorbable suture. But these differences were not significant at the 0-05 level and might well disappear with more operative experience, particularly the modified technique of knot-tying. In handling qualities Vicryl was superior to both 10-0 monofilament nylon and 8-0 virgin silk with the exception of knot-tying, in which it was inferior to 8-0 virgin silk. Vicryl is considered to be a useful absorbable suture in cataract surgery but knot-tying requires modification of technique.


The covalent structure of cartilage collagen. Amino acid sequence of the NH2-terminal helical portion of the alpha 1 (II) chain. The amino acid sequence of 162 residues from the NH2-terminal region of bovine alpha 1 (II) is reported. Automated sequence analysis of chains from pepsin-treated type II collagen indicated the sequence and order of two CNBr peptides, alpha 1 (II)-CB2 and alpha 1 (II)-CB3, at the beginning of the repetitive triplet sequence of alpha 1 (II). The sequences of alpha 1 (II)-CB6, alpha 1 (II),-CB12, and 39 residues of alpha 1 (II)-CB11 were determined largely by automated Edman degradation. Comparative sequence data are reported which indicate that the level of homology between alpha 1 (I) and alpha 1 (II) chains in the NH2-terminal region is about 80%. A similar level of homology was reported for the central portions of these chains (Butler, W.T., Miller, E.J., Finch, J.E., Jr., and Inagami, T. (1974), Biochem. Biophys. Res. Commun. 57 190). The degree of intraspecies variability between chain types is thus greater than the interspecies variability for a single chain type. Within the sequence reported here, the alpha 1 (II) chain contains glucosylgalactosylhydroxylysine at three positions. The corresponding sequence of alpha 1 (I) contains only one clycosylated hydroxylysine with the other two positions occupied by lysyl residues.


Reexamination of the conformation of muscle proteins by optical activity. The circular dichroism and optical rotatory dispersion of muscle proteins are reexamined. By the method of Chen et al. (Chen, Y.H., Yang, J.T., and Chau, K.H. (1974), Biochemistry 13, 3350), the estimated helical contents of myosin (78%), heavy meromyosin (HMM) (70%) subfragment 1 (SF-1) (60%), and G-actin (45%) are higher than hitherto reported. Tropomyosin (TM) and light meromyosin fraction I (LMM Fr. I) possess more than 90% helix in agreement with the values based on the bo method. HMM, SF-1, and G-actin also contain about 8, 16, and 27% beta form. The three troponins (TN) and three light chains (LC) of myosin have moderate amounts of helices (29 to 51%) and some beta form (13 to 23%). If the light chains are intact in HMM and SF-1, myosin would have 3-5% beta form, which is difficult to detect with the present method. For comparison, the predictive method based on amino acid sequence gives similar estimates for TM, G-actin, and TN-C with bound calcium, but slightly higher helical contents than our results for TN-I and the light chains.


Regions of tRNA important for binding to the ribosomal A and P sites. Studies on the enzymatic inhibition of phenylalanyl-tRNAPhe and formylmethionyl-tRNAFMet binding to the ribosomes by defined tRNA fragments indicate that beside the anticodon the following regions of tRNA are important for ribosomal A-site interaction: the TppsipCp sequence, the CpCpA end, and hU loop. In contrast, binding to the ribosomal P site is not inhibited by the fragments of uncharged yeast tRNAPhe containing the hU or the TpsiC loop of the molecule. Comparative studies on the inhibitory effect of the oligonucleotides TppsipCpGp and UpUpCpGp indicate that the presence of the minor bases in TpsiC loop is not an essential prerequisite for the binding of tRNA to the ribosomal A site. Furthermore, evidence is presented that shows that the binding of the TppsipCpGp oligonucleotide to the ribosomes influences the ribosomal P site and increases there the efficiency of the codon-anticodon interaction. It is suggested that the TppsipCpGp binds to the ribosomal A site and competes there with the TpsiC loop of the aminoacyl-tRNA for the same binding site. A model for the interaction between tRNA and the ribosomal A site is proposed that involves partial unfolding of hU and TpsiC loops of the tRNA and, therefore, suggests the dynamic involvement of tRNA in protein synthesis.


A comparison of kinetic parameters of polypeptide substrates for protein methylase II. Kinetic properties of protein methylase II (S-adenosymethionine:protein O-methyltransferase, EC 2.1.1.24) which methylates (esterifies) the free carboxyl side chains of amino acids in proteins was studied using various polypeptides as methyl acceptor substrates. Bovine pancreatic ribonuclease, a model substrate for the enzyme, was subjected to specific cleavage by cyanogen bromide, trypsin, and performic acid oxidation. Several polypeptide fragments derived were then separated by molecular sieve chromatography on a column of Sephadex G-25. The method was found to be very simple and gave good yields. Km values for these polypeptides as well as a few other protein substrates were determined. While Km values for the isolated peptides range generally between 4.8 and 0.7 X 10-3 M, those of native bovine panreatic ribonuclease, luteinizing hormone, and follicle-stimulating hormone were determined to be 4.0 X 10-4, 5.0 X 10-5, and 0.77 X 10-5, respectively. Sites of enzymatic methylation of the native ribonuclease were also investigated. Although polypeptides derived from the C-terminal and N-terminal regions of the molecule were found to accept methyl groups, they were unable to under go enzymatic methylation when native molecule was used as the substrate indicating that within the native ribonuclease these regions are in a conformation which do not allow them to be methylated by protein methylase II under the present assay conditions.


Transition from noncooperative to cooperative and selective binding of histone H1 to DNA. A transition from noncooperative to cooperative binding of DNA and histone h1 occurs between 20 and 40mMNaCI in 5 mM Tris-HCI, pH 7.5. Below 20mM NaCI in mixtures in H1 and excess DNA, H1 binds to all of the DNA molecules, causing them to sediment faster, and does not distinguish between DNA molecules that differ in size or base composition. However, at NaCI concentrations above the narrow transition range, H1 binds to only some of the DNA molecules and leaves the rest free. If the DNA molecules in a mixture are the same size, H1 selectively binds those that have the highest content of adenosine (A)+thymidine (T). By means of competion experiments at salt concentrations  spanning the transition range, it is demonstrated that H1 selectivity requires cooperativity. A high degree of selectivity based on A + T content can be produced by cooperative binding: for average DNA sizes of 2 X10(6) daltons, more than ten molecules of calf lymphocyte DNA (57% a+ t) are chosen per molecule of Escherichia coli DNA (50% A+T).


Conformational transition of Escherichia coli RNA polymerase induced by the interaction of sigma subunit with core enzyme. The isolated sigma  subunit of Escherichia coli RNA polymerase has been labeled covalently with a fluorescent probe, N-(1-pyrene)maleimide. The labeled sigma subunit (PM-sigma) still retained its biological activity in stimulating transcription of T7 DNA by core enzyme. When a stoichiometric amount of core enzyme was added to a solution of PM-sigma, there was a decrease in fluorescence intensity without shifts in emission maxima of PM-sigma. The kinetics of the interaction between the sigma subunit and core enzyme was investigated with the stopped-flow technique by monitoring the fluorescence quenching. A biphasic change of fluorescence intensity with respect to time was observed when PM-sigma was rapidly mixed with an excess of core enzyme.  The kinetic data can be analyzed in terms of a mechanism in which a fast bimolecular binding of sigma to core enzyme is followed by a relatively slow isomerization of the holoenzyme formed. From the best-fit kinetic parameters, an overall binding constant of less than or equal to 3X10(-10)M was estimated for the PM-sigma core complex, in agreement with that obtained by the fluorimetric titration. In addition, we have studied the effect of temperature on the rate constant associated with the conformational change of the holoenzyme, which shows a temperature transition around 20 degrees C. The nonlinear Arrhenius plot obtained implies that the conformational transition is complex and may be composed of several processes. The activation energy for the "overall" conformational change was estimated to be 6.7 kcal/mol. The kinetic evidence for the conformational transition of holoenzyme induced by the interactions of sigma subunit with core enzyme presented here further supports the proposition that the sigma subunit acts on core enzyme to trap a unique conformation of RNA polymerase which recognizes the proper promoters and initiates the synthesis of specific RNA chains.


Nuclear magnetic resonance investigation of the base-pairing structure of Escherichia coli tRNATyr monomer and dimer conformations. The structures of the Escherichia coli tyrosine tRNA monomer and dimer have been investigated by high-resolution nuclear magnetic resonance (NMR). At 23 degrees C the monomer contains 26 +/- 2 base pairs and the low-field NMR spectrum (11.7-15 ppm) can be accounted for in terms of the cloverleaf structure (23 base pairs) and three additional resonances that are assigned to tertiary structure base pairs. Assignments suggested for the various resonances are consistent with thermal denaturation studies in low-salt solutions. Under these conditions the temperature dependence of the spectrum can be interpreted in terms of sequential unfolding of the cloverleaf structure with the minor and dihydrouridine stems melting first, followed by the T psi C stem, the anticodon stem, and finally the amino acceptor stem. Certain features of the tertiary structure of tRNATyr are similar to other tRNA, but some details of the folding must be different, since no resonance from the S4U8-A14 tertiary base pair is observed. The tRNATyr dimer contains only 20 +/- 2 base pairs per tRNA (40/dimer) at 23 degrees C and a good account of the low-field NMR spectrum can be given in terms of a secondary structure in which bases of the T psi C stem and loop are involved in inter-molecular base pairing. Formation of the dimer requires opening of the hU and T psi C stems, but not the anticodon or amino acid acceptor stems, and this fits well with relative stabilities observed for these stems in the monomer. The model also provides an explanation for the formation of 2n-mers, that were stable enough to be separated by gel electrophoresis at room temperature (10 mM Mg2+). Experimental conditions required for interconversion of monomer and dimer are also described.


Mechanims of stimulation of in vitro protein synthesis by some copolymers of styrene, vinyluracil, and vinyladenine with maleic acid and acrylic acid. Copolymers of vinyl bases with acrylic acid and styrene or 1-vinyluracil with maleic acid were found to stimulate in vitro polyphenylalanine synthesis using a system extracted from Escherichia coli MRE600. Poly(styrene-maleic acid) was found to inhibit a ribosomal bound ribonuclease. Poly(1-vinyluracil, maleic acid), poly(1-vinyluracil, acrylic acid), and poly(9-vinyladenine, acrylic acid) were not inhibitors of the ribosome bound ribonuclease. The potent (up to fivefold) stimulation by these three polymers is due to the action of the polymers to interfere with ribosomal bound inhibitory protein. A protein, removed by washing ribosomes with 1 M ammonium chloride, characterized by M.J. Miller, A. Niveleau, and A.J. Wahba ((1974) J. Biol. Chem. 249, 3803) has been described as a potent inhibitor of in vitro poly(U)-coded protein synthesis using extracts of Escherichia coli MRE 600.


Photoinduced cross-linkage, in situ, of Escherichia coli 30S ribosomal proteins to 16S rRNA: identification of cross-linked proteins and relationships between reactivity and ribosome structure. The kinetics of photoinduced cross-linkage of Escherichia coli 30S ribosomal proteins to the 16S-rRNA molecule in the intact Escherichia coli 30S ribosomal subunit was studied in this report. All of the 30S ribosomal proteins become cross-linked to the 16S rRNA before changes in the sedimentation characteristics of the 30S ribosomal subunit can be detected. The proteins exhibit different reactivities in the cross-linkage reaction. One group of proteins-S3, S7-S9, S11, S12, and S15-S19-is cross-linked to the 16S rRNA by single-hit kinetics, or by photoprocesses of nonunity but low multiplicities. A second group of proteins--S1, S2, S4-S6, S10, S13, S14, and S21--is cross-linked to the 16S rRNA by photoprocesses of a complex nature. A comparison of these data with other properties of the individual 30S ribosomal proteins related to ribosome structure indicated that most of the 30S ribosomal proteins cross-linked to the 16S rRNA by photoprocesses of low multiplicities had been classified rRNA-binding proteins by nonphotochemical methods, and most of the proteins cross-linked to the 16S rRNA by photoprocesses of large multiplicities had been classified as nonbinding proteins. There were certain exceptions to these correlations. Proteins S4 and S20, both RNA-binding proteins, become cross-linked to the 16S rRNA by photoprocessses of large multiplicities, and proteins S3, S11, S12, and S18, none of which have been classified RNA-binding proteins, exhibited low multiplicities in the cross-linkage reaction. All of these exceptions could be explained in terms of limitations inherent in the photochemical methods used in this study and in other types of methods that have been used to study RNA-protein interactions in the 30S ribosomal subunit. The data presented here also suggest that labile RNA-protein cross-links are present in the uv-irradiated 30S ribosomal subunits, and that neither peptide-bond cleavage nor photoinduced modification of the charged side-chain groups in the ribosomal proteins accompanied the cross-linkage reaction. However, some photoinduced RNA-chain breakage might have occurred.


Properties of tRNA species modified in the 3'-terminal ribose moiety in an eukaryotic ribosomal system. Phe-tRNAPhe species modified on the 3'-terminal ribose residue were investigated for their ability to participate in individual steps of the elongation cycle using eukaryotic ribosomes from reticulocytes. None of the Phe-tRNAs used, namely Phe-tRNAPhe-C-C-3'dA, Phe-tRNAPhe-C-C-3'-NH2A, and Phe-tRNAPhe-C-C-Aoxi-red, can function in the overall process. All modified Phe-tRNAPhe species can be bound nonenzymatically to ribosomes. Phe-tRNAPhe-C-C-3'NH2A exhibits exceptionally high binding at low Mg2+ concentration compared with Phe-tRNAPhe-C-C-A binding. Ac-Phe-tRNAPhe species prepared from the three modified tRNAs, when bound to the donor site, were devoid of donor activity. The enzymatic binding of both Phe-tRNAPhe-C-C-3'dA and Phe-tRNAPhe-C-C-3'NH2A is less efficient than that of Phe-tRNAPhe-C-C-A but these Phe-tRNAPhe species have acceptor activity. Phe-tRNAPhe-C-C--Aoxi-red is not a substrate for the EF-I promoted binding reaction and has no acceptor activity. The nonaminoacylated species, tRNAPhe-C-C-A, tRNAPhe-C-C-3'dA, and tRNAPhe-C-C-3'NH2A, bind to the ribosome to a larger extent than the corresponding aminoacylated tRNAs, both in the presence and in the absence of poly(U). Peptidyl-tRNAPhe-C-C-3'dA bound to the donor site cannot activate the acceptor site for EF-I promoted binding of Phe-tRNAPhe as does peptidyl-tRNAPhe-C-C-A. Further, it was observed that a correct codon-anticodon interaction influences the recognition of the 3' terminus of tRNA. Specificity of eukaryotic ribosomes for the 2'- and/or 3'-aminoacylated tRNA species is discussed and compared with the properties of Escherichia coli system.


The effect of Mg(II) on the spectral properties of Co(II) alkaline phosphatase. Alkaline phosphatase of Escherichia coli, isolated by procedures which do not alter its intrinsic metal content, contains 1.3 +/- 0.3 g-atom(s) of magnesium and 4.0 +/- 0.2 g-atoms of zinc per mol of molecular weight 89 000 (Bosron et al., 1975). Substitution of Co(II) for Zn(II) and/or Mg(II) results in spectral properties which can be correlated with enzymatic activity. Magnesium does not activate the apoenzyme but augments the activity of 2-Co(II) enzyme almost 3-fold and that of the 4-Co(II) enzyme 1.3-fold. The magnesium-induced increase in activity of the 2-Co(II) enzyme is accompanied by spectral changes which are consistent with an alteration from largely octahedral-like to pentacoordinate-like coordination geometry. Magnesium increases the intensity of the absorption and magnetic circular dichroism (MCD) signals of the 4-Co(II) enzyme but without evidence of changes in coordination geometry. Cobalt when bound to the magnesium sites results in octahedral-like EPR spectra, unperturbed by phosphate which significantly affects cobalt at the pentacoordinate-like sites. In the absence of magnesium, 6 g-atoms of cobalt are required to maximize the spectral properties, but activity does not increase further after the addition of only 4 g-atoms of cobalt, while activity is optimal with only 2 g-atoms of cobalt. Hydrogen-tritium exchange measurements indicate that magnesium also stabilizes the dynamic structural properties of the apo- and 2-Co(II) enzymes but has little effect on the structure of 4-Co(II) phosphatase. The response to magnesium of both the spectral properties and enzymatic activities of cobalt alkaline phosphatase demonstrates that magnesium regulates cobalt (and zinc) binding and modulates the activity of the resultant products.


Coumermycin A1: A preferential inhibitor of replicative DNA synthesis in Escherichia coli. I. In vivo characterization. Coumermycin A1, an antibiotic related to novobiocin, inhibited nucleic acid synthesis in intact Escherichia coli with replication being slightly more sensitive to this drug than transcription. The ultraviolet-induced repair synthesis of DNA was only partially inhibited under conditions where replication was eliminated by coumermycin A1. Inhibition of protein synthesis was a secondary effect. Coumermycin A1-resistant E. coli were isolated and the mutation was mapped near dnaA. Chromatography of crude protein extracts of sensitive and resistant bacteria on drug affinity columns implicated a soluble protein of approximately 37,000 molecular weight as the target site for coumermycin A1. Depending on the medium used, this antibiotic had either a bacteriocidal or a bacteriostatic effect on E. coli. Results showed that the effect of coumermycin A1 cannot be explained by the degradation of DNA under bacteriocidal growth conditions.


Coumerimycin A1: A preferential inhibitor of replicative DNA synthesis in Escherichia coli. II. In vivo characterization. In vitro inhibitions by coumermycin A1 of DNA and RNA synthesis in toluenized cells were studied. In a sensitive strain, 50% inhibitions of replication and transcription were observed at 0.035 and 0.600 mug/ml, respectively. DNA synthesis in a toluenized-resistant mutant was 50% inhibited at 0.140 mug/ml of coumermycin A1, whereas RNA synthesis was unaffected at all concentrations tested. Studies with a mixture of toluenized-sensitive and -resistant bacteria ruled out the presence of a diffusable activator or inhibitor of coumermycin A1 action. Density label studies with toluenized pol A+ and pol A- strains indicated that replicative DNA synthesis was specifically inhibited, in agreement with the in vivo studies in the preceding paper of this issue (Ryan, M. J. (1976), Biochemistry 15). Highly purified Escherichia coli DNA polymerase III and RNA polymerase both were inhibited by this antibiotic. However, the high concentrations necessary for these inhibitions suggest that they are not biologically relevant. No interaction between DNA and coumermycin A1 was observed with the following analytical procedures: ultraviolet difference spectra, DNA absorbance-temperature transitions, equilibrium buoyant density centrifugation, and DNA cross-linking determinations.


Proteolytic and chemical modification of colicin E3 activity. Proteolyses of colicin E3 by both trypsin and subtilisin yield fragments of various molecular weights. On the basis of sodium dodecyl sulfate gel electrophoresis, tryptic cleavage yields peptides of molecular weight about 42 000 and 18 000, while the comparable pieces in a subtilisin digest have apparent weights of about 36 000 and 24 000. The digests lose almost all of their in vivo cell killing activity but the in vitro activity leading to ribosomal inactivation is augmented. Trypsin-treated colicin E3 shows a 20-30-fold increase in its ability to release the 52 nucleotide fragment from the 16S ribosomal ribonucleic acid (rRNA) and this activity is associated with the smaller fragment. Subtilisin-treated colicin E3 is only about two to three fold more active than the native protein in vitro, and the peptides obtained upon cleavage cannot be separated by gel filtration or polyacrylamide gel electrophoresis without sodium dodecyl sulfate. However, in the presence of 0.1% sodium dodecyl sulfate, subtilisin-treated E3 shows a 20-30-fold augmentation in in vitro activity which is again associated with the smaller fragment extracted from the sodium dodecyl sulfate gel. Amino terminal end-group studies showed that the two larger fragments and intact E3 have the same N-terminal residue, valine. These fragments presumably originate from the amino end of the native protein. The smaller tryptic fragment has an N-terminal alanine, while the smaller subtilisin piece has an N-terminal leucine. In addition, modification of a single carbosyl group in intact colicin E3 abolishes more than 90% of the in vivo activity with a simultaneous increase in in vitro activity. This carboxyl group is located in the larger fragments obtained in both trypsin and subtilisin cleavage. Binding of E3 to sensitive cells is drastically reduced or eliniated by this chemical modification and by both of the limited proteolytic cleavages.


Pepsin-solubilized collagen of human nucleus pulposus and annulus fibrosus. Human nucleus pulposus and annulus fibrosus, obtained at autopsy from patients 7-30 years of age, were extracted with 2 M guanidine-HCl (pH 5.82) to remove proteoglycans, then stirred with pepsin in 0.5 M acetic acid, followed by three 24-h extractions with 1 M NaCl (pH 7.5) and one 24-h extraction with 2 M KSCN (potassium thiocyanate) (pH 7.2). Pepsin and NaCl solubilized an average of about 30% of nucleus pulposus collagen and 18% of annulus fibrosus collagen. KSCN extracted a further 34% of nucleus pulposus collagen and only 4% of annulus fibrosus collagen. CM-cellulose chromatography of nucleus and annulus collagen purified from the pepsin, NaCl and KSCN supernatants consistently revealed only one peak, always appearing slightly ahead of the alpha1 position for rat tail tendon type I collagen. Polyacrylamide and SDS-gel electrophoresis consistently revealed only one band with the mobility of alpha1 chains. Amino acid composition of collagen from nucleus and annulus is comparable to those of mammalian and avian cartilage type II collagen, and distinctly different from those of rat tail tendonand guinea pig skin type I collagens. Periodate oxidation of nucleus and annulus collagens showed that 81% and 67%, respectively, of the hydroxylysine residues survive treatment, compared to 71% for bovine articular cartilage collagen and 17% for guinea pig skin collagen. Total hexose analysis revealed 1.8 muM and 2.0 muM hexose per muM periodate-stable hydroxylysine in nucleus and annulus collagens, respectively. Ion exchange chromatography showed the presence of glucose and galactose in a ratio of 0.92:1 in nucleas collagen and 1.07:1 in annulus collagen. Pepsin-solubilized, NaCl-extracted collagen from nucleus and annulus formed native-type fibrils in vitro. The banding patterns of ATP-induced segment-long-spacing precipitates of nucleus and annulus collagens were identical to each other and indistinguishable from those of cartilage (type II) collagen, but distinctly different from those of rat tail tendon (type I) collagen. These data suggest that the collagen which can be extracted after limited pepsin attack of human nucleus and annulus is of the form [alpha1 (II)]3.


The properties of ribosomal proteins from a moderate halophile. The ribosomes from the extreme halophile Halobacterium cutirubrum are unusual in that their ribosomal proteins are acidic rather than basic as is the case with almost all bacterial ribosomes (Bayley, S.T. (1966) J. Mol. Biol. 15, 420-427). To determine whether the ribosomes of a moderate halophile show similar properties the ribosomal proteins from an unidentified moderate halophile, which grows over a wide range of NaCl concentrations (0.04-4.3 M), were compared to those of Escherichia coli and H. cutirubrum. The proteins are slightly more acidic than those of E. coli but much less acidic than those from the extreme halophile as judged by their mobility on polyacrylamide gels and their amino acid composition. The electrophoretic profile on polyacrylamide gels of the ribosomal proteins from the moderate halophile is similar whether the cells are grown in 0.5 M or 4.25 M NaCl.


Membrane-associated reactions in ubiquinone biosynthesis in Escherichia coli. 3-Octaprenyl-4-hydroxybenzoate carboxy-lyase. A sensitive and quantitative assay for 3-octaprenyl-4-hydroxybenzoate carboxy-lyase has been developed. This enzyme, which catalyses the third reaction in ubiquinone biosynthesis in Escherichia coli, was partially purified and some of its properties determined. It was found that a considerable proportion of the carboxylyase activity could be separated from the membrane fraction in cell extracts prepared using a French press. Gel filtration showed the molecular weight of the enzyme to be about 340 000. For optimal activity the carboxy-lase was shown to require Mn2+, washed membranes or an extract of phospholipids, and an unidentified heat stable factor of molecular weight less than 10 000. The carboxy-lyase reaction was also shown to be strongly stimulated by dithiothreitol and methanol. The properties of the carboxy-lyase are compared with the three other enzymes concerned with ubiquinone biosynthesis in E. coli which have been studied in vitro. The fact that the substrate of the carboxy-lyase is membrane-bound and the enzyme is stimulated by phospholipid suggests that it normally functions in association with the cytoplasmic membrane in vivo.


Stimulaiton of insulin biosynthesis in isolated mouse islets by L-leucine, 2-aminonorbornane-2-carboxylic acid and alpha-ketoisocaproic acid. An immune binding technique was used for measuring the effects of certain amino acids on the rate of insulin biosynthesis. [13H]phenylalanine served as the radioactive precursor for insulin synthesized by isolated mouse pancreatic islets. L-Leucine was found to stimulate the insulin biosynthesis and this effect was observed already at a physiologic concentration in contrast to the much higher concentrations needed to stimulate insulin secretion in vitro. Furthermore, it was found that 2-aminonorbornane-2-carboxylic acid and alpha-ketoisocaproic acid shared with glucose and L-leucine the ability to stimulate insulin biosynthesis. In contrast, L-alanine, L-arginine adn D-leucine had no stimulatory effect in the absence of glucose, while in the presence of 5 mM glucose L-arginine decreased and L-alanine increased the incorporation rate of tritiated phenylalanine. The fact that many of those compounds which stimulated insulin biosynthesis have also been shown elsewhere to be metabolized by the B-cells supports the view that the rate of insulin biosynthesis may be substrate dependent.


Carbohydrate components of the glycopeptides of ovine lutropin. The three tryptic glycopeptides from ovine lutropin, in which two were from the alpha-subunit (alpha-56 and alpha-82 glycopeptides) and one from the beta-subunits (beta-13 glycopeptide), have been isolated and their carohydrate compositions analyzed. The results indicate that the alpha-56 glycopeptide has the highest amount of carbohydrate, whereas the beta-13 glycopeptide has the least. In general, each of the glycopeptides has similar distribution of various sugars, i.e. high in mannose and glucosamine and low in fucose, sialic acid, galactose and galactosamine. Within the limit of experimental error, the sum of their carbohydrate composition is in agreement with the published data on the intact hormone or separated subunits.


Kinetic properties and the effect of substrate analogues on 5'-methylthioadenosine nucleosidase from Escherichia coli. 5'-Methylthioadenosine nucleosidase (EC 3.2.2-) from Escherichia coli has been purified 220-fold. A molecular weight of 31 000 for the enzyme was estimated from gel filtration on Sephadex G-150. The Km for 5'-methylthioadenosine was 3.1-10(-7) M. In addition to 5'-methylthioadenosine, the nucleoside analogues 5'-ethylthioadenosine, 5'-n-propylthioadenosine, and S-adenosyl-homocysteine also served as substrates for the enzyme. These substrate analogues acted as competitive inhibitors of the reaction with 5'-methylthioadenosine. The Ki values for 5'-ethylthioadenosine, 5'-n-propylthioadenosine, and S-adenosylhomocysteine were determined to be 1.3-10(-7) M, 4.6-10(-8) M, and 1.92-10(-7) M respectively.


Purification and partial characterization of a protein proteinanse inhibitor isolated from eggplant exocarp. A protein proteinase inhibitor was isolated and purified from eggplant exocarp by heat treatment, ammomium sulfate fractionation, column chromatography on DEAE-cellulose, and gel filtration on Sephadex G-25 and G-50. The final purified preparation of the inhibitor was found homogeneous by electrophoretic analysis. The inhibitor showed strong and stoichiometric inhibition on trypsin whereas it showed weak inhibition on alpha-chymotrypsin. It displayed no inhibiting characteristics on pepsin. The molecular weight of the inhibitor was estimated to be approximately 6000. This finding, with the trypsin inhibition data, suggested that the inhibitor combined trypsin in the molar ratio of 1:1. The amino acid analysis indicated that the inhibitor is rich in half-cystine, glycine and aspartic acid, and contains no tryptophan, histidine, methionine or valine.


Distribution of lipids in cytoplasmic and outer membranes of Escherichia coli K12. The lipid composition of cytoplasmic and outer membranes of Escherichia coli K12 was studied. Compared with the cytoplasmic membrane, the outer membrane is enriched in both saturated fatty acids and phosphatidylethanolamine. This is also the case when the fatty acid composition of the phospholipids is changed, either by varying the growth temperature or by using mutants with alterations in their fatty acid metabolism. Phosphatidylethanolamine of the outer membrane contains relatively more saturated fatty acids than phosphatidylethanolamine of the cytoplasmic membrane.


Exchange of phospholipids between mitochondria and microsomes in vitro stimulated by yeast cell cytosol. Yeast cell cytosol stimulated the exchange of phospholipids between yeast mitochondria and microsomes in vitro, and also between organelles isolated from rat liver. The major phospholipids exchanged in both cases were phosphatidylinositol and phosphatidylcholine, together with smaller amounts of phosphatidylethanolamine. Evidence was also obtained that interconversion of phospholipids occurred during the incubation, probably via base exchange mechanisms.


Isolation and characterization of the liponucleotides of Saccharomyces cerevisiae. The liponucleotide fraction of Saccharomyces cerevisiae was isolated from cells grown on tritiated uracil and identified as CDPdiacylglycerol on the basis of (1), its behavior as a single compound upon DEAE-cellulose and thin-layer chromatography; (2), its extreme lability to mild alkaline methanolysis; and (3), its hydrolysis by the CDPdiacylglycerol hydrolase of Escherichia coli to yield tritiated 5'-CMP. At most, only about 5% of yeast liponucleotide could be dCDPdiacylglycerol, in contrast to the presence of nearly equimolar amounts of CDP-and dCDPdiacylglycerols in E. coli. Although no CDPceramide could be detected in the liponucleotide fraction of this organism, the possibility still exists that it may be an intermediate in the biosynthesis of sphingolipids in systems yet to be examined.


The sequence of ppGpp and pppGpp in the reaction scheme for magic spot synthesis. The kinetics of ppGpp (guanosine 5'-diphosphate, 3'-diphosphate) ahd pppGpp (guanosine 5'-triphosphate, 3'-diphosphate) synthesis, at the onset of amino acid starvation, and of their decay after inhibiting synthesis, were analysed in Escherichia coli. The pentaphosphate, but not ppGpp, is the first product of the stringent response to amino acid starvation. The pentaphosphate is rapidly converted with first order kinetics (t 1/2 = 6 s) to ppGpp which is broken down less rapidly (t 1/2 = 20 s).


Synthesis of DNAs complementary to human ribosomal RNAs polyadenylated in vitro. Conditions are described under which poly(A) polymerase from Escherichia coli ribosomes will catalyse the addition of AMP residues onto the 3'-ends of human 18 S and 28 S ribosomal RNAs at an average rate of 40 AMP residues per 1000 nucleotides in 20 min. Single-stranded complementary DNAs (cDNAs) can then be transcribed from the polyadenylated RNAs with RNA-directed DNA polymerase from avian myeloblastosis virus. All of the sequences in the RNAs are represented in the cDNAs; measurements of the rates at which the cDNAs hybridize with their template RNAs showed that, when appropriate adjustments for differences in lengths and G + C contents of the reacting sequences are taken into account, the Rot 1/2 values of homologous RNA-cDNA hybridization reactions are directly proportional to the base-sequence complexity of the RNAs.


A study of DNA spontaneous degradation.

A study of DNA spontaneous degradation. The intact apurinic site half-life in DNA molecules, at 37 degrees C and pH 7.21, in a medium approximating the intracellular ionic composition, was found to be between 288 and 335 h. The spontaneous degradation of 3H-labelled T7 phage DNA, at 37 degrees C and pH 7.21, in the same medium, was followed by sedimentation analysis on sucrose gradients after denaturation with NaOH; it was found to be equivalent to 0.009 break per h per DNA strand. The number of intact apurinic sites found in this incubated T7 phage DNA was much lower than the theoretical value calculated on the assumption that depurination was the only cause of DNA degradation. We concluded that depurination was a minor cause of DNA degradation in our experimental conditions.


2'(3')-O-L-(O-methyl-3-phenyllactyl) adenosine. A potential analog of 2'(3')-O-aminoacyl ribonucleosides. The title compound and the corresponding diacyl derivative has been prepared by the reaction of 5'-O-(4-methoxytrityl)adenosine with L-(O-methyl-3-phenyl)lactic acid in the presence of dicyclohexylcarbodiimide in pyridine and subsequent detritylation in 80% acetic acid. Neither compound accepted the N-acetyl-L-phenylalanyl residue from N-acetyl-L-phenylalanyl-tRNA in an Escherichia coli ribosomal system nor did either inhibit the puromycin reaction in the same system.


New types of streptomycin-resistant mutants of Escherichia coli. Mutagenesis of Escherichia coli K12 cells with ethyl methanesulfonate and selection of streptomycin-resistant mutants after a long delay for phenotypic expression allowed us to isolate new types of streptomycin-resistant ribosomes. Misreading patterns of the ribosomes in the presence of streptomycin revealed that most of the streptomycin-resistant mutants isolated under these conditions differed from the four classical types of streptomycin-resistant mutants studied and characterized by Strigini, P. and Gorini, L. (1970) J. Mol. Biol. 47, 517-530.


Consequences of modification on the association of DNA polymerase with the cell membrane. 1. This is a report of a possible causal relation between the structure of the membrane-bound DNA polymerase and the mutator characteristic of Exherichia coli, mut T1. The characteristics of the membrane-bound polymerase are compatible with its identification as DNA polymerase II, and enzyme which has been determined to be genetically closely linked to the mut T1 gene. Several genes concerned with membrane structure are also known to be linked to the mutator locus. 2. Differences exist between membrane-bound polymerase activity of a wild-type E. coli K-12 and of an isogenic strain harboring the mutator gene. (a) The cell membranes of the mutator strain retain 5--6 times as much activity as do membrane complexes from wild-type cells. (b) The DNA polymerase activity of the membranes from the mut T1 strain is less sensitive to inhibition by the sulfhydryl-binding reagent, N-ethylmaleimide. (c) The membrane-DNA polymerase complex of mut T1 cells uses endogenous, membrane-bound DNA for replication-repair in preference to exogenous DNA. 3. The differences described are specific to structural differences in the membrane complex. When DNA polymerase activity is solubilized from the complexes, the enzymes of the two strains exhibit similar characteristics. These results are consistent with the thesis that an alteration in membrane structure is the basis of mut T1 activity. The results do not support any hypothesis that mut T1 phenotype is a reflection of mutations in the structural gene for DNA replicase (polymerase) or its components.


Specific inhibition of the termination process of protein synthesis by negamycin. The effect of negamycin and its derivatives on protein synthesis in an Escherichia coli cell-free system was determined. (1) Unlike tetracycline and streptomycin, negamycin did not inhibit codon-specific binding of aminoacyl-tRNA to ribosomes. (2) The miscoding activity of the genamycin family compounds was not parallel with the activity inhibiting the termination reaction. (3) Although negamycin strongly inhibited the overall termination reaction, it inhibited only slightly the two substeps involved; the formation of releasing factor 1 - U-A-[3H]G - ribosome complex and the peptidyl-transferase action (release of formylmethionine from the initiation complex by puromycin). (4) The termination reaction performed with ribosomes from either streptomycin- or kanamycin-resistant E. coli cells was sensitive to negamycin. These results indicate that the inhbitory effect of negamycin on the termination reaction is specific to negamycin and distinct from that of tetracycline and streptomycin.


The effects of ionophore A23187 on erythrocytes. Relationship of atp and 2,3-diphosphoglycerate to calcium-binding capacity. The divalent cation ionophore, A23187, was employed as a means to load fresh human erythrocytes with calcium, and the capacity for accumulation was characterized. Erythrocytes exposed to A23187 in calcium-containing media rapidly accumulated calcium in millimolar quantities. The final cellular concentration was dependent upon medium calcium concentration and the size of the cellular organophosphate pool. When ATP and 2,3-diphosphoglycerate contents were depleted or repleted, the cellular calcium content changed proportionally. Calcium loading of fresh erythrocytes produced no discernible change in the cellular concentrations of ATP or 2,3-diphosphoglycerate. Calcium accumulation was also accompanied by loss of cellular potassium and H2O, deterioration of cell filterability, and spheroechinocytic transformation.