Surveillance of communicable diseases in tropical Africa. In tropical Africa, parasitic and infective diseases remain the predominant causes of mortality and morbidity, and health and epidemiological services are poorly developed. Experience elsewhere has shown the value of surveillance in the control of communicable diseases and this paper contains a brief review of the use of surveillance in tropical African countries and considers the priorities, techniques and constraints of the major programmes.


Monitoring communicable disease: vaccination programmes. The success of vaccination as a means of disease prevention depends on its appropriate application and its reputation with the profession and the public. Surveillance, by providing accurate information on effectiveness and the frequency and nature of hazards, is essential to this purpose. Several factors that influence the success and safety of vaccination programmes are discussed. The essential elements of a vaccine surveillance scheme are outlined with special reference to experience derived from monitoring vaccines in current use.


Nutritional surveillance of young children in developing countries. This paper discusses simple but effective methods of surveillance of growth and nutrition of children in developing countries where resources are scarce and food shortages most likely to occur. In the youngest age group, the simplest weight-for-age chart, already used, is undoubtedly the best method. Where the age of children is not known, arm circumference provides an acceptable alternative. In older children, arm circumference alone may not be reliable and can be improved by measuring height as well and combining the two results.


Surveillance in developed countries with particular reference to child growth. Surveillance of biological function rather than of disease events is a possible method of detecting early changes in population health. The reasons for and methods of a pilot surveillance scheme of the growth of schoolchildren in Britain are discussed. The study, started in 1972, supports the feasibility of surveillance using simple growth measurements and information provided by parents on self-administered questionnaires. Other possible applications of this type of surveillance include growth and nutrition at other stages of life as related to nutrition intervention programmes, and respiratory function as related to environmental changes or the introduction of new substances such as 'safer' cigarettes.


Surveillance of tuberculosis.

Surveillance of tuberculosis. Surveillance must be an integral part of any effective tuberculosis programme and should be concerned with two distinct subjects--surveillance of the tuberculosis situation and surveillance of control measures applied. This paper describes and discusses these two aspects with regard to both developed and developing countries, and makes recommendations for the collection of reliable surveillance data.


Surveillance and monitoring of cardiovascular disease: assessment of trends. This paper describes the current state of surveillance and monitoring of acute, chronic and asymptomatic cardiovascular disease with particular reference to the situation in Czechoslovakia. It goes on to discuss some of the difficulties involved in obtaining adequate data on which to base surveillance and of creating a coherent central information system. The importance of combining registration or notification with active intervention is emphasized, and the possible suggested of introducing simple techniques of cardiovascular investigation into health screening procedures or consultations established primarily for another purpose.


Against Popperized epidemiology.

Against Popperized epidemiology. The recommendation of Popper's philosophy of science should be adopted by epidemiologists is disputed. Reference is made to other authors who have shown that the most constructive elements in Popper's ideas have been advocated by earlier philosophers and have been used in epidemiology without abandoning inductive reasoning. It is argued that Popper's denigration of inductive methods is particularly harmful to epidemiology. Inductive reasoning and statistical inference play a key role in the science; it is suggested that unfamiliarity with these ideas contributes to widespread misunderstanding of the function of epidemiology. Attention is drawn to a common fallacy involving correlations between three random variables. The prevalence of the fallacy may be related to confusion between deductive and inductive logic.


New isoprenologs of vitamin K2 series in Sarcina lutea. Four new menaquinone homologs have been isolated and characterized from Sarcina lutea. The use of purified silica gel G in the isolation of pure menquinones from bacterial nonpolar lipids has been developed. During this procedure, fractionation of any naturally occuring menaquinones with sidechain variation takes place. The structures of the isolated menaquinones have been elucidated by UV-, and mass spectrometry, and by their corresponding hydroquinones and layer chromatography properties. Thus, Sarcina lutea has been shown to produce four homologs of dihydromenaquinones, i.e., dihydromenaquinone-6,-7,-8, and -9.


The "ideal" daily vitamin A intake.

The "ideal" daily vitamin A intake. This approach suggests that the healthier the sample, the greater the daily vitamin A intake. Under the conditions of this experiment, approximately 33,000 I.U. of vitamin A may be designated as the "ideal" daily allowance. This is almost seven times the RDA of 5000 I.U. for the adult male and eight times higher than the 4000 I.U. RDA for adult females. It is recognized that the "ideal" is nonexistent as a theoretic end-point since there is biochemical individuality and because the art of measuring leaves much to be desired. Nonetheless, the technique utilized here provides a mechanism and a goal not previously considered.


Pyrithiamine-induced thiamine deficiency: effects on rat myelination. Pyrithiamine was administered to newborn rats throughout the vulnerable period for myelinogenesis. A major metabolic defect was produced in the cerebral activities of the thiamine-dependent enzymes, transketolase and pyruvate decarboxylase. In spite of a defect in carbohydrate metabolism which is lethal in adult rats, overall development, and myelination as indicated by biochemical and morphological criteria, proceeded at an essentially normal rate. These findings indicate alternative metabolic pathways may be operational in newborn rat brain enabling it to circumvent major blockage in thiamine-dependent reactions.


Absence of influence of high doses of biotin on reproductive performance in female rats. Biotin at 2 doses totalling 5 and 50 mg/kg s.c. does not disturb the reproductive performance of the female rats 1-6 weeks after treatment. The number of implantations and resorptions as well as the weights of placentas and foetuses remain normal. The ovaries do not show weight or histological alterations. Vaginal cycle and readiness to mate remain undisturbed.


Interaction of erythorbic acid with ascorbic acid catabolism. After a vitamin C depletion period of 12 days supplementing erythorbic acid (50 mg/day/kg bodyweight) over 16 days significantly accelerated the catabolism of a newly ingested tracer dose of (1-14C) ascorbic aicd in guinea pigs in comparison to animals supplemented with ascorbic acid (5 mg/day/kg bodyweight), but this effect of erythorbic acid could not be normalized by additional supplementation with ascorbic acid. Half-lives for (1-14C) ascorbic acid (50% of the dose excreted) were drastically reduced from 97 h to 50 h and 59 h, respectively. Also figures on the retention of radioactivity showed the availability of ascorbic acid to be reduced. This is in accordance with the significant reduction in size of ascorbic acid bodypool, and in weight gain in the groups receiving erythorbic acid, or erythorbic and ascorbic acids. The bioavailability was calculated to be strongly depressed to nearly 50% of the total ingested biologically active material (ascorbic plus erythorbic acids), assuming erythorbic acid to have only one-twentieth of the biological activity of ascorbic acid.


The "ideal" daily niacin intake.

The "ideal" daily niacin intake. This approach indicates that the healthier the sample, the greater the niacin intake. Under the conditions of this experiment, approximately 115 mg of niacin may be designated as the "ideal" daily allowance. This is six or seven times the RDA of 12 to 20mg for adult males and females. It is recognized that the "ideal" as a theoretic endpoint is nonexistent since there is biochemical individuality and because the art of measuring leaves much to be desired. Nonetheless, the technique utilized here provides a mechanism and a goal not previously considered.


Blood values of some vitamins in long-stay psycho-geriatric patients. A group of 18 long-stay hospital psycho-geriatric patients (aged 64-101 years) and a group of 10 healthy elderly subjects (aged 53-67 years) who were living in their own residence were studied. The group of geriatric patients were suffering from some degree of pressure sores, dementia, irritability and anorexia. Biochemical tests on vitamin adequacy with particular references to ascorbic acid, thiamine and retinal were performed in the subjects. The thiamine status was measured by determining the thiamine pyrophosphate stimulating effect of transketolase enzyme activity in whole blood. The ascorbic acid and retinal nutritures were determined by measuring their levels in plasma. From the biochemical data of the vitamins it would appear that a significant proportion of the long-stay hospital psycho-geriatric patients may be at risk of thiamine, ascorbic acid and retinol deficiencies.


Hypocalcemic development in high and low calcium-adapted chicks during acute calcium deficiency. Chickens were adapted to either a high or low calcium intake and then subjected to an acute calcium deficiency by feeding ground yellow corn +/- phosphate. Hypocalcemia developed within 3-5 h in those chicks given the corn plus phosphate. The response was about equal in the high and low adapted animals suggesting that the effect was not mediated via regulatory factors such as parathyroid hormone or thyrocalcitonin intervention. Rather, it is suggested that the influence of phosphate is direct and may be due to an enhancement of calcium movement from the fluids to the bone. The high calcium adapted chicks also exhibited some hypocalcemia in response to eating corn without phosphate supplementation. This development, contrary to the other cases, appeared to be due to a lag in calcium regulating mechanisms. The low calcium adaptation did not seem to influence growth within the timespan used, but there was a significant reduction in bone ash values and of adrenal enlargement. Thus, it was evident that the low and high calcium diets were different in regards to calcium metabolism and it appears that the low calcium intake may have been stressful.


An evaluation in rats of nutritional status and reproduction performance post oral contraceptive therapy. Adult female rats were orally administered either 0.052, 0.26 mg of Enovoid E (OC) or the vehicle, propylene glycol. Thereafter they were mated six, thirteen or twenty-seven days after the discontinuation of the drug. Chemical analyses i.e. cholesterol levels in plasma, liver and adrenals; tocopherol levels in plasma and liver; fatty acid patterns of cholesteryl esters of plasma liver, and adrenals, and plasma lipoproteins distribution, were performed on the pre-breeding (post-OC) females, mothers (following weaning of the young) and young female progeny, 10 weeks after weaning. Weanlings derived from mothers previously exposed to OC weighed more than their counterparts from mothers who had not been given the drug. A slight decrease of the survival time of the young during lactation was also observed in groups treated with higher levels of OC. Liver weights were increased in mothers as a result of pregnancy and lactation. Observed elevations of plasma cholesterol levels and cholesteryl arachidonate in the livers of the progeny were probably due to prolonged exposure to the 15% corn oil diet. In addition, previously reported metabolic effects of OC, such as increases in liver cholesteryl oleate, decreases in plasma and adrenal cholesteryl arachidonate, decreases in the alpha/beta lipoprotein ratio and alpha-tocopherol levels in plasma were confirmed.


[Absorption and metabolic efficiency of iron in nickel deficiency]. Absorption and Metabolic Efficiency of Iron During Ni Deficiency. Ni deficiency leads to reduced iron contents in organs and to greatly reduced Hb levels and erythrocyte counts. Using models it was studied whether this Ni-dependent Fe anemia can be attributed to an impaired absorption of iron or to its metabolic efficiency. An experiment with seven 30-day-old rats from each of two generations were used for this. In Ni deficiency (0.015 ppm dietary nickel) iron absorption was clearly impaired at both 50 ppm and 100 ppm iron in the diet. Compared to the groups given 20 ppm nickel, the amount of iron absorbed fell two-thirds and one-third, respectively. By comparison, the influence on the metabolic efficiency of the iron was relatively small; at high iron supply, however, it was reduced by 8% in the Ni-deficient animals. Therefore, the reduced levels of hemoglobin, erythrocytes and hematocrit must essentially be caused by the impared absorption.


Visceral angiography. A community hospital experience. Selective visceral angiography is well within the ability of small community hospitals. If carefully performed by a well-trained interested radiologist, the procedures carry negligible morbidity and mortality. These studies can provide an exact diagnosis, a working differential or at least enough data to plan a surgical approach with accuracy. Nine cases in which the angiographic diagnosis was vital to the patients' care have been presented.


Concomitant prophylactic inferior vena caval clipping. Fatal pulmonary embolism is a common complication of any major surgery, especially in high risk patients. Experimental work in animals showed definite impairment of myocardial function after inferior vena caval ligation, but no changes after clipping. During the last four years, we have used concomitant vena caval clipping as a prophylactic measure in major abdominal vascular procedures. No added mortality or morbidity has occurred because of clipping per se and no deaths were due to pulmonary embolism. We recommend concomitant prophylactic clipping as an adjunctive procedure for all laparotomy patients who are prone to pulmonary embolism.


Jejunal mucosal graft with transhepatic intubation. Rodney Smith's technic for very high strictures of the hepatic duct. Very high hepatic strictures present a difficult surgical problem. Restenosis frequently occurs even when the best method has been applied. Rodney Smith developed a method of transhepatic intubation in association with a jejunal mucosal graft through which the transhepatic tube is passed and fixed. We applied this technic in two cases and consider the results obtained to be very satisfactory. The patients have been followed for four and 3-1/2 years respectively.


Clinical review of 106 consecutive carotid endarterectomies. We have reviewed 106 consecutive carotid endarterectomies performed from 1966 through 1973. During this period, 99 elective procedures and seven emergency procedures were done on 84 patients, 22 of whom had bilateral endarterectomies. Indications for elective procedures included transient ischemic attacks, emboli from ulcerative plaques, high grade stenotic plaques on the same or opposite side in patients with old cerebrovascular accidents and asymptomatic bruits. Seven patients had emergency endarterectomies performed for acute strokes and these demonstrated a higher risk. They were grouped together as emergency procedures for comparison with the 99 elective procedures. Previous histories, risk factors, complications and outcome in the two groups are presented and discussed. Mortality in the elective cases was 2%, compared to 30% in the emergency cases with acute stroke. Surgical indications, operative technic and immediate and late results are discussed.


Unusual problems in renal hypertension.

Unusual problems in renal hypertension. Ten cases of renal hypertension with uncommon causes were presented and unusual problems in their diagnosis and surgical management were discussed. These causes included segmental stenosis involving aberrant renal artery, renal artery stenosis in association with renal arteriovenous fistula, postoperative renal hypertension and hypertension secondary to renal tuberculosis. A high index of clinical suspicion and increasing awareness will help in diagnosing such rare and unusual cases of renal hypertension.


The value of endoscopic retrograde opacification of biliary-pancreatic ducts in icterus. Important progress in the last few years in fiberscopic instrumentation for digestive endoscopy has made possible duodenoscopy with catheterization of the papilla to obtain retrograde biliopancreatic contrast x-rays. Up to now the only possible way to explore biliary ducts, in icterus, was surgically. This new method makes their study possible without any surgical intervention and with a simple neuroleptanalgesia. It is most advantageous in icterus where clinical tests, laboratory tests and other complementary examinations such as laparoscopy do not help to determine the cause. This examination is not devoid of dangers, particularly of the risk of cholangitis and pancreatitis and therefore certain technical precautions are necessary, e.g., immediate surgical intervention in case of organ obstruction and well defined indications.


Sacrococcygeal teratoma in the neonate.

Sacrococcygeal teratoma in the neonate. Three cases of sacrococcygeal teratoma of the neonate occurred during a two-year period in which approximately 6,000 live births occurred in our hospital. Two infants were delivered vaginally and one by cesarean section. One infant underwent immediate surgery and survived, whereas the other two were in too poor condition to warrant surgery. Early excision is the treatment of choice; radiotherapy and chemotherapy are not effective in the malignant variety of sacrococcygeal lesion.


The effects of ophthalmic drugs, vehicles, and preservatives on corneal epithelium: a scanning electron microscope study. Scanning electron microscopy was found to be of considerable use in evaluating the effects of various commonly used drugs, vehicles, and preservatives on the corneal surface. Of the preparations tested 0.3 per cent gentamicin caused many central cellular microvilli to stand up prominently. Moderate losses of peripheral microvilli, with mild superficial cellular desquamation was noted with 0.25 per cent phospholine iodide, 2 per cent pilocarpine, 2 per cent fluorescein, and Fluor-I-Strip. The top layer of epithelial cells desquamated with 4 per cent cocaine or neopolycin treatment. The top two layers of cells were lost when 0.01 per cent benzalkonium chloride was instilled. When cell death occurred severe membrane disruption was accompanied by loss of microvilli and rupture of intercellular tight junctions. These studies show that the cytotoxicity of topical ocular preparations can be tested in an in vivo model and evaluated by scanning electron microscopy.


Human alpha-crystallin. I. The isolation and characterization of newly synthesized alpha-crystallin. Studies of the incorporation of 14C amino acids into human lens proteins demonstrate that an alpha-crystallin fraction takes up more than six times as much radioactivity as any other lens protein. Based on analyses with a calibrated Bio-Gel A-1.5 m column, a molecular weight of 4.9 x 10(5) +/- 5 per cent was obtained for this protein while sedimentation equilibrium analyses indicated a weight average molecular weight, Mw, of 7.5 x 10(5) +/- 4 per cent at 10,000 r.p.m. Gel electrophoresis in sodium dodecyl sulfate revealed two components with molecular weights of 22,000 and 20,000, values similar to those found with calf alpha-crystallin. Alkaline urea gel electrophoresis indicated one major polypeptide with a mobility similar to the B2 chain of calf alpha-crystallin and two major bands with mobilities between those of the calf alpha-crystallin A2 and A1 chains. Amino acid analyses of this newly synthesized alpha-crystallin gave a composition which with a few exceptions is very similar to that of calf alpha-crystallin. All three major polypeptides contained 14C amino acids. However, from the present data, it cannot be determined whether the three polypeptides were independently synthesized or a rapid transformation produced one of the labeled polypeptides in the A region. There appears to be between three and four times as many presumptive A as B polypeptides.


Cytochemical studies on endogenous peroxidase in conjunctival and corneal epithelial cells. Endogenous peroxidase activity in conjunctival and corneal epithelial cells was studied cytochemically by the diaminobenzidine method. Many peroxidase-positive cells were found in the fornical conjunctiva. They were also seen, but less frequently, in the tarsal conjunctiva, limbus, and cornea. Three types of peroxidase-positive (cytochemically reactive) cells were differentiated besides goblet cells: (1) the most superficial cells with several reactive secretory granules (2) moth-eaten cells near the surface with abundant reactive microgranules, and (3) deeper cells with reactive microgranules and a few densely reactive granules. Some goblet cells shared the same characteristics with other types of cells. The relationship among these cells, functional differences between granules, and the function of peroxidase are discussed.


Corneal membrane water permeability as a function of temperature. Osmotically driven water flow across the corneal epithelium and endothelium has been measured as a function of temperature. For both membranes deviations from a single straight-line relationship are found in a logarithmic plot of hydraulic conductivity against 1/T. Both membranes show a high (14 to 16 kcal per mole) apparent activation energy at temperatures between 23 degrees C. and 5 degrees C. At higher temperatures, between 23 degrees C. and 37 degrees C., the apparent activation energy falls to 5.7 and 9.3 kcal per mole for the epithelium and endothelium, respectively. The low value for the apparent activation energy for water crossing the epithelium and endothelium at normal corneal temperatures presumably reflects water passage through water-filled channels.


Objective evaluation of experimental ocular irritation. After topically administered nitrogen mustard the irritative effect on the pupil, intraocular pressure, protein concentration of the aqueous humor and on the vessels of the iris and conjunctiva was quantified. The protein level in aqueous was determined by refractometry. The degree of hyperemia of conjunctiva and iris was determined by measuring the amount of Evans blue dye present in the tissue minutes after intravenous administration. A reproducible relationship was obtained using these methods to evaluate the degree of irritation produced by nitrogen mustard.


Intraocular lysozyme in experimental uveitis in rabbits: aqueous and vitreous assay. We determined normal aqueous and vitreous lysozyme levels in rabbit eyes and induced experimental uveitis to record the uppermost aqueous and vitreous lysozyme levels. The normal aqueous humor of the rabbit eye contained 1.05 mug per milliliter lysozyme and the normal vitreous humor contained 0.45 mug per milliliter. After the intravitreal administration of a foreign protein, the aqueous and vitreous lysozyme levels rose within one day, reaching maximum values of 38.4 mug per milliliter and 114 mug per milliliter, respectively, at 14 days, and subsequently declining to minimal values by 28 days after injection.


Congenital total external ophthalmoplegia associated with infantile spinal muscular atrophy. Fine structure of extraocular muscle. A case of total congenital external ophthalmoplegia associated with infantile spinal muscular atrophy is presented. In the first 29 months of life, ophthalmoplegia has remained complete. Ultrastructure of lateral rectus extraocular muscle indicates a neurogenic process as the basis of the ophthalmoplegia. Light microscopy alone is insufficient to distinguish primary "myopathic" from "neurogenic" disease in external eye muscles.


Organic cation uptake in vitro by the rabbit iris-ciliary body, renal cortex, and choroid plexus. The uptake in vitro of radioactively labeled test substances was studied in tissues from albino rabbits. Choroid plexus, slices of outer renal cortex, and iris-ciliary body were incubated in a K-rich medium containing one of the cations 14C-Emepronium (Cetiprin), 14C-tetraethylammonium, 14C-choline, or 125I-o-iodobenzyltrimethylammonium and sometimes the anions 131I-o-iodohippurate and 125I-iodipamide. Choroid plexus and renal cortex accumulated all test substances, some to very high tissue-medium ratios. The iris-ciliary body preparation accumulated the anions well but the organic cations only weakly. The only convincing uptake was that of Emepronium. The affinity of this uptake system seemed to be similar to that in the kidney, half-saturating around 10(-4)M Emepronium.


Studies of aqueous humor dynamics in man. V. Effects of acetazolamide and isoproterenol in young and old normal volunteers. Young and old volunteers received acetazolamide systemically or isoproterenol topically. Although the responses of the two groups of volunteers were quite similar for acetazolamide, they differed significantly for isoproterenol. It is suggested that a normal aging change is altered responsiveness of the site of aqueous production and of the outflow pathways to topical isoproterenol. The site of aqueous production becomes less responsive; the outflow pathways become more responsive.


The rates of movement of Na+, Cl-, and HCO-3 from plasma to posterior chamber: effect of acetazolamide and relation to the treatment of glaucoma. The rates of accession of sodium and chloride, and the formation of bicarbonate into the posterior chamber have been studied in normal dogs, and following complete inhibition of carbonic anhydrase by acetazolamide, 50 mg. per kilogram. Sodium accession approximated fluid formation rate and was reduced 30 per cent by acetazolamide. Bicarbonate formation was reduced to one-third the control value by the inhibitor; chloride was unaffected. Reduction of accession was 1.9 mM per minute for sodium and 1.6 mM per minute for bicarbonate, showing that these are stoichiometrically linked. These results, together with other data in fish and rabbit, support the idea that bicarbonate formation is a general function of ciliary epithelia and, through its effect on sodium, is the basis for a considerable component of fluid movement into the eye. Inhibition of bicarbonate formation appears to be the pharmacological basis for the treatment of glaucoma by carbonic anhydrase inhibitors.


Aqueous humor dynamics in glaucomato-cyclitic crisis. The transfer coefficients of fluorescein in the anterior chamber by flow, kfa, and by diffusion, kdpa, can be calculated for an individual human eye, through an analysis of fluorescein concentrations in the anterior chamber, pupillary aqueous, and serum ultrafiltrates following intravenous injection. Using this technique, aqueous humor dynamics was studied in eight patients with glaucomato-cyclitic crisis. In seven patients, the coefficients determined during the attacks averaged 1.23 +/- 0.08 (S.E.M.) X 10(-2) min.-1 for kfa and 3.51 +/- 0.81 X 10(-3) min.-1 for kdpa in the involved eye, and 0.91 +/- 0.12 X 10(-2) min.-1 for kfa and 1.36 +/- 0.39 X 10(-3) min.-1 for kdpa in the fellow eye. The differences in the coefficients between the two eyes were statistically significant (P less than 0.05). In six patients, the coefficients were measured during the remission, giving average values of 0.81 +/- 0.08 X 10(-2) min.-1 for kfa and 1.36 +/- 0.16 X 10(-3) min.-1 for kdpa in the involved eye, and 0.78 +/- 0.07 X 10(-2) min.-1 for kfa and 1.20 +/- 0.10 X 10(-3) min.-1 for kdpa in the fellow eye. The differences in the coeficients between the two eyes were not significant. In five patients, the determinations were repeated during the attack and remission, and the differences in the coefficients between both phases were statistically significant (P less than 0.05) in the involved eye.


The effect of dopamine on the intraocular pressure and pupil of the rabbit eye. The presence of a dopamine-specific receptor that can influence intraocular pressure in the rabbit eye is suggested by these experiments: intravitreal, systemic, or topically administered dopamine solutions can produce a decrease in intraocular pressure that is dose-dependent. In high doses the decrease in intraocular pressure is accompanied by mydriasis, and alpha-adrenergic pupillary response. This alpha-response may be caused by dopamine as a direct, but weak alpha-stimulator or by displacement of norepinephrine onto the receptor from its storage site. At lower doses the decrease in intraocular pressure occurs in the absence of mydriasis and in spite of blockade of beta-adrenergic receptors. Further, a specific dopaminergic blocker, haloperidol, prevents this decrease in intraocular pressure. The decreases are small, but reproducible and suggest that dopamine can influence the adrenergic regulation of intraocular pressure.


Human alpha-crystallin-III isolation and characterization of protein from normal infant lenses and old lens peripheries. Alpha-crystallin isolated from the peripheries of old normal or cataractous lenses appears to be identical, consisting of eleven polypeptides, five B, and six A chains. In contrast, alpha-crystallin isolated from normal six-week-old human lenses has only three major polypeptides, corresponding to B1, A1, and A2 of the old human lens protein as well as small amounts of some of the other components. Comparisons with bovine alpha-crystallin are also reported. Based on gel filtration experiments with Bio-Gel A-1.5m, two distinct populations of alpha-crystallin were found in old lens periphery, one containing species greater than 1.5 X 10(6) daltons and another of approximately 9 X 10(5) daltons. In the cataract preparations, the higher molecular weight fraction is predominant. This fraction is not present in young lenses.


Differential sensitivity at the cellular level in primary open-angle glaucoma: prednisolone and ouabain. The concentration of drug necessary to inhibit lymphocyte transformation by 50 per cent (I50 value) was determined for the glucocorticoid prednisolone-21-phosphate, and for the sodium-potassium adenosinetriphosphatase inhibitor ouabain, in 37 persons representing the full range of ocular and cellular glucocorticoid sensitivity. The lymphocytes of patients with primary open-angle glaucoma, and of high ocular responders to glucocorticoids, were more sensitive to prednisolone-21-phosphate than were those of ocular low and intermediate responders. By contrast, there was no significant difference in sensitivity to ouabain. There was also no significant correlation of lymphocyte sensitivity to prednisolone-21-phosphate with that to ouabain. These results provide indirect evidence that the increased cellular sensitivity to glucocorticoids in primary open-angle glaucoma is a specific effect, and not merely representative of a general vulnerability of "sick cells."


Effect of long-wave ultraviolet light on the lens. I. Model systems for detecting and measuring effect on the lens in vitro. Rat mouse, and chick lenses incubated with 3-aminotriazole under long-wave ultraviolet (UV) show reduced accumulation and incorporation of leucine and a loss of glutathione. The effect on leucine incorporation is strikingly enhanced when capsule-epithelium pools are incubated. The procedure may identify photosensitizers or metabolic inhibitors which are cataractogenic when acting in conjunction with UV.


Xerophthalmia.

Xerophthalmia. Some characteristics of children with xerophthalmia are described and the difference in age between those with serious corneal xerophthalmia and those showing milder conjunctival xerophthalmia is noted. The various public health measures instituted to prevent xerophthalmia are examined. The particular importance of protein in food given to severely malnourished children suffering from xerophthalmia is stressed and studies on enzyme activities which are enhanced in the xerophthalmic cornea are briefly noted.


Lens growth in the Nakano mouse.

Lens growth in the Nakano mouse. The growth curve of the lens of the Nakano mouse was compared to that of the normal mouse. There was no obvious difference for the first two weeks of age. After this period the growth of the normal lens continued while that of Nakano mouse lens slowed abruptly and eventually ceased. Studies with labeled leucine, however, showed that even after the appearance of the "pin-head" opacity the protein synthesis, although depressed, continued. These findings combined with previous histological observations suggest that new fiber formation is probably unaffected in the early stages of the Nakano cataract. The apparent cessation of lens growth is probably associated with the extensive liquefaction that is observed to occur at the posterior nuclear region.


The thickness of the human lens cortex in the different types of senile cataract. In every type of beginning senile cataract the cortex of the lens measured with the slit lamp appears thinner than the normal lens cortex in the same age group. The decreased rate of fiber formation and of proteosynthesis precedes the first appearance of lens opacities.


Incorporation of glutamic acid into soluble protein as a function of age. The incorporation of U-14C glutamic acid into soluble bovine and human lens protein fractions has been studied as a function of age. Culture technique and DEAE cellulose fractionation has been used. In bovine lenses the incorporation of glutamic acid into alpha and gamma crystallins decreased with age whereas in the beta crystallin group the specific activities remained constant. All protein fractions of the human lenses showed a gradual reduction of the incorporation of the radioactive amino acid between 40 and 74 years of age. The highest specific activity was found to correspond to Spector's HL protein and its existence is confirmed throughout the whole human life-span.


A study of the distribution and origin of myoinositol in the cornea of the rabbit. The concentration of inositol in the cornea of the rabbit is about one millimole per kilogram tissue water, a level eight- to tenfold that of the aqueous humor. The present study shows that its concentration in the stroma is about the same as in the epithelium and endothelium. Uptake of 2-3H inositol by the corneal layers above the level in the medium was demonstrated in vitro, but the low ratios obtained do not exclusively indicate active transport of the compound. Synthesis of inositol from glucose by the cornea was shown and the estimated rate is compatible with the hypothesis that inositol originates in the cornea and diffuses across the endothelium to the aqueous humor.


Ultrastructural effects of Conray 60 and Pantopaque on ependyma and choroid plexus following intraventricular injections. Electron microscope evaluation of choroid plexus and ependyma following single cerebral intraventricular injections of Conray 60 and Pantopaque was carried out on 35 rats and 4 dogs. The animals were sacrificed at periods ranging from 1 hour to 4 months. Conray was not detected with the light or electron microscope; however, Pantopaque was presumptively localized as electron-dense masses associated with lipid-like bodies at the ventricular interface of both choroidal and ependymal epithelium. Conray 60 injections consistently induced convulsions in rats. Histological studies demonstrated moderate cellular damage and multilayered proliferation of ependymal epithelium. Morphological damage following Pantopaque was more severe and widespread in both choroid plexus and ependyma suggesting that, of the two agents, Conray may have the greater clinical potential provided that the associated convulsions are controlled by appropriate measures.


The nephrogram of normovolemic, renal artery hypotension. Experimental hemorrhagic hypotension results in a prolonged nephrogram during excretory urography. We evaluated the effect of reduced renal perfusion pressure on the nephrogram in normovolemic dogs. Renal artery perfusion pressure to one kidney was reduced to 40-50 mm Hg by inflating a renal artery balloon catheter. Subsequent urograms were qualitatively assessed for nephrographic density. Plasma and urinary iodine concentrations were determined by fluorescent excitation analysis. Total renal blood flow (TRBF) and its intrarenal distribution were assessed with isotopically labeled microspheres. In the majority of dogs severe oliguria and a prolonged and increasingly dense nephrogram were produced in the kidney exposed to reduced perfusion pressure. The nephrogram of the contralateral, normotensive kidney rapidly diminished in density, associated with the excretion of a large volume of dilute urine. The total urinary iodine excreted in 60 min was significantly less than that excreted by both kidneys in control experiments. TRBF to the hypotensive kidney was significantly reduced from its control level, and the percentage of TRBF perfusing the outer cortex decreased.


Metrizamide in experimental urography. IV. Effects of bilateral ureteric stasis on urine iodine concentration after intravenous infection of an ionic and a non-ionic contrast medium. The non-ionic contrast medium metrizamide was compared to the sodium salt of diatrizoate to investigate the potential usefulness of metrizamide in clinical urography when ureteric stasis is applied. After intravenous injection of the contrast media to rabbits at dose level 175 mg I/kg, the kidneys were subjected to temporary bilateral ureteric stasis. Urine was collected through ureteric catheters and analyzed for its concentration of iodine, sodium and potassium. After metrizamide injection the urine iodine concentration was twice as high as after sodium diatrizoate injection. This difference between one non-ionic and one ionic contrast medium was larger than what has been reported earlier during free flow of urine at the same dose. Furthermore, during the periods of ureteric stasis metrizamide was excreted faster than diatrizoate. When diatrizoate was given as its sodium salt, the sodium given was excreted at about the same rate as the diatrizoate given.


Experimental renal artery thromboembolism. The natural history of experimental renal artery embolism with autogenous thrombus in 16 mongrel dogs has been investigated by sequential selective renal angiography and correlated with examination of the kidneys following autopsy. Angiography in the early (0-24 hr) post-thromboembolism period shows evidence of arterial obstruction and/or spasm which progressively disappears leaving no apparent permanent damage. However, within one or two weeks arterial and nephrographic changes and loss of renal size are discernible by careful comparison with the pre-embolism angiogram. At autopsy multiple disseminated microscopic infarcts involving the convoluted tubules were demonstrable in all cases. Occasionally large wedge-shaped infarcts involving the full thickness of the cortex were superadded. The potential importance of these findings relating to angiographic technique and assessment of treatment of renal thromboembolism is discussed.


In vivo measurement of hepatic iodine concentration using fluorescent excitation analysis. Hepatic iodine concentration was measured in the live dog by external use of fluorescent excitation analysis. The number of characteristic photons produced by interaction of exciting radiation from an americium-241 source with iodine within the tissue is proportional to the tissue iodine concentrations. A correction is made for absorption of radiation by the abdominal wall and other tissues lying between the volume of liver being assayed and the detector collimator. The technique is applicable to the in vivo measurement of iodine concentrations from 0.5 to 40 mg/g. Accuracy of the technique is approximately +/- 10%, which is within the range of variation in iodine concentration at various sites within the liver. Radiation dose is low, and radiolabeled tracer compounds need not be used.


Comparison of left ventricular ejection fraction and regional contractility fraction pre and post coronary angiography. Thirty patients [four normals, eight with mild and 18 with severe coronary artery disease (CAD)] were studied to evaluate left ventricular function type and post coronary angiography. The end diastolic volume (EDV), end systolic volume (ESV), ejection fraction (EF) and the regional contractility fractions (RCF) of eight ventricular segments were evaluated by left ventriculography. The RCF's were analyzed, in addition to the EF's to ascertain the effect of contrast material upon normal and scarred or ischemic segments of the left ventricle, and to rule out spurious findings in the EF due to coexistent reciprocal changes in RCF's. There was good correlation (r = .45 to .97 and p less than .01 to less than .001) in the above parameters of left ventricular function in patients with and without CAD before and after angiography. Thus, although intraventricular and intracoronary injections of contrast material produce transient changes in myocardial contractility, in a clinical setting left ventriculography may be performed prior to or following coronary angiography without danger of spurious results in normals and in patients with CAD.


Measurement of histamine, complement components and immune complexes during patient reactions to iodinated contrast material. Studies were performed on nine patients who had just suffered urticarial or bronchospastic reactions following injection of iodinated contrast material (ICM). The parameters studied were white cell histamine, serum complement components C'3 and C'4 and circulating immune complexes. The white cells of the nine patients demonstrated an average increase of 125% in intracellular histamine over the 3-4 week period following the ICM reaction. Control volunteers averaged only a 10% change. The data implies that there was a loss of white cell histamine during the ICM reaction with replenishment during the following 3-4 week period. Complement components C'3 and C'4 were normal. Circulating antigen-antibody complexes were sought via precipitin bands in agar gel using Clq. None could be isolated following seven ICM reactions. Thus these ICM reactions appeared to release histamine without detection of circulating immune complexes and with normal levels of two major complement components.


High renin activity accompanying angiotensin II inhibition in rats with ischemic renal failure. Acute renal failure was produced in rats by right nephrectomy and total occlusion of the left renal artery for 70 min. Angiotensin II competitive inhibitor, P113 (1-sar-8-ala-angiotensin II), was administered intravascularly for 100 min, starting 15 min before the clamping of the renal artery. A marked rise in plasma renin activity was observed 15 min after declamping and was significantly higher in the P113-treated rats than in saline-treated animals. The rise in plasma renin activity was observed 15 min after declamping and was similar in the two groups, indicating that P113 does not prevent the development of acute renal failure in this experimental model. It is suggested that the marked rise in plasma renin activity may be due to interruption of the normal feedback mechanisms which suppress renin release, as a result of occupation of the angiotensin II receptor sites by P113.


Heat-enhanced radioresponse in HeLa cells. The response of HeLa cells to X-irradiation increases when the cells are irradiated at elevated temperatures or when irradiation is followed by hyperthermia. The magnitude of this thermal effect increases with increasing temperature and varies inversely with dose rate during exposure of the cells to the combined treatment. Enhancement of the radioresponse by postirradiation incubation at elevated temperatures increases with increased incubation time. The results suggest that in HeLa cells the synergistic interaction between hyperthermia and irradiation is due to interference with the repair of potentially lethal damage and/or enhanced expression of lethal damage.


Human chorionic gonadotropin stimulation of Leydig cell function in puberty. Comparison of testosterone response in plasma and urine. The plasma and urinary  testosterone response to one i.m. injection of 5,000 IU human chorionic gonadotropin (HCG) was tested in 45 children and adolescents of several diagnostic entities: testosterone was measured before the injection and on the second, fourth and sixth days thereafter. It was found that there was a better correlation between the testosterone concentrations and the different pubertal stages than between testosterone concentration and either chronological or skeletal age. In the children with normal pubertal development, there was a slight rise in basal testosterone concentration with the progression of the pubertal stages. HCG caused a rise in plasma and urinary testosterone: this response was more marked in pubertal stages 4 and 5. The urinary response was variable whereas the changes in plasma testosterone concentration were more constant. In children and adolescents with primary or secondary hypogonadism, the basal levels of testosterone were low or undetectable and there was only a slight response to the single dose of HCG. It was concluded that a single i.m. injection of 5,000 IU HCG and the determination of plasma testosterone before, and two and four days after injection is a useful screening test for Leydig cell function.


High incidence of ventricular septal defects in a family. A family is described in which eight children, belonging to two sibships, had ventricular septal defects. The diagnoses were based on the clinical, radiological and ECG findings. The findings are compatible with a multifactorial mode of inheritance carrying a recurrence risk of 25 to 50%.


Osteochondroma following irradiation. Case report and review of the literature. A case of an osteochondroma of the distal radius appearing 10 years after irradiation for hemangioma in infancy is described. The literature is reviewed and the effects of irradiation on bone growth, and possible malignant change, are discussed.


Herpes simplex encephalitis simulating systemic embolism. A case of herpes simplex virus encephalitis is described in a patient known to suffer from chronic atrial fibrillation and whose clinical symptoms suggested cerebral embolism of cardiac origin. The final diagnosis was based on the presence of herpes simplex virus type 1 antibodies in the cerebrospinal fluid and a significant rise in antibody titers in the serum. The patient regained consciousness after craniotomy and treatment with idoxuridine. The case emphasizes that the possibility of herpetic encephalitis should be considered in patients presenting with typical signs of a cerebrovascular accident.


High-purity water supplies for biomedical research laboratories. A quantitative bacteriological survey of high-purity water systems used in biomedical research laboratories at the National Institutes of Health revealed great variations among the systems. In general, distilled water had significantly lower contamination levels than deionized water. Interviews with investigators revealed their conceptions about the importance of bacterial contamination in high-purity water and some expectations about bacterial quality. Guidelines are suggested for the use of water from systems of varying quality.


A stable liquid human reference serum.

A stable liquid human reference serum. A procedure is described for the preparation of a liquid reference control suitable for use in calibration of both automated and manual chemical procedures. Pooled human serum was partially dehydrated under vacuum from the frozen state. Ethylene glycol was added to replace the water to produce a 30% solution. The stability of a liquid reference control so prepared was investigated by examining aliquots stored at room, refrigerator, and freezer temperatures for various storage times up to 568 days. Eighteen components were assayed with the Technicon Auto Analyzers SMA 6/60 and 12/60 in a tandem mode with creatinine and creatine phosphokinase channels replacing the glucose and urea nitrogen of the standard SMA 12/60. Initially specimens were examined daily, then at weekly, monthly, and longer intervals. Standard deviations and coefficients of variation were compared with those obtained on commercial lyophilized controls assayed during the same time interval. The results indicated that the ethylene glycol treatment can be used to produce a liquid reference control with stabilized enzyme activities and stable component levels.


Evaluation of tests for infectious mononucleosis through proficiency testing. Two standard tests for infectious mononucleosis (the Davidsohn differential and the ox cell hemolysin test) and ten slide tests; Monotest (Wampole), Monospot (Ortho), IM Kit (Micro. Res. Corp.) Monosticon (Organon), Diagluto IM (Beckman), Monosticon Dri-Dot (Organon), Bacto-Hetrol (Difco), Mono-Diff (Wampole), Monophile (Bio-Diagnostic Systems) and Rythrotox (BMC) were evaluated as they were used in 50 laboratories within the State of Utah and by two referee laboratories outside the state. The state proficiency testing program for infectious mononucleosis was modified so that the desired evaluation survey data could be obtained. Sensitivity, specificity, reproducibility (within and between shipments) and percent agreement with target results were determined for each test; factors which might have affected the test results were evaluated to determine which were correlated with good performance.


Hepatoencephalopathy associated with situs inversus of abdominal organs and vascular anomalies in a dog. Throughout its life, a 1-year-old Doberman Pinscher dog had had gastrointestinal disorders and episodes of circling. It was less active than its littermates, and it usually seemed lethargic. When 3 months old, the pup had been anesthetized for ear cropping and it had remained anesthetized for 32 hours. Behavioral problems were apparent when the dog was 11 months old. Two weeks later the behavioral problems accentuated to the point that the dog developed "temper tantrums" and became aggressive toward its owner. On the basis of clinicopathologic and contrast radiographic findings, hepatoencephalopathy due to persistent patent ductus venosus was diagnosed and the dog was euthanatized. Situs inversus abdominalis and multiple vascular anomalies were found at necropsy. Gross and microscopic studies of the viscera were correlated with corrosion casts of the vasculature. In addition to changes in organ position, 3 spleens and malformation of the pancreas were observed. Vascular anomalies were: (1) The portal vein was contiguous with the common hepatic vein by way of a patent ductus venosus; branches of the portal vein were not given off to the liver; (2) the caudal vena cava was continued by the azygos vein, and a cranial segment of the caudal vena cava was absent; (3) the hepatic artery was larger than normal; (4) the left gastric artery arose anomalously from the cranial mesenteric artery; and (5) histologic and radiographic study of the liver did not reveal any interlobular branches of the portal vein; however, hypoplastic branches of the gastroduodenal vein did enter the liver and were demonstrated on a corrosion cast.


Primary hyperparathyroidism in a dog.

Primary hyperparathyroidism in a dog. Anorexia, severe skeletal demineralization, and muscular weaknesss developed in a 9 year-old Collie. Hypercalcemia, hypophosphatemia, and nephrocalcinosis were found on clinical evaluation. The surgical removal of a parathyroid adenoma was followed by profound hypocalcemia that was unresponsive to calcium therapy, and the dog died. The persistent hypocalcemia was attributed to increased deposition of calcium in mineral-deficient bone.


Diagnostic abdominal paracentesis and lavage: experimental and clinical evaluations in the dog. Diagnostic abdominal paracentesis and saline lavage, using a peritoneal dialysis catheter, was evaluated experimentally and clinically in dogs. Use of the catheter enabled detection of abdominal fluid in volumes as low as 0.2 ml/kg of body weight. In paracentesis, the catheter proved to be a more reliable means of detecting hemoperitoneum than did a needle. In 14 clinical cases of blunt trauma, use of the catheter provided reliable evidence of intraabdominal injury.


Diagnostic abdominal paracentesis and lavage in the evaluation of abdominal injuries in dogs and cats: clinical and experimental investigations. Abdominal paracentesis and lavage, using a peritoneal dialysis catheter, was evaluated clinically for use in detecting intraabdominal injury in 20 dogs and 3 cats. The technique was also evaluated experimentally in 12 dogs in which various volumes of autogenous blood were infused into the abdomen. The procedure was found to be safe, well tolerated, and reliable in the detection of intraabdominal injuries. The volume of free blood in the abdomen of the experimental dogs was accurately determined. The procedure was superior to physical examination and to needle paracentesis for the early detection and evaluation of intraabdominal injuries.


Idiopathic hypoparathyroidism in a dog.

Idiopathic hypoparathyroidism in a dog. Idiopathic hypoparathyroidism in a 3-year-old dog was manifested clinically as periodic episodes of depression, vomiting, and tetanic convulsions. Hypocalcemia and hyperphosphatemia were detected late in the course of the illness, but the dog died before corrective measures could be instituted. The pathologic features were similar to those reported in dogs in which hypoparathyroidism was induced by injection of homologous parathyroid tissue, thus suggesting an auto-immune pathogenesis.


Antimetabolites produced by microorganisms. XV Synthesis of 2-methyl-L-arginine, 2-methyl-L-ornithine and their enantiomers. 5-(3-Azidopropyl)-5-methyl-2, 4-imidazolidinedione was prepared either from 5-(3-chloropropyl)-5-methyl-2,4-imidazolidinedione or 5-methyl-5-(3-[(4-methylphenyl)sulfonyloxy]-propyl)-5-methyl-2, 4-imidazolidinedione and hydrogenolyzed to the corresponding amine which, after carbamimidoylation, afforded 2-methyl-DL-arginine upon acid hydrolysis. Racemic 2-methylarginine was converted enzymically to a mixture of 2-methyl-D-arginine and 2-methyl-L-ornithine. 2-Methyl-L-arginine was reconstructed from 2-methyl-L-ornithine via its Cu(II) chelate with O-methyl-isourea and 2-methyl-D-ornithine was obtained by alkaline hydrolysis of 2-methyl-D-arginine.


Antitumor activity of derivatives of mycophenolic acid. One hundred and eight derivatives of mycophenolic acid (MA) have been prepared by modifications at the phenolic hydroxyl and/or carboxyl sites. None of these compounds was as effective as MA in suppressing cell growth of L-518Y cell in vitro, whereas several compounds with changes at both the hydroxyl and carboxyl groups were more effective than MA against Ehrlich solid carcinoma and L-1210 leukemia in mice.


Carbamoyl mycophenolic acid ethylester, an oral antitumor agent. Several derivatives of mycophenolic acid (MA) were tested for their antitumor activity against leukemia L-1210 when administered orally and carbamoyl mycophenolic acid ethylester (CMAE) was selected as the most active antitumor agent in these tests. An oral administration of CMAE also inhibited the growth of Ehrlich solid carcinoma, NF sarcoma, myeloma X-5563 and sarcoma 180 in mice. In comparison with antitumor activity of MA, the improvement in activity of CMAE against leukemia L-1210 and Ehrlich solid carcinoma was indeed remarkable, CMAE seems to be less immunosuppressive than MA.


Inhibition of the initial dipeptide synthesis of globin chains by the antibiotic enomycin in the reticulocyte lysate. During inhibition of protein synthesis by the antibiotic enomycin at less than 5nm in the reticulocyte lysate system, polyribosomes disaggregated and the 80S ribosomes accumulated. At these concentrations little inhibition of chain elongation and release from the ribosomes was demonstrated. Enomycin caused an increase in the amount of 80S initiation complex as well as the 40S ribosomal subunit-Met-tRNA complex. The former complex could react with puromycin under the inhibiting conditions. Val-tRNA  binding to the 80S ribosomes was not decreased by the antibiotic. However pactamycin-induced accumulation of the initial dipeptide (fMet-Val) was inhibited when the system was preincubated with enomycin and and fMet-tRnaf. Thus the preferential inhibition of the initial phase of protein synthesis by enomycin is made evident by its inhibition of the initial dipeptide synthesis.


Evidence for diverse types of large plasmids in tumor-inducing strains of Agrobacterium. Homology between the large plasmids of 15 pathogenic Agrobacterium strains isolated from various parts of the world has been measured and was found to vary over a wide range, from 3 to 100%. Two genetically distinct groups of plasmids can be identified: one closely related to the plasmid of A. tumefaciens A6, an octopine-utilizing strain, and the other closely related to the plasmid of A. tumefaciens C-58, a nopaline-utilizing strain. The plasmids of four Agrobacterium strains do not belong to either group. One of these four strains utilizes octopine, one utilizes nopaline, and two utilize neither. Three strains contained two large plasmids. In one of these strains, the two plasmids were not homologous to one another. Chromosomal homologies for the Agrobacterium strains surveyed also vary over a wide range, but do not correlate with plasmid homologies. Neither do plasmid homologies correlate with any numerical classification scheme. The significance of these plasmid homology studies for crown gall tumorigenesis is considered.


Distribution of peptidoglycan synthetase activities between sporangia and forespores in sporulating cells of Bacillus sphaericus. Sporulating cells of Bacillus sphaericus 9602 containing fully engulfed forespores at different stages of maturity were broken by ultrasonic disruption, followed by grinding with alumina. In this way soluble enzymes derived mainly from the sporangial or from the forespore cytoplasms were obtained. Diaminopimelate ligase activity is required exclusively for cortical peptidoglycan synthesis, is absent during vegetative growth, and is synthesized during forespore maturation. It is found exclusively in the sporangial cytoplasm. L-lysine ligase is required for vegetative cell wall peptidoglycan synthesis but not for cortex synthesis. It is found in both fractions, but it has a fourfold higher specific activity in the forespore cytoplasm. Other enzymes that are required for synthesis of the nucleotide-pentapeptide precursors of both cortical and vegetative cell wall peptidoglycans are found in similar specific activities in both compartments. Mature spores, free of any residual sporangial material, have specific activities of all of these enzymes and of L-lysine ligase similar to those in forespores and in vegetative cells and are devoid of diaminopimelate ligase activity. Thus, the differential expression of at least one gene required for spore cortex synthesis in B. sphaericus occurs exclusively in the sporangial cytoplasm.


Nitrate reductase activity in heme-deficient mutants of Staphylococcus aureus. Mutants H-14 and H-18 of Staphylococcus aureus require hemin for growth on glycerol and other nonfermentable substrates. H-14 also responds to delta-aminolevulinate. Heme-deficient cells grown in the presence of nitrate do not have lactate-nitrate reductase activity but gain this activity when incubated with hemin in buffer and glucose. Lactate-nitrate reductase activity is also restored to the membrane fraction from such cells by incubation with hemin and dithiothreitol; addition of adenosine 5'-triphosphate has no effect upon the restoration. Cells grown with nitrate in the absence of hemin have two to five times more reduced benzyl viologen-nitrate reductase activity than do those grown with hemin. The activity increases throughout the growth period in the absence of hemin, but with hemin present enzyme formation ceases before the end of growth. There was no evidence of enzyme destruction. The distribution of nitrate reductase activity between membrane and cytoplasm was similar in cells grown with and without hemin; 70 to 90% was in the cytoplasm. It is concluded that heme-deficient staphylococci form apo-cytochrome b, which readily combines in vitro with its prosthetic group to restore normal function. The avaliability of the heme prosthetic group influences the formation of nitrate reductase.


Measurement of growth and iron deposition in Sphaerotilus discophorus. Aspects of the physiology of iron deposition of an iron-precipitating strain of Sphaerotilus were investigated in laboratory culture to characterize the process. Measurement of growth (incorporated L-[3H]alanine) and iron deposition (incorporated 59Fe) demonstrated that Sphaerotilus exhibit a characteristic temporal pattern of iron deposition, which is delayed until the latter portion of the exponential or the onset of the stationary growth phase. The growth rate (mu=0.17 h-1) was apparently independent of the iron concentration in the medium. There was, furthermore, no direct correlation between the iron concentration and final cell protein yield. It was concluded from experiments involving growth on artificial substrata (glass cover slips) that sessile populations derived no phsiological advantage (manifested as differences in growth rates) over free-living cells. There was no difference in the rate or onset of iron deposition of attached compared to suspended cells. Blocking of protein synthesis by the addition of chloramphenicol suggested that, once iron deposition was initiated, continued protein synthesis was not required for full expression of this capability. The results suggested that iron deposition may be possibly mediated by certain of the constituents of the organism's sheath.


Constitutive synthesis of enzymes of the protocatechuate pathway and of the beta-ketoadipate uptake system in mutant strains of Pseudomonas putida. Mutant Pseudomonas putida strains that produce constitutive levels of the beta-ketoadipate uptake system are selected by the sequential transfer of cultures between mineral growth media supplemented with the noninducing growth substrate succinate and growth media containing beta-ketoadipate as the sole carbon and energy source. The mutant strains also produce constitutively three catabolic enzymes that give rise to beta-ketoadipate from the metabolic precursor beta-carboxy-cis, cis-muconate, and thus a single regulatory gene appears to govern the expression of the enzymes as well as the uptake system. The three enzymes that convert beta-carboxy-cis, cis-muconate to beta-ketoadipate are induced to higher levels when the orgainisms are grown with p-hydroxybenzoate (a compound that is catabolized via beta-ketoadipate); the beta-ketoadipate uptake system is partially repressed when the cells are grwon at the expense of p-hydroxybenzoate. The transferase that acts upon beta-ketoadipate remains inducible in the constitutive mutant strains. Thus a minimum of three biosynthetic controls must be exerted over the expression of the five genes. Since the regulatory mutation does not alter the expression of the gene for the transferase, the physiological target of the selection procedure appears to be mutant strains that produce the uptake system constitutively. Levels of the uptake system are higher in uninduced constitutive mutant cultures than in induced cultures of the wild type. Hence procedures analogous to the one we employed may be of general use in obtaining mutant strains that produce high levels of uptake systems.


Role of arginine deiminase in growth of Mycoplasma hominis. Arginine has been considered as the major energy source of nonglycolytic arginine-utilizing mycoplasmata. When three strains of Mycoplasma arginini, and one strain each of Mycoplasma arthritidis, Mycoplasma fermentans, Mycoplasma gallinarum, Mycoplasma gallisepticum and Mycoplasma hominis were grown in the medium with high arginine concentration (34 mM) compared with low arginine (4 mM), both the protein content of the organisms and the specific activity of arginine deiminase increased. M. fermentans, the one arginine-utilizing species included in the survey which is also glycolytic, showed an increase in protein content but no increase in specific activity of the enzyme. The glycolytic non-arginine-utilizing M. gallisepticum did not show an increase in either parameter. The Km for arginine deiminase from crude cell extracts was 1.66 X 10(-4)M. The enzyme demonstrated a hyperbolic activation curve subject to substrate inhibition and was not affected by the presence of L-histidine. When mycoplasmic protein and arginine deiminase were determined for M. hominis under aerobic and anaerobic conditions, aerobically grown cells exhibited no detectable enzymatic increases until late in log phase. Higher levels of arginine deiminase were observed earlier in the anaerobic growth cycle. The rate of 14CO2 evolution from [guanido-14C]arginine was not altered in arginine-supplemented cells compared with cells grown in low arginine. In addition, CO2 production did not parallel increased arginine deiminase activity. These observations argue that arginine is used only as an alternate energy source in these organisms.


Characteristics of Bacillus stearothermophilus mutants blocked in catabolic function. With polyacrylamide disc gel electrophoresis and specific staining, it was demonstrated that one mutation involving the alcohol dehydrogenase of a double mutant of Bacillus stearothermophilus 1503 apparently prevented enzyme synthesis, and another lesion in the same organism resulted in synthesis of an inactive form of aconitase. Some properties of the double mutant and two fumarase mutants are discussed in relation to similar mutants derived from Bacillus subtilis.


Formation of indole-3-carboxylic acid by Chromobacterium violaceum. l-Tryptophan is converted to indole-3-carboxylic acid by growing cultures and resting cell suspensions of Chromobacterium violaceum


Evidence for autogenous regulation of Pseudomonas putida tryptophan synthase. Studies of a trpA mutant constitutive for tryptophan synthase production support the hypothesis of autogenous regulation (R. F. Goldberger, 1974; A. R. Proctor and I. P. Crawford, 1975) of the Pseudomonas putida trpAB loci.


Evidence that the isomerization of D-ribose and L-rhamnose is catalyzed by the same enzyme in Mycobacterium smegmatis. D-Ribose isomerase was purified and crystallized from cells of Mycobacterium smegmatis grown on either D-ribose or L-rhamnose. Isomerase activity for both of these sugars remained together throughout the purification. The isomerase from L-rhamnose-grown cells had the same chemical and physical properties as the enzyme isolated from D-ribose grown cells. In addition, immunological studies indicated that both activities were in the same protein since antisera prepared against either of the crystals cross-reacted with the other and gave lines of symmetry by the agar gel diffusion method.


Regulation of L-phenylalanine ammonia-lyase from Rhizoctonia solani. Maximal levels of L-henylalanine ammonia-lyase activity were observed when the mycelial felts of Rhizoctonia solani were grown for 4.5 days on Byrde synthetic medium containing 3.5% glucose and 0.3% L-phenylalanine, Differential centrifugation studies have indicated that the enzyme is localized in the soluble fraction. The time course of induction of L-phenylalanine ammonia-lyase activity by L-phenylalanine showed a lag period of 1 to 1.5 h and reached a maximum around 4 to 6 h after the addition of the inducer to the medium. L-Phenylalanine, L-tyrosine, and L-tryptophan were nearly equally efficient inducers of the enzyme. D-Phenylalanine was as efficient as the L-isomer, whereas D-tyrosine was a poor inducer. Light, gibberellic acid, indole 3-acetic acid, and kinetin had no effect on the induction of L-phenylalanine ammonia-lyase activity. Cycloheximide did not inhibit the uptake of amino acids by the mycelia but completely blocked the incorporation of radioactive amino acids into soluble proteins and the development of L-phenylalanine ammonia-lyase activity. Actinomycin D inhibited both the incorporation of 32P into ribonucleic acid and the enzyme activity. Conclusive evidence for de novo synthesis of L-phenylalanine ammonia-lyase was obtained by the incorporation of radioactive amino acids into the enzyme. Electrophoretic analysis of the purified preparation showed a single protein band that coincided with radioactivity and L-phenylalanine ammonia-lyase activity. Glucose and intermediates of the tricarboxylic acid cycle, like citric acid, alpha-ketoglutaric acid, and succinic acid, and the metabolites of L-phenylalanine, like o-coumaric acid, o-hydroxyphenylacetic acid, and protocatechuic acid, significantly repressed L-phenylalanine ammonia-lyase activity. The observed repression was not relieved by cyclic adenosine 5'-triphosphate.


Repair of ultraviolet light-induced damage in Micrococcus radiophilus, an extremely resistant microorganism. Repair of ultraviolet radiation damage was examined in an extremely radioresistant organism, Micrococcus radiophilus. Measurement of the number of thymine-containing dimers formed as a function of ultraviolet dose suggests that the ability of this organism to withstand high doses of ultraviolet radiation (20,000 ergs/mm2) is not related to protective screening by pigments. M. radiophilus carries out a rapid excision of thymine dimers at doses of ultraviolet light up to 10,000 ergs/mm2. Synthesis of deoxyribonucleic acid is reduced after irradiation, but after removal of photodamage the rate approaches that in unirradiated cells. A comparison is drawn with Micrococcus luteus and M. radiodurans. We conclude that the extremely high resistance to ultraviolet irradiation in M. radiophilus is at least partly due to the presence of an efficient excision repair system.


Map of genes for carotenoid and bacteriochlorophyll biosynthesis in Rhodopseudomonas capsulata. The recently discovered gene transfer system of Rhodopseudomonas capsulata was used to construct a genetic map of a region concerned with bacteriochlorophyll and carotenoid production. Mutants blocked in the biosynthesis of these compounds were isolated, and each was characterized on the basis of pigments accumulated during growth under low pO2. One-point, two-point, three-point, and ratio test crosses were performed between various mutant strains, and the results were amenable to conventional genetic analyses. A mapping function was found that related cotransfer frequency to map distance. Seven clusters of mutations, five affecting carotenoid and two affecting bacteriochlorophyll biosynthesis, were arranged in one linkage group. Each cluster of mutations is thought to represent a gene. The length of the mapped region is estimated to be less than 1% of the genome. Cotransfer is observed between markers separated by about 5 to 10 genes.


Influence of exogenous sugars and polyols on C1-influx and efflux by the ascomycete Neocosmospora vasinfecta. Glucose and other transportable sugars and polyols inhibited Cl- influx very soon after addition to mycelium in the process of Cl- accumulation. Under the usual experimental conditions (0.1 mM KCl, glucose greater than or equal to 2 mM) the mean percentage of inhibition of Cl- influx by glucose was 54.1 +/- 8.0 (+/- standard error; N = 26). Transport of the exogenous carbohydrate was necessary for inhibition of Cl- influx. Thus, the estimated Ki for glucose inhibition of Cl- influx (28 muM) was close to the Km for glucose transport; glycerol did not inhibit Cl- influx unless it was itself transported, and the degree of inhibition exerted by various carbohydrates correlated with their uptake rates. Inhibition was not caused by the accumulated sugar itself, as high levels (ca. 60 mM) of intramycelial 3-O-methylglucose gave rise to a stimulation of Cl- influx when the exogenous sugar was removed. It is suggested that interaction of Cl- and carbohydrate transport arises from competition for a common energy-coupling mechanism in the cell membrane. Both glucose and 3-O-methylglucose elicited Cl- efflux, but the maximal Cl- efflux rates were observed only after 40 min of incubation and only in the presence of the readily metabolizable glucose. Removal of the exogenous glucose, even after maximal Cl- efflux had been established, resulted in the rapid cessation of efflux. Studies under anaerobic conditions gave further evidence that glucose uptake was necessary and that efflux was not due to temporary depletion of energy reserves. It is proposed that glucose-induced leakage of Cl- is due to reversal of the Cl- uptake system, even though the Km for efflux is much greater than that for influx.


Purification and properties of chorismate mutase-prephenate dehydratase and prephenate dehydrogenase from Alcaligenes eutrophus. Chorismate mutase and prephenate dehydratase from Alcaligenes autophus H16 were purified 470-fold with a yield of 24%. During the course of purification, including chromatography on diethylaminoethyl (DEAE)-cellulose, phenylalanine-substituted Sepharose, Sephadex G-200 and hydrogyapatite, both enzymes appeared in association. The ratio of their specific activities remained almost constant. The molecular weight of chorismate mutase-prephenast dehydratase varied from 144,000 to 187,000 due to the three different determination methods used. Treatment of electrophoretically homogeneous mutase-dehydratase with sodium dodecyl sulfate dissociated the enzyme into a single component of molecular weight 47,000, indicating a tetramer of identical subunits. The isoelectric point of the bifunctional enzyme was 5.8. Prephenate dehydrogenase was not associated with other enzyme activities; it was separated from mutasedehydratase by DEAE-cellulose chromatgraphy. Chromatography on DEAE Sephadex, Sephadex G-200, and hydroxyapatite resulted in a 740-fold purification with a yield of 10%. The molecular weight of the enzyme was 55,000 as determined by sucrose gradient centrifugation and 65,000 as determined by gel filtration or electrophoresis. Its isoelectric point was pH 6.6. In the overall conversion of chorismate to phenylpyruvate, free prephenate was formed which accumulated in the reaction mixture. The dissociation of prephenate allowed prephenate dehydrogenase to compete with prephenate dehydratase for the substrate.


Ammonia-sensitive mutant of Klebsiella aerogenes. We have isolated a temperature-sensitive mutant of Klebsiella aerogenes unable to grow aerobically at 42 C in standard glucose minimal medium containing 0.03 M ammonium sulfate as a source of nitrogen. This strain, MK810, will grow at this temperature in significantly lower concentrations of ammonia (1 mM) or when ammonia is replaced by a growth rate-limiting source of nitrogen such as histidine or glutamate. A detailed physiological characterization and preliminary biochemical tests support the contention that the mutant has an altered alpha-ketoglutarate dehydrogenase that at the restrictive condition fails to manufacture sufficient succinyl-coenzyme A. We explain the ammonia sensitivity by the dual role of alpha-ketoglutarate as substrate for the formation of succinyl-coenzyme A and glutamate. A defect in the enzyme necessary for the production of succinyl-coenzyme A makes ammonia an overly effective competitor for alpha-ketoglutarate.


Ultrastructure of the cell walls of two closely related clostridia that possess different regular arrays of surface subunits. Cell walls of Clostridium thermohydrosulfuricum and C. thermosaccharolyticum have a two-layered structure, consisting of a thin, lysozyme-sensitive murein layer and a surface (S) layer composed of hexagonally or tetragonally arranged subunits. The subunits can be removed from the murein layer by treatment of cell wall preparations, are composed of a fragile, pH-sensitive monolayer of macromolecular subunits. In both organisms the first stage of the cell division process involves only the plasma membrane and the murein layer. During the subsequent cell separation, a surplus of S-layer subunits appears at the site of division, and consequently the newly formed cell poles remain completely covered by the s layer throughout the separation process. In autolyzed cells an additional layer of subunits assembles on extended areas of the inside of the mucopeptide layer. These observations indicate that the biological function of the S layer depends on its ability to maintain a complete covering of the cell surface at all stages of cell growth and division.


Ultrastructure of developing ascospores in Sordaria brevicollis. The ultrastructure of ascospore wall formation in the pyrenomycete Sordaria brevicollis was studied in developing asci at progressive time intervals. From early spore delimitation through final stage of maturation, the wall of the ascospore differentiated into four composite layers, the periascosporium the delineation ascosporium, the subascosproium, and the endoascosproium, While ascospores were at the hyaline stage of development,they possessed only the periascosporium and delineation ascosporium as their wall components. At about 7 to 8 days from the initiation of the cross, the spores developed a yellow color, and this coloration was always associated with the elaboration of the subascorsporium just internal to the ascosporium. Asthe spores continued to progressively darken in color, the subascosporium was seen to increase in complexity, electron density, and thickness. Soon after the formation of the subascosporium, the endoascosporium began to develop de novo and was, therefore, the last wall layer formed as the spore approached maturity.


Cortex content of asporogenous mutants of Bacillus subtilis. A method for the measurement of muramic lactam, which is specifically located in the cortical peptidoglycan of bacterial spores, was developed as a quantitative assay method for spore cortex content. During sporulation of Bacillus subtilis 168, muramic lactam (i.e., spore cortex) began to appear at state IV of sporulation and continued to increase over most of the late stages of sporulation. Spore cortex contents of various spo mutants of B. subitils were surveyed. Cortex was not detected in mutants in which sporulation was blocked earlier than stage II sporulation. Spores of spo IV mutant had about 40% of the cortex content of the wild-type spores. One spo III mutant had a low amount of cortex, but four others had none.


Genetic mapping of leucine and isoleucine-valine loci in Rhizobium japonicum. Leucine and isoleucine-valine loci have been mapped in Rhizobium japonicum. Transformation analysis suggests a common pathway for isoleucine-valine biosynthesis. Three-point reciprocal crosses indicated that all the leucine loci are not genetically linked.


Demonstration and possible function of NADH:NAD+ transhydrogenase from ascaris muscle mitochondria. Mitochondria from the muscle of Ascaris lumbricoides var. suis function anaerobically. NADH is generated in the intermembrane space as a consequence of the "malic" enzyme reaction. It has been suggested that this reducing equivalent in the form of hydride ion, would be translocated across the inner membrane in order to mediate ATP generation via the fumarate reductase reaction. In accord with this suggestion, intact Ascaris mitochondria showed appreciable NADH oxidase activity. Sonication resulted in an approximately 2-fold increase in NADH oxidase activity, whereas "malic" enzyme, fumarase, and NADH:NAD+ transhydrogenase activities increased approximately 7- to 14-fold, respectively. Phosphorylation capabilities and permeability toward pyridine nucleotides also indicated the intactness of the mitochondria. Ascaris mitochondria incubated anaerobically in the presence of fumarate, and [14C]NADH catalyzed a rapid reduction of the fumarate to succinate with the concomitant formation of equivalent quantities of extramitochondrial NAD+. However, very little isotope was recovered from the washed mitochondria, indicating the possibility of hydride ion translocation in the absence of nucleotide translocation. NADH:NAD+ transhydrogenase has been isolated from the muscle mitochondria of the intestinal nematode, Ascaris lumbricoides var. suis. The enzyme seems to have been solubilized from the mitochondrial membrane fraction by treatment with sodium deoxycholate followed by dialysis and subsequent adsorption by and elution from alumina C gamma. No NADPH:NAD+ transhydrogenase activity was detectable, making the Ascaris system unique over others reported. Activity was protected by L-cysteine, reduced glutathione and dithioerythritol, but strongly inhibited by low concentrations of p-chloromercuribenzoate or silver nitrate. The thionicotinamide derivative of NAD+ (thioNAD+) was employed to accept hydride ions from NADH in order to assay spectrophotometrically at 398 nm. Apparent Km values for thioNAD+ and NADH were 1 X 10(-4) M and 8 X 10(-6) M, respectively. That the physiological nucleotide, could act as hydride ion acceptor from NADH was indicated by the findings that NAD+ competitively inhibited the reduction of thioNAD+ when assayed at 398 nm. The additional finding of a noncompetitive inhibition between NAD+ and NADH suggested at least two binding sites on the enzyme, one for NADH and another common site for NAD+ and thioNAD+. More conclusive evidence indicating the participation of NAD+ as acceptor was obtained by incubation of the enzyme with NADH and [14C]NAD+ and demonstrating a rapid formation of [14C]NADH. These findings, in conjunction with those discussed above, suggest a physiological function of this enzyme in hydride ion translocation.


Characterization of proteins from Ascaris lumbricoides which bind specifically to carboxypeptidase. Inhibitors of the peptidase and esterase activities of carboxypeptidases A and B have been isolated from extracts of Ascaris lumbricoides var suis. These proteins were obtained by treatment of the aqueous extracts at low pH, precipitation with ammonium sulfate, molecular sieving on Bio-Gel P-4, and chromatography on DEAE-cellulose. The inhibitors were resolved into three homogeneous peaks on CM-cellulose. These components, CM-A, CM-B, and CM-C, have constant specific activity and were recovered in a 41% yield. They moved as single bands when subjected to electrophoresis at high or low pH on polyacrylamide gels and they have similar amino acid compositions. Methionine, tyrosine, and cysteine are absent from each of the inhibitors. The 65 residues of CM-B suggest a minimum molecular weight of 7530, in close agreement to the value of 7600 +/- 200 determined on a Bio-Gel P-100 column. Each of the proteins has the same NH2-terminal residues, NH2-Asx-Glx-Val-Glx- and the same COOH-terminal residue, leucine. A plot of per cent acrylamide versus log relative mobility suggests that the three proteins are charge isomers. CM-B appears to be stable to high NaCl concentrations, extremes of pH, high temperatures, and digestion by intestinal proteases. Carboxypeptidase C, carboxypeptidase N, and yeast protease C are not inhibited by CM-B. However, the exopeptidase and esterase activities of human carboxypeptidase A are inhibited. The inhibitors appear to bind to bovine carboxypeptidase A with an atypical stoichiometry. Two moles of CM-B inhibitor bind to 1 mol of enzyme. The evidence is: (a) a demonstrated purity of bovine carboxypeptidase A, (b) minimal and maximal inhibitor molecular weights by different methods, of 7600 and 8300, and (c) a maximum specific activity of apparently homogeneous inhibitors which is 50% of that predicted for unit stoichiometry.


Pigeon liver amidophosphoribosyltransferase. Ligand-induced alterations in molecular and kinetic properties. Amidophosphoribosyltransferase (EC 2.4.2.14) has been partially purified from pigeon liver, and ligand-induced alterations in molecular and kinetic properties have been studied. In Tris-HCl buffer the predominant form of the enzyme has an s20,w of 5.9 +/- 0.7, Stokes radium of 42 A, and estimated molecular weight of 102,000. Incubation with phosphoribosylpyrophosphate (PP-ribose-P) results in an increase in the s20,w to 7.9 +/- 0.6, Stokes radius to 53 A, and estimated molecular weight to 172,000. Incubation of this larger form with purine ribonucleotides leads to a decrease in the molecular weight of amidophosphoribosyltransferase that is proportional to the concentration of purine ribonucleotide. Purine ribonucleotides produce sigmoidal kinetics with respect to the substrate PP-ribose-P, with Hill coefficients of 1.4 to 1.6 and 1.8 to 2.0 in the presence of AMP and GMP, respectively. Incubation with 0.6 M KCl leads to sigmoidal kinetics. Hill coefficient of 1.8 and dissociation of the larger form of amidophosphoribosyltransferase. Inorganic phosphate has complex effects upon the enzyme. In 25 mM potassium phosphate buffer the enzyme aggregates to a large form with an s20,w of 8.3 +/- 0.2, Stokes radius of 53 A, and estimated molecular weight of 181,000. Inorganic phosphate and PP-ribose-P both stabilize the enzyme to storage in vitro at 4 degrees. However, inorganic phosphate is 4 times more effective than PP-ribose-P in preventing inactivation of the enzyme by sodium dodecyl sulfate. Inorganic phosphate produces sigmoidal kinetics with respect to PP-ribose-P, Hill coefficient of 1.5. The interaction coefficients for AMP and GMP are reduced from 1.8 to 1.2 and 2.2 to 1.4, respectively, in the presence of 25 mM potassium phosphate. It is concluded that pigeon liver amidophosphoribosyltransferase is a complex allosteric protein whose activity is regulated by a series of conformational changes induced by a number of ligands.


Biohydrogenation of unsaturated fatty acids. Hydrogenation by cell-free preparations of Butyrivibrio fibrisolvens. Hydrogenation of cis-9,trans-11-octadecadienoic acid to yield trans-11-octadecenoic acid by cell-free preparations of Butyrivibrio fibrisolvens has been obtained under strictly anaerobic conditions. Reduced methyl viologen, NADH, and an endogenous electron donor each can serve as a reductant. Inhibition studies and gel filtration patterns reveal the presence of at least two hydrogenation systems, one of which is coupled through a flavin, possibly FMN. Although the enzymes comprising the biohydrogenation pathway, the fatty acid reductases and linoleic acid isomerase, are part of the bacterial membrane, they do not appear to be constituted as a multienzyme complex.


13C NMR spectroscopy of labeled pyridoxal 5'-phosphate. Model studies, D-serine dehydratase, and L-glutamate decarboxylase. Pyridoxal 5'-phosphate labeled to the extent of 90% with 13C in the 4' (aldehyde) and 5' (methylene) positions has been synthesized. 13C NMR spectra of this material and of natural abundance pyridoxal 5'-phosphate are reported, as well as 13C NMR spectra of the Schiff base formed by reaction of pyridoxal 5'-phosphate with n-butylamine, the secondary amine formed by reduction of this Schiff base, the thiazolidine formed by reaction of pyridoxal 5'-phosphate with cysteine, the hexahydropyrimidine formed by reaction of pyridoxal 5'-phosphate with 1,3-diaminobutane, and pyridoxamine 5'-phosphate. The range of chemical shifts for carbon 4' in these compounds is more than 100 ppm, and thus this chemical shift is expected to be a sensitive indicator of structure in enzyme-bound pyridoxal 5'-phosphate. The chemical shift of carbon 5', on the other hand, is insensitive to these structure changes. 13C NMR spectra have been obtained at pH 7.8 and 9.4 for D-serine dehydratase (Mr = 46,000) containing natural abundance pyridoxal 5'-phosphate and containing 13C-enriched pyridoxal 5'-phosphate. The enriched material contains two new resonances not present in the natural abundance material, one at 167.7 ppm with a linewidth of approximately 24 Hz, attributed to carbon 4' of the Schiff base in the bound coenzyme, and one at 62.7 Hz with a linewidth of approximately 48 Hz attributed to carbon 5' of the bound Schiff base. A large number of resonances due to individual amino acids are assigned. The NMR spectrum changes only slightly when the pH is raised to 9.4. The widths of the two enriched coenzyme resonances indicate that the coenzyme is rather rigidly bound to the enzyme but probably has limited motional freedom relative to the protein. 13C NMR spectra have been obtained for L-glutamate decarboxylase containing natural abundance pyridoxal 5'-phosphate and 13C-enriched pyridoxal 5'-phosphate. Under conditions where the two enriched 13C resonances are clearly visible in D-serine dehydratase, no resonances are visible in enriched L-glutamate decarboxylase, presumably because the coenzyme is rigidly bound to the protein and the 300,000 molecular weight of this enzyme produces very short relaxation times for the bound coenzyme and thus very broad lines.


Raman spectra of a solid antifreeze glycoprotein and its liquid and frozen aqueous solutions. Raman spectroscopy was used to study the anomalous decrease in the freezing temperature of water produced by an antifreeze glycoprotein obtained from the sera of an Antarctic fish. An active fraction of this glycoprotein has a molecular weight of approximately 18,000 by equilibrium sedimentation compared to an apparent weight of 20 by freezing temperature depression. The Raman spectra of water present in a 1% antifreeze glycoprotein solution and of ice frozen from this solution were indistinguishable from the spectra of pure water and ice, respectively. These results indicate that the bulk properties of water and ice are unaffected by the presence of the antifreeze glycoprotein. Raman measurements on ice grown slowly, using as seed an oriented single crystal of ice in contact with 1% glycoprotein solutions, showed that the active glycoprotein was not excluded from the ice phase. On the other hand, we found that a smaller, inactive glycoprotein was excluded. Comparison of the Raman spectra of active and inactive glycoprotein components as solids, in 5% solutions, and rapidly frozen 5% solutions, showed that the two components differ in conformation and possibly in the environment of their carbohydrate hydroxyls. These observations suggest that hydrogen bonding of the carbohydrate hydroxyls of the active glycoprotein at the ice-solution interface may physically prevent growth of the ice lattice.


The effect of superhelicity on the interaction of histone f1 with closed circular duplex DNA. A set of covalently closed circular duplex simian virus 40 DNA preparations of varying superhelical densities was prepared by closure of nicked duplex DNA with polynucleotide ligase in the presence of varying amounts of ethidium. The resulting molecules were tested for complex formation with the lysine-rich histone f1. The results confirmed earlier experiments in demonstrating that f1 histone reacts preferentially with superhelical DNA compared to relaxed circular DNA. Furthermore, the extent of the reaction is demonstrated to depend on the superhelical density. At the relatively low ratios of histone to DNA used in these experiments, the product of the interaction of f1 histone with superhelical DNA does not precipitate. At higher ratios of histone to DNA, an insoluble aggregate is formed.


alpha-L-Fucopyranosylceramide, a novel glycolipid accumulated in some of the human colon tumors. A fucosylceramide was isolated from a ceramide monohexoside fraction of human colon carcinomas by acetylation followed by preparative thin layer chromatography on Silica Gel G developed with butylacetate. It contained only fucose as the carbohydrate component and the ceramide moiety was characterized as N-palmitoyloctadecasphingenine as a predominant component. The structure of the compound was determined as alpha-L-fucopyranosylceramide by methylation analysis, mass spectrometry of permethylated and reduced glycolipid, and by enzymatic hydrolysis with alpha-L-fucosidase. The chemical concentration of this compound in colon tumor tissue depends on degree of malignancy: highly malignant metastatic deposits from colon cancers were 3 to 70 ng/mg of residue protein, whereas that of localized colon tumor was 0 to 2 ng/mg of protein residue. The present observation offers additional support for the occurrence of metabolic abnormality of fucolipids as membrane phenotype of malignancy as was suggested in previous studies (Yang, H-J., and Hakomori, S. (1971) J. Biol. Chem. 246, 1192-1200; Steiner, S., Brennan, P.J., Melnick, J. L. (1973) Nature New Biol. 245, 19-21).


Terminal deoxynucleotidyltransferase. Serological studies and radioimmunoassay. Mouse antisera against calf terminal deoxynucleotidyltransferase (terminal transferase) have been prepared. The sera have been used to characterize terminal transferase both by studying inhibition of enzyme activity and by developing a competition radioimmunoassay using highly purified 125I-labeled terminal transferase. By either assay, anti-terminal transferase serum did not cross-react significantly with calf DNA polymerases alpha and beta, Escherichia coli DNA polymerase I, or the reverse transcriptase of Moloney mouse leukemia virus. The calf terminal transferase did, however, share cross-reactive but not identical determinants with human and murine terminal transferase. The radioimmunoassay could detect as little as 2 ng of terminal transferase/mg of soluble protein in a tissue extract. Thymocytes were found to contain 280 ng of terminal transferase/mg of cell protein or about 1 X 10(5) molecules/cell; bone marrow had about 1% of the level of enzyme found in thymus. Extracts of spleen, peripheral white blood cells, lymph nodes, liver, muscle, and kidney all lacked detectable antigenicity of terminal transferase. These data indicate that terminal transferase is a tissue-specific enzyme and is not related to other DNA polymerases.


Purification and properties of proline reductase from Clostridium sticklandii. Proline reductase of Clostridium sticklandii is a membrane-bound protein and is released by treatment with detergents. The enzyme has been purified to homogeneity and is estimated by gel filtration and sedimentation equilibrium centrifugation to have a molecular weight of 298,000 to 327,000. A minimum molecular weight of 30,000 to 31,000 was calculated on the basis of sodium dodecyl sulfate-acrylamide gel electrophoresis and amino acid composition. Amino acid analysis showed a preponderance of acidic amino acids. No tryptophan was detected in the protein either spectrophotometrically or by amino acid analysis. A total of 20 sulfhydryl groups measured by titration of the reduced protein with 5,5'-dithiobis(2-nitrobenzoic acid) is in agreement with 20 cystic acid residues determined in hydrolysates of performic acid-oxidized protein. No molybdenum, iron, or selenium was found in the pure protein. Although NADH is the physiological electron donor for the proline reductase complex, the purified 300,000 molecular weight reductase component is inactive in the presence of NADH in vitro. Dithiothreitol, in contrast, can serve as electron donor both for unpurified (putative proline reductase complex) and purified proline reductase in vitro.


Differentiation of lymphoid cells. A selective anti-mitogenic component of normal serum which inhibits the induction of immunoglobulin production. Rabbit lymph node cell populations cultured in vitro in the presence of fetal calf serum are induced to produce immunoglobulin M-secreting cells. The induction of such immunoglobulin production, measured by the capacity of the cell population to secrete immunoglobulins, was inhibited when cells were cultured with sera from a variety of species despite the presence of fetal calf serum. The addition of such inhibitory serum 36 hours after initiation of the cell culture or thereafter was without effect on the extent of induction of immunoglobulin production. On the other hand, the presence of inhibitory serum in culture during only the first 24 hours yielded the same inhibition as when serum was present throughout the 72-hour culture period. Inhibitory sera also suppressed the incorporation of thymidine into DNA. The induction of immunoglobulin production and the incorporation of thymidine into DNA were essentially equally inhibited by the same range of serum concentrations. Unlike conventional inhibitors of DNA synthesis, the inhibitory sera exhibited selective specificity with regard to the kind of cells that could be affected. Thus, such sera inhibited the DNA synthesis of lymph node cells cultured in the presence of fetal calf serum but did not inhibit concanavalin A-stimulated DNA synthesis of such cultured cells and, similarly, serum did not inhibit DNA synthesis of thymus cells cultured in the presence of fetal calf serum. The sera of all species examined were inhibitory except for fetal sera. As judged from a quantitative assay, bovine and porcine serum contained the highest titer of inhibitor, whereas sera from human, rat, mouse, and rabbit were clustered in a group exhibiting less inhibitor. Ascites fluid and lymph node extracellular fluids contained less inhibitor than found in the serum of the same animal and lysates of washed lymph node cells were devoid of inhibitor. Although fetal bovine serum and newborn bovine serum did not contain the inhibitor, it was detectable within 24 hours of parturition. The inhibitor is of relatively large apparent molecular weight (about 300,000) and has been purified about 70-fold.