Clinical use of topical cortisteroids.

Clinical use of topical cortisteroids. The action of topical corticosteroids is anti-inflammatory and antimitotic. By the use of laboratory and clinical trials they can be divided into four grades of potency. Weak steroids may be made more potent by better penetration which can be achieved by plastic occlusion or by alteration of the vehicle in which they are suspended. Local side-effects of topical corticosteroids are described and attention is drawn to the face as a particularly vulnerable area. Use of large amounts of the most potent steroids has produced Cushingoid states normally associated only with systemic steroids, and this is more likely if the patient has liver damage. Practical advice on the choice of steroid for particular disorders is given and the importance of intermittent treatment both for safety and effectiveness is stressed.


Pulmonary embolism: current therapeutic concepts. Pulmonary thromboembolism is a widespread problem and is an important cause of death in patients with a variety of medical and surgical conditions. There have been few significant advances in the understanding of the aetiology beyond additional evidence confirming the importance of Virchow's triad. An impressive list of epidemiological associations has been compiled, however. Some knowledge of the natural progression of the disease is required as an aid in the understanding of the application of the therapeutic and prophylactic measures available in the management of pulmonary embolism. It would seem that at least two-thirds of pulmonary emboli are non-fatal, and in these cases the natural resolution, even of comparatively large embolic masses, is very efficient in patients without pre-existing cardiopulmonary disease. Diagnosis may prove difficult and most ancillary investigations are of questionable value. On the other hand, pulmonary radio-isotope scanning is far more specific and pulmonary angiography is a comparatively simple and complication-free diagnostic procedure. Prophylaxis is a real and practical aim, especially following surgery or myocardial infarction. In these groups widespread clinical trials of prophylactic measures have been made possible by the objective radio-iosotope screening techniques. Mechanical means of preventing venous stasis and anticoagulation appear effective. In addition, low-dose subcutaneous heparin seems to be as useful as heparin in conventional dosage. Apart from conventional supportive therapy, there are three major approaches to the treatment of pulmonary embolism. Heparin remains the mainstay, particularly in the less severe cases, hopefully preventing propogation of thrombosis and recurrence of embolism, thus allowing resolution to take place. Thrombolytic therapy with streptokinase or urokinase is capable of producing far more rapid dissolution of pulmonary emboli with consequent theoretical advantages over heparin. No reduction in mortality has been shown using thrombolytic therapy. Patients who fail to respond satisfactorily to acute resuscitative measures may require pulmonary embolectomy.


Clinical and haemodynamic effects of minoxidil in refractory hypertension. Thirteen patients whose hypertension had been resistant to conventional drug therapy, had minoxidil added to their regimen in doses from 5 to 60 mg/day. All responded with satisfactory reductions of blood pressure to mean values of 149/90mm Hg (supine) and 143/89mm Hg (standing). A significant portion of their previous antihypertensive therapy was either greatly decreased or withdrawn completely. Although fluid retention occurred in most patients as the dose of minoxidil was increased, this could be successfully checked by the use of diuretics. It was considered that in addition to beta-adrenoreceptor blocking drugs to control the reflexly induced cardiac stimulation, sufficiently aggressive diuretic therapy is mandatory to ensure the successful use of this drug. Haemodynamic evaluations in seven patients clearly showed that lowering of blood pressure was the result of decreases in peripheral vascular resistance as significant increases in the cardiac index occurred in all patients.


Borderline hypertension discovered during screening: effect of twelve months' treatment. The frequency of borderline hypertension (diastolic BP 95 to 109 mm Hg) is described in 5,877 men and 1,125 women attending a screening clinic. Borderline hypertension is more frequent in each 5-year age group in men than women. The results of the initial 12 months treatment show a greater fall in mean systolic and diastolic pressures in those on active treatment in comparison with placebo. Over the same 12 months the sum of selected ECG precordial lead voltages shows a significant fall in those on active treatment as compared with those on placebo.


[Structure and function of the microtubules]. The article the author reviews the substantial data from the literature on the morphology, biochemistry and physiology of these subcellular structures, which are the object of intensive studies during the recent ten years. He describes and presents his own figures of electrone microtubules in single cariotic cells and their subcellular organels. Ticrotubules in single cariotic cells and their stbcellular organels. The author pays attention to the most omportant processes, in which microtubules play substantial role: movement of chromosomes during the division of cells, intracellular organization and transport of the materials, maintenance of cellular form, movement of cells, formation of sensory apparatuses. In discussing these processes he pays special attention to the metabolic processes, with which the participation of microtubules is connected as well as to the mechanisms, by means of which various drugs, hormones and other biologic substances act.


[Effect of antilymphocyte serum on the antigen-binding cells of the blood and the survival of skin grafts]. The author carried out comparative studies on rats to examine the effect of antilympholytic serum on the survival of skin transplants and the amount of natural rosette, which formed lymphocytes from the peripheral blood in vitro with sheep erythrocytes. There was a correlation dependence on the duration of the survival of transplants and inhibition of rosette formation during the first reaction for the rejection of the transplant.


[Thermovision applied to relaxation].

[Thermovision applied to relaxation]. Dynamic  telethermography (thermovision) affords at distance picking up on a T.V. screen (black and white or color) of the thermal body map. Its use in relaxation (autogenic training) enabled us to study in their overall, the thermal modifications observed during the relaxation. We have been able to find changes occurring in the release of heat at the front of the neck and at the inner corner of the eye socket; up to now, this had been unnoticed. Finally, it has allowed a dynamic work: the progression in heat release at the extremities of the limbs, at the thorax and the abdomen level was subject to an accurate and detailed study which leads to future prospects of interest by showing vascular exchanges, the thermal changes of which being only reflecting them.


[A new synthetic antiparkinsonian drug, tropatepine hydrochloride in extrapyramidal syndromes induced by neuroleptics]. Tropatepine hydrochloride was given per oral route to 184 patients and per injections to 34 patients. Average prescribed doses were about 20 mg (2 tablets). This clinical study has shown that tropatepine hydrochloride has an antiparkinson activity against neuroleptic-induced extra-pyramidal syndrome. In comparison to the activity of the other synthetic antiparkinson drugs, the activity of tropatepine hydrochloride is: -- similar on akineto-hypertonia and on tremor; -- better on akathisia and, though less frequently, on anormal dyskinetic movements due to long term neuroleptic treatment. Tolerance is good ; furthermore clinical and biological performed examinations have shown that the drug seems free of toxic effects In more than 200 treated patients no severe mental aberration and no habituation have been reported.


[Convulsive incidents and EEG changes during treatment with tricyclic antidepressive agents]. The study of the action of tricyclic antidepressant on epilepsy and E.E.G. leads to a separation between two groups of patients treated with these drugs : 1. The epileptic patients with personnal or family history of epilepsy have a higher risk of epileptic seizures during these treatments. However, a certain number of them get better as far as seizures and personnality disturbances are concerned. On these patients E.E.G. appears an activation of the pattern. We must precise that, with a proper anti-epileptic treatement, there are only rare seizures and, in any case, epilepsy is not a contra-indication to antidepressant treatments. E.E.G. gets even better in a few cases. 2. In patients without history of epilepsy, seizures are rare. If they appear, one finds several predisposing factors, alcoholism being the most frequent. High dosages are not a constant factor. Previous E.C.T. could increase the risk of seizures. On these patients E.E.G., the action of tricyclic antidepressant is not constant : we found important changes as well as no changes at all. The changes would be an aggravation with an epileptic pattern or slow rythms, as well as the pattern getting better. There is no correlation between clinical manifestations and E.E.G. patterns.


On the rapidity with which LRH induces release of LH and FSH from perifused anterior pituitary tissues of the rat. In a series of perifusion (superfusion) experiments, data were obtained which indicated that LRH and K+, when effective, induced increase in LH and/or FSH release within about one minute after arriving in the vicinity of the test rat pituitary tissues. Hormone release stimulating effects of LRH or high K+ continued for some time after washout of test agent. To secure these data perifusion flow was set at 2.5 or more ml/min., each test solution was pulsed for two minutes only, effluent samples were collected at 15 second intervals shortly before, during and after each pulse, and both mixing and dead space time were minimized by introducing each new test solution directly in front of the tissue chamber, via a 4-way or 5-way pulse control stopcock through which two streams of perifusion fluid (Control and Test) passed continually. LH and FSH in effluent samples were estimated by radioimmunoassay.


Action of steroids on LH-RH provoked gonadotropin release. The action of mesterolone (1 alpha-methyl-17-beta-hydroxy-5 alpha-androsten-3-one) mesterolone cipionate, testosterone, 17 alpha-methyl-testosterone, 17 beta-hydroxy-5 alpha-androstane-3-one, cyproterone actetate and D-norgestrel on basal and LH-RH stimulated, gonadotropin release was studied in vitro. Hemi-gland preparation of the anterior pituitary were incubated in the presence or absence of steroids and/or LH-RH. Steroid hormones were added at a concentration of 25, 5 and 1 mug per ml medium. Basal levels of LH and FSH discharge were not influenced by mesterolone, whereas inhibition of LH-RH stimulated gonadotropin release was noted at 25 mug mesterolone/ml medium. The addition of mesterolone cipionate to the incubation medium did not significantly change basal FSH levels, whereas an inhibition of LH-RH provoked FSH discharge was noted. Basal LH release was stimulated by the presence of 1 mug mesterolone cipionate per 1 ml medium. LH-RH stimulated LH release in the presence of 25, 5 and 1 mug mesterolone cipionate per 1 ml medium was indistinguishable from control levels. Basal levels of LH and FSH were not affected by testosterone, whereas an impaired gonadotropin response to LH-RH was observed in the presence of testosterone. At 1 mug testosterone per 1 ml medium only LH response to LH-RH was found. Basal  gonadotropin levels in the presence of 17 alpha-methyl-testosterone were indistinguishable from control values. The only inhibition of LH-RH action was found for stimulation of LH release at 25 mug 17 alpha-methyl-testosterone/ml medium. On the other hand, 17 beta-hydroxy-5 alpha-androstane-3-one stimulated basal LH and FSH discharge, and LH-RH actions were not impaired. Results of our studies with cyproterone acetate indicate that this steroid stimulates basal pituitary gonadotropin discharge at high concentration, without blunting responses to LH-RH. No action of D-norgestrel on basal and LH-RH stimulated gonadotropin release was observed. These experiments add further evidence that steroids modulate LH-RH actions at the pituitary level. It is concluded that the fine control of gonadotropin release resides at the adenohypophysis whereas the coarse control of LH and FSH discharge is at the hypothalamus and/or other centers of the central nervous system.


Control of pituitary FSH synthesis by hypothalamic factors in rats. Electrolytic lesions placed in the median eminence of adult male rats are followed, in 7--10 days, by a significant decrease in pituitary FSH concentration. The intravenous injection of a hypothalamic extract prepared from normal animals induces, in 45 minutes, a significant increase in the amounts of FSH stored in the pituitary of median eminence-lesioned animals. The concentration of FSH in the pituitary of lesioned animals is also increased to normal by the intravenous injection of an extract prepared from the isolated hypothalamic island of animals previously submitted to a total "hypothalamic deafferentation", in spite of the fact that this does not contain any FSH-RF activity. It is suggested that the hypothalamus possesses separate humoral factors for the regulation of the release and of the synthesis of pituitary FSH.


Phentolamine blocks the somatostatin-mediated inhibition of insulin secretion. This study was undertaken to determine the influence of alpha-adrenergic blockade upon the somatostatin-mediated inhibition of insulin secretion. Somatostatin inhibited both the basal and glucose-stimulated pancreaticoduodenal vein insulin output of dogs. This effect was not observed during a simultaneous infusion of phentolamine, an alpha-adrenergic blocker. It is proposed that alpha-adrenergic mechanisms play a role in the inhibition of insulin secretion by somatostatin.


Localization of LHRH in neurons in frog brain (Rana pipiens and Rana catesbeiana). Hypothalamic extracts from frogs (Rana pipiens) were found to contain a significant quantity of immunoreactive LHRH (3.27 +/- 0.63 ng/hypothalamus) (mean +/- SE) measured by radioimmunoassay. In additional radioimmunoassay studies of gross brain LHRH distribution in R. pipiens and R. catesbeiana, 16% of the total frog brain LHRH was located within the telencephalon-septum-optic chiasm regions while the remainder was distributed within the infundibular hypothalamic-pituitary complex. Immunohistochemical studies using the peroxidase-anti-peroxidase (PAP) unlabeled antibody enzyme technique demonstrated the presence of LHRH selectively within some neuronal perikarya located primarily in the median septal nucleus. Fibers containing immunoreactive LHRH were seen in the vicinity of these neuronal cell bodies and in the medial and lateral septal nuclei. In addition, LHRH-containing fibers extended to the median eminence and posterior pituitary, transversing a course beneath the preoptic recess or through the medial forebrain bundle, and then through the lateral infundibular hypothalamus to enter the median eminence bilaterally. LHRH within the median eminence was located in both the inner subependymal and outer glandular zones. On the basis of earlier physiological studies, it is proposed that this LHRH-peptidergic septo-infundibular pathway is involved in control of cyclic gonadotropin activity in the frog.


Distribution of gonadotropin-releasing hormone in the mouse brain as revealed by immunohistochemistry. The distribution of gonadotropin-releasing hormone (GnRH) was studied immunohistochemically in the brain of the adult mouse with the peroxidase-antiperoxidase (PAP) method. Primary antisera were prepared against unconjugated synthetic GnRH and GnRH conjugated to limpet hemocyanin or bovine serum albumin. GnRH was localized in the organum vasculosum of the lamina terminalis (OVLT), with the greatest amount being found ventral to the cephalic end of the third ventricle. In the cephalic region of the median eminence, it was concentrated bilaterally in longitudinal bands located dorsal to the tuberoinfundibular sulci. More caudally, near the junction of the infundibulum with the brain, GnRH accumulated over the apex of the tuberoinfundibular sulci, with several foci being scattered from this region medially to the ependyma of the third ventricle. The greatest aggregation of GnRH occurred in the psotinfundibular median eminence in an area extending from the floor of the third ventricle to the ventral surface of the brain. In the caudal median eminence, GnRH was arranged in a narrow ventral band that crossed the midline. GnRH appeared to be located in axonal processes and terminals of the OVLT and median eminence; the structures observed were granular. GnRH was not localized within the neuronal cell bodies of any hypothalamic nuclei. When one antiserum to conjugated GnRH was used at high concentration, the cytoplasm of ependymal cell bodies and tanycyte processes was stained more intensely than the general background, but, with absorption and/or dilution of the antiseru, this staining was shown to be nonspecific.


Delayed effects of in vivo LHRH immunoneutralization on gonadotropins and prolactin secretion in the female rat. A single ip injection (0.2 ml) of a rabbit antiserum to LHRH was given at noon on proestrus (day 0) to 4-day cycling rats. A blockade of the preovulatory surge of LH and FSH was observed, as well as inhibition of ovulation; the prolactin (PRL) surge was unaffected. Four days later, palsma LH and FSH were still low and hypoprolactinemia appeared, while the estrous cycle was unaffected. Then, after a transient diestrous period, vaginal smears indicated a pattern of persistent estrus for at least 240 days starting from day 8 after treatment. Hypothalamic LHRH remained low throughout this period, with the exception of a transient rise around day 36; hypothalamic serotonin and dopamine were unchanged. A peak of FSH in serum appeared on day 28 and an elevation of serum LH on day 36; this latter rise was concomitant with the increase of hypothalmic LHRH and with ovarian luteinization. Except for this short period, the ovaries showed a picture of persistent estrus, with large, cystic follicles. During the whole experimental period, basal serum FSH levels were higher than those of LH. Pituitary FSH and LH contents remained in the range usually found during the normal estrous cycle. Starting from the 8th day after treatment, a marked hyperprolactinemia appeared. Serum estradiol and progesterone were assayed on days 10 and 40 after injection; no significant increase in either steroid was observed on day 10, but on day 40 the levels of estradiol increased to values similar to those of progestrus, while progesterone showed only a small, but significant, increase. Thus, a single injection of LHRH antiserum (AS) in a normal cycling rat provoked a long-term alteration in hypothalamic function. The results so far obtained suggest that the long-lasting hyperprolactinemia induced by this treatment, might be due to an abnormal ovarian secretion.


[In vitro studies on the islands of Langerhans. XI. Insulin secretion and content of isolated islands of Langerhans in the Wistar rat under short term incubation and under organ culture conditions]. Insulin (IRI) secretion pattern of collagenase isolated islets from Wistar rats were investigated in a batch type incubation (60 min) and under organ culture conditions (up to 7 days) with different concentrations of glucose as stimulus. The B-cell response within 60 min of incubation was determined in Krebs-Ringer bicarbonate buffer with 1 mg/ml bovine serum albumin and 16 mM HEPES (KRB-HEPES) and compared with the hormone release in a culture medium (TC 199) containing 10% calf serum. Additionally the insulin content of islets before and after 7 days of culture was assayed radioimmunologically. In the presence of culture medium the insulin secretion was enhanced by 5.8 mM glucose whereas this glucose concentration does not stimulate the insulin secretion in KRB-HEPES. During organ culture the insulin secretion was identical in media with 1.0 and 5.8 mM glucose, respectively, but 15 mM glucose raised the hormon output more than 10-fold. The insulin content of islets, cultured for 7 days was decreased in the presence of 15 mM glucose up to 30% and in the presence of 5.8 mM glucose up to 13% of that of islets after isolation. The recovery rate of insulin calculated as the sum of secretion and content after cultivation at 15 mM glucose was higher than 100%, whereas the experiments in the presence of 1.0-5.8 mM glucose are characterized by a recovery rate of 25%. The results are discussed in connection with a altered intracellular breakdown of insulin in the B-cells.


Effect of nickel chloride on primary antibody production in the spleen. Mice immunized intraperitoneally with sheep erythrocytes were treated with nickel chloride, a common particulate air pollutant. Primary antibody production in the spleen was examined using a hemolytic plaque technique. A negative linear dose-response relationship (p is less than 0.05) was observed between the logarithm of (plaques/10(6) cells) and the nickel concentration administered. Mice  injected with 3.09 mug Ni2+/g body weight displayed lymphocyte function similar to that of control mice. However, injection of 9.26-12.34 mug Ni2+/g caused significant immunosupression (p is less than 0.05).


On genetically determined human serum cholinesterases. The existence of genetically determined human serum cholinesterase variants (actlcholine acyl-hydrolase; EC 3.1.1.8) has been recognized by the use of a short acting muscle relaxant, suxamethonium. This polymorphism is now explained by at least four alleles belonging to an autosomal locus Ch1 or E1 depending on the nomenclature. These are: the usual gene, the dibucaine-resistant gene, the fluoride-resistant gene and the silent gene. At present, the modes of transmission for the silent genes is confusing and lack proper understanding. Another variant, not an allele of the first group, is recognized by gel electrophoresis, belong to a second locus Ch2 or E2. Evidence has been presented that many more rare serum cholinesterase variants may exist. Besides these genetic variants, normal serum cholinesterase is known to exist in multiple molecular forms. The present article attempts to discuss the polymorphism of human cholinesterase in relation to their chemical and genetic characteristics and their possible function.


Fluorescent whitening agents in the environment. An assessment of the ecological situation regarding the use of fluorescent whitening agents (FWAs) is given. Firstly a survey is made of the existing literature with references to the contributions on the subject in this EQS supplement volume, then the effects of waste water treatment with respect to the elimination of FWAs in the different stages of treatment are discussed. The final load of FWAs in receiving water is estimated. These figures are brought into relation with the actual content of receiving water residues of natural and manmade origins. The significance  of the FWA load is discussed. All the arguments lead to the strong suggestion that these residues are finally metabolized by natural systems of elimination, so that no ecological risks exist when FWAs are properly used in the main fields of application, i.e. in the textile, paper and soap and detergent industry.


History of whitening.

History of whitening. Man's earliest endeavours to enhance the brightness of white objects in everyday use, especially clothing, led to the invention of various bleaching processes. KRAIS'S discovery in 1929 that the natural substance esculin could be applied to textiles to give whites of unprecedented brilliance led to the industrial development of fluorescent whitening agents (FWA's). The limited effectiveness of esculin, a glycoside of 6,7-dihydroxycoumarin prompted research which led to the synthesis of other compounds of the same class, many of which have proved useful as whiteners; though the really broad developments of the 1940's stemmed from the synthesis of the 4,4'-diaminostilbene-2,2'disulfonic acid derivatives which are sill the most important groups of FWAs.


The chemistry of fluorescent whitening agents. Major structural types. This gives a summary of those fluorescent whitening agents (FWAs) that have industrial importance. Firstly, a survey of the general physical and applicational properties is given, and then the chemistry of FWAs is dealt with. The FWAs are classified in six major structural classes: (A) stilbenes (B) coumarin and carbostyril compounds, (C) 1,3-DIPHENYL-2-PYRAZOLINES, (D) naphthalimides, (E) benzoxazolyl substitution products of conjugated systems, and (F) combined products of other heteroaromatics with ethylene or aromatic systems, owing to their methods of preparation and to their optical characteristics.


Specific inhibition of translocation by tubulosine in eukaryotic polysomes. The alkaloid tubulosine inhibits the process of peptide chain elongation by eukaryotic polysomes by specifically preventing the elongation-factor-2-dependent step of translocation. Tubulosine does not affect either the elongation-factor-1-dependent binding of aminoacyl-tRNA or peptide bond formation. The site of action of tubulosine appears to be independent from the ribosomal site involved in cycloheximide action, since the alkaloid is active in blocking polymerization and enzymic translocation by polysomes from a yeast mutant resistant to cycloheximide. Furthermore tubulosine does not affect the non-enzymic translocation which takes place in the presence of high potassium ion concentrations, whereas this reaction is strongly inhibited by cyclohemimede. The different steps of translocation are discussed to explain the reactions blocked by tubulosine and cycloheximide.


Globin mRNA species containing poly(A) segments of different lengths. Their functional stability in Xenopus oocytes. Rabbit globin mRNA species containing poly(A) segments of different lengths were prepared by partial phosphorolysis of mRNA with Escherichia coli polynucleotide phosphorylase. By varying the salt concentration and the time of incubation of the phosphorolysis mixture, as well as performing oligo(dT)-cellulose chromatography at 22 degrees C and at 4 degrees C, globin mRNA preparations containing poly(A) segments of approximately 122, 95, 68, 39, 32, 21, and 16 adenylate residues were obtained. It was found that the functional stability of the mRNA species containing 32 or more adenylate residues after injection into Xenopus laevis oocytes equaled that of the native globin mRNA. On the other hand, the functional stability of mRNA containing an average number of 21 adenylate residues was about 30% of the native mRNA, while that of mRNA containing 16 adenylate residues was as low as poly(A)-free globin MRNA.


Structural properties of Escherichia coli RNA polymerase Subunits. 1. The surface of the RNA-polymerase-DNA complex possesses an exposed polypeptide loop. 2. Proteinases with differing specificities (trypsin, chymotrypsin, subtilisin and clostripain) preferentially cleave the exposed region. 3. The cleaved polypeptide is reassembled into RNA polymerase by renaturation from a solvent which promotes a random coil conformation. 4. Isolated beta subunit has a proteolytically resistant nucleus of approximately 70000 molecular weight. This resistant polypeptide may be generated by trypsin, chymotrypsin, subiilisin or clostripain. 5. Isolated alpha subunits are comparatively resistant to proteolysis. 6. Although of similar molecular weights beta and beta' appear to have unrelated primary sequences and markedly different conformations in free solution. 7. Digestion of the beta subunit may be blocked by formation of the alpha2beta subassembly. 8. Evidence is presented suggesting that beta' in the intact enzyme (alpha2beta beta') possesses the exposed polypeptide loop.


Purification of protease II from Escherichia coli by affinity chromatography and separation of two enzyme species from cells harvested at late log phase. N-Acetyl-D-arginine linked to an agarose matrix has been used to purify protease II from Escherichia coli by affinity chromatography. The specific adsorption of protease II to this absorbent was achieved in 220 mM potassium phosphate buffer pH 7.6, and the enzyme was eluted with L-arginine. Enzyme preparations from cells harvested at late log phase have been resolved into two molecular species which differ in specific activity, kinetic constants and carbohydrate content. Both species appeared homogeneous by electrophoresis in conventional buffers and also in the presence of sodium dodecyl sulfate. Only one enzyme species was obtained by the same procedure using bacteria harvested at the middle of exponential growth.


Transglycosylic reactions of nucleotides of deoxy sugars. Biosynthesis of deoxy analogues of alpha,alpha'-trehalose. The biosynthesis of alpha,alpha'-trehalose was investigated in a reaction catalyzed with yeast UDP-Glc :glucose-6-phosphate 1-glucosyl transferase. It was revealed that the hydroxyls at positions C-2, C-3, C-4 and C-6 are not essential for preserving its substrate properties in this reaction. The affinity of the enzyme and the rate of hexose incorporation into trehalose decreases in following sequence: UDP-glucose, UDP-2-deoxy-D-glucose, UDP-6-deoxy-d-glucose, UDP-4-deoxy-D-glucose and UDP-3-deoxy-D-glucose.


Small-angle x-ray and light-scattering study of native and trypsin-modified methionyl-tRNA synthetase from Escherichia coli. Small-angle X-ray scattering experiments were performed on an absolute scale on solutions of methionyl-tRNA synthetase from Escherichia coli in its native and trypsin-modified forms. A light-scattering study was performed on the same solutions to verify monodispersity. The structural parameters for the trypsin-modified enzyme, radius of gyration (2.48 nm), volume (90 nm3), surface/volume (1.5 nm-1) and the distribution of chords can account for an equivalent prolate ellipsoid of revolution having an axial ratio 2.3 and a maximum length of 9 nm, with a creviced surface. The rsults obtained for the native enzyme [i.e. radius of gyration (4.3 nm), volume (244 nm3), distribution of the scattering intensity and distribution of chords] exclude the possibility of a very compact quaternary structure and suggest that the enzyme consists of at least two globular parts, probably the two protomers, linked together by interactions involving a limited region of the structure.


The binding of indole to the alpha-subunit and beta2-subunit and to the alpha2beta2-complex of tryptophan synthase from Escherichia coli. Identification of a second indole-binding site on the alpha-subunit. The binding of indole and indolepropanol phosphate, an analogue of the substrate indoleglycerol phosphate, to the individual alpha and beta2-subunits and to the alpha2beta2-complex of tryptophan synthase was studied by equilibrium dialysis. The use of [14C]indole and indolepropanol [32P]phosphate permitted simultaneous binding studies to be carried out. Competition between indole and indolepropanol phosphate in binding to a particular site was taken as evidence for that site being part of the active site of the alpha-subunit. The binding of indole to the active site of the alpha-subunit is weak (Kd = 18mM). A second distinct site binds indole more strongly (Kd = 1.5 mM) and interacts with the active site indirectly. It is therefore designated an effector site. Furthermore, the binding of indole and/or indolepropanol phosphate appears to stabilize different conformations of the alpha-subunit. The beta2-subunit binds indole only weakly (Kd = 12 mM) to many (n = 10) sites per polypeptide chain. The alpha2beta2-complex retains one or two sites per alphabeta-equivalent of relatively high affinity (Kd = 1.2 mM). The active sites of the component alpha and beta-subunits probably belong to the second class of many (n = 40) sites of low (Kd = 30 mM) affinity for indole. These findings support conclusions from the literature that both bi-substrate reactions involving indole catalyzed by tryptophan synthase and its subunits must follow strictly ordered addition mechanisms with the respective other substrate adding first.


Studies on the environment of protein S7 within the 30-S subunit Escherichia coli ribosomes. Analyses are described of three types of ribosomal fragment, all derived from the well-characterized ribonucleoprotein species consisting of proteins S7, S9, S10, S14 AND S19, together with RNA from sections O'-D-E'-K-P-P'-E-A of the 16-S sequence. 1. When 30-S subunits were hydrolysed with ribonuclease T1 in presence of deoxycholate in addition to the components previously described, a fragment could be isolated which contained only proteins S7 and S19, together with minor amounts of S13 or S14. Oligonucleotide analysis of this fragment showed that it contained RNA from sections O'-E' and P-A of the 16-S RNA, but that section K (from the middle of this area) was missing. 2. It has previously been shown that when 30-S subunits are irradiated with ultraviolet light, protein S7 is the primary target of cross-linking of protein to RNA. By making use of this reaction, ribonucleoprotein fragments were isolated from irradiated 30-S subunits the unbound proteins were removed, and RNA fragments containing covalently linked protein S7 were identified. It was possible to demonstrate that the site of cross-linking lies within the O'-A region of the 16-S RNA, and more precise experiments showed that this site almost certainly is in the P-A region. 3. When the parent five-protein ribonucleoprotein fragment (above) was deproteinized under very mild conditions, RNA complexes could be isolated which consisted of non-contiguous sequences, but which migrated as a single species into a polyacrylamide gel. Analysis of one of these complexes showed that it contained sequences from sections O'-E' and P-A, but that section K was missing (cf. first paragraph above). This demonstrated that these two separate regions of RNA interact within the 30-S subunit, independently of the presence of protein.


Phenylalanyl-tRNA and seryl-tRNA synthetases from baker's yeast. Substrate specificity with regard to ATP analogs and mechanism of the aminoacylation reaction. Eighteen analogs of ATP have been tested in the aminoacylation reaction of phenylalanyl-tRNA and seryl-tRNA synthetases from baker's yeast. Four compounds are substrates for phenylalanyl-tRNA synthetase, five for seryl-tRNA synthetase, one compound is an inhibitor for both enzymes; their Km and Ki and V values have been determined. The substrate specificity shows that for the catalytic action of both enzymes with these substrates positions 6, 7, 8 and 9 of the purine moiety and positions 2' and 3' of the ribose moiety are important.


Phenylalanyl-tRNA synthetase from baker's yeast. Salt dependence of steady-state kinetics indicates two molecular forms of the enzyme. Steady-state kinetic data of aminoacylation of tRNAPhe by phenylalanyl-tRNA synthetase depend on salt concentration. At 5 mM KCl and 20 mM MgSO4 a non-linear curve is found in the double-reciprocal plot for ATP and phenylalanine, while at 200 mM KCl and 50 mM MgSO4 a linear curve is observed. KCl and MgSO4 dependence of the reaction also show biphasic curves with intersection points of the two extrapolated linear parts at 50 mM and 10 mM, respectively. A biphasic curve is also found if the concentration of CTP is varied at constant low ATP concentration. Extrapolations of the linear parts of the curves for ATP as well as for CTP at 5 mM KCl and 20 mM MgSO4 intersected the 1/[NTP] axis at 1.2 +/- 0.1 mM. Hence the existence of a non-linear curve for ATP as well as phenylalanine does not necessarily indicate two non-equivalent binding sites for these substrates. A more likely explanation is the existence of two different molecular forms of phenylalanyl-tRNA synthetase which are interconvertible by salt. This explanation is substantiated by the observation that proteolytic digestion of phenylalanyl-tRNA synthetase is more easily achieved at low than at medium ionic strength. In addition mischarging of tRNAIle with phenylalanine by phenylalanyl-tRNA synthetase occurs at a moderate rate at 5 mM KCl and 20 mM MgSO4 whereas it is largely depressed by addition of either 5 mM CTP or 150 mM KCl.


Thermal melting curves of tRNAPhe from yeast lacking different numbers of nucleotides from the 3'-end. The thermal melting curves of tRNAPhe and tRNALys from yeast lacking different numbers of nucleotides from the 3'-end were recorded. The removal of the first nucleotide had no effect on the melting profile. The stepwise removal of the following six nucleotides caused changes in the character of the derivative melting curves: they became rather broad and melting started at lower temperatures. These results indicate that the nucleotides at the 3'-end contribute to the stability of the molecular structure of tRNAs.


The specific role of ribosomal protein S1 in the recognition of native phage RNA. The previously reported requirement of ribosomal protein S1 for translation of phage RNA is now shown to be related to the involvement of the protein in initiation complex formation. The structure of the messenger RNA appears to be uniquely related to S1 function, since translation and initiation and midly unfolded phage RNA (by modification with formaldehyde) are independent of S1. It is proposed that S1 functions in conjunction with initiation factor IF-3 by recognizing and unfolding elements of the tertiary structure of phage RNA. A model is suggested for S1 function in both initiation of protein synthesis and initiation of phage RNA replication.


The subcellular distribution and state of the elongation factor Tu in extracts of Escherichia coli B. The concentration of elongation factor Tu (EF-Tu) is about 8 times that of ribosomes in extracts of Escherichia coli B grown in glucose-minimal medium. 90% of the EF-Tu is found in the ribosome-free fraction of the cell and 75% of this is free of other macromolecules as judged by chromatography on Sephadex G-100; about 10% is bound to the elongation factor EF-Ts and the remaining 10-15% is eluted from Sephadex G-100 earlier than expected for its molecular weight. Tryptic peptide maps of the three forms of EF-Tu are essentially indistinguishable.


Cytidine diphosphate diglyceride of bovine brain. Positional distribution of fatty acids and analysis of major molecular species. A method is described for the isolation of CDP-diglyceride from bovine brain. Yields of the product ranged from 9.2-15.5 mumol per kilogram of tissue, which corresponds to about 1% of the level of phosphatidic acid. Mild alkaline hydrolysis of the product gave three water-soluble phosphate esters which had the same electrophoretic mobilities as CMP, CDP-glycerol and glycerol 3-phosphate. The liponucleotide was quantitatively hydrolysed by CDP-diglyceride hydrolase from Escherichia coli to phosphatidic acid and CMP. No dCMP was recovered in enzymatic or alkaline hydrolysates and it is concluded there can be little or no dCDP-diglyceride in bovine brain. Brain CDP-diglyceride was similar to phosphatidylinositol in that in both lipids stearate was the major saturated fatty acid and arachidonate the most abundant unsaturated fatty acid. This differed significantly from the fatty acid patterns of other metabolically related phospholipids, phosphatidic acid and cardiolipin. Brain CDP-diglyceride was hydrolysed with phospholipase C from Clostridium welchii with the liberation of the diglyceride moiety in high yield. Treatment of the diglyceride with pancreatic lipase showed CDP-diglyceride with the asymmetric distribution of fatty acids characteristic of most mammalian phospholipids, saturated fatty acids being found mostly at position 1 and polyunsaturated fatty acids at position 2. The derived diglyceride acetates were separated into different molecular species by argentation thin-layer chromatography. These analyses showed that 1-stearoyl, 2-arachidonoyl was the major species of brain CDP-diglyceride.


Maltose transport in Escherichia coli K12. A comparison of transport kinetics in wild-type and lambda-resistant mutants as measured by fluorescence quenching. The kinetic parameters for the maltose transport system in Escherichia coli K12 were determined with maltose and maltotriose as substrates. The system exhibits an apparent Km of 1 muM for maltose and 2 muM for maltotriose. The V of entry was determined as 2.0 and 1.1 nmol substrate/min per 10(8) cells. Mutations in lamB, the structural gene for the receptor protein of phage lambda, increased the Km for maltose transport by a factor of 100-500 without influencing the maximal rate of transport. Maltotriose is no longer transported in these lamB mutants. The maltose-binding protein, an essential component of the maltose transport system, was found to exhibit substrate-dependent fluorescence quenching. This phenomenon was used to determine dissociation constants and to estimate the rate of ligand dissociation. A Kd of 1 muM for maltose and of 0.16 muM for maltotroise was found. From the comparison of the kinetic parameters of transport of maltose and maltotriose in wild-type and lambda-resistant mutants with the binding constants for both sugars to purified maltose-binding protein, we conclude that the lambda receptor facilitates the diffusion of maltose and maltodextrins through the outer membrane.


Subunit composition of Escherichia coli RNA polymerase during transcription in vitro. The subunit composition of Escherichia coli RNA polymerase during the transcription in vitro of bacteriophage T7DNA was analysed at several steps of RNA synthesis. RNA-polymerase . DNA complexes were sedimented through a glycerol gradient and the RNA polymerase subunits present in each fraction of the gradient were separated by dodecylsulfate-polyacrylamide gel electrophoresis and quantified colorimetrically on the gels. RNA polymerase selectively bound to T7 DNA in the absence of nucleoside triphosphates has the same subunit composition as free RNA polymerase holoenzyme (beta'betaalpha2) omicron. Addition of the nucleoside triphosphate combinations ATP, GTP, UTP or ATP, CTP, UPT, or GTP, CTP UTP to the binding reaction does not alter the subunit composition of RNA polymerase holenzyme bound to DNA. In contrast, in the presence of ATP, GTP and CTP up to 3 pmol of omicron-subunit are released from a complex containing RNA polymerase and 1 pmol of T7 DNA. In the presence of the four nucleoside triphosphates about 90% of the RNA polymerase associated with DNA and nascent RNA has the subunit composition of RNA polymerase core enzyme (bet'betaalpha2). The omicron-subunit is released from the complex and is recovered near the top of the gradient. The transition from the binding complex to the elongation complex and the incorporation of gamma32P-labeled ATP and GTP at the 5' end of RNA molecules were followed in parallel. In the purified elongation complex about 1 pmol of ATP or GTP is incorporated into RNA per pmol RNA polymerase core enzyme engaged in RNA synthesis.


Histidyl-transfer-ribonucleic-acid synthetase from Salmonella typhimurium. Studies of the sulfhydryl groups. The reactivity of the sulfhydryl groups of histidyl-t RNA synthetase from Salmonella typhimurium and the effect of substrates on the reactivity has been studied using p-hydroxymercuribenzoate and 5, 5'-dithiobis (2-nitrobenzoic acid) as reagents. It has been found that 5, 5'-dithiobis (2-nitrobenzoic acid) titrates only two sulfhydryl groups per molcule of enzyme and the reaction is essenaitlly monophasic, while p-hydroxymercuribenzoate titrates four sulhydryl groups. As observed kinetically the reaction with p-hydroxymercuribenzoate is strongly biphasic, each phase corresponding to about two sulfhydryl groups per enzyme molecule. With both reagents no detectable difference in sulfhydryl group reactivity was observed when ATP, histidine and tRNA specific for histidine were added individually or in combination to the enzyme. The enzyme activity slowly changes after two or four sulhydryl groups are blocked by 5, 5'-dithiobis (2-nitrobenzoic acid) or p-hydroxymercuribenzoate respectively. A new, stable level of activity is reached that is characterized by a different Km value for the aminoacylation reaction. The results indicate that the sulfhydryl groups reacting with the two reagents used here are neither directly involved in the binding of the substrates nor in the catalytic process. The ultimate change in enzyme activity after reaction of the sulfhydryl groups suggests a transition to an alternative enzyme structure.


Preparation and properties of an analogue of poly(A) and poly(G): poly(isoguanylic acid). Isoguanosine-5'-pyrosphosphate, in the presence of an oligonucleotide primer, was polymerized by Escherichia coli polynucleotide phosphorylase under conditions analogous to those required for polymerization of 5'-GMP. The resulting poly(isoguanylic acid), poly(isoG), was a multistranded helix with a stability considerably higher than that of poly(G), and fully resistant to various nucleolytic enzymes. The polymer exhibited a two-step temperature transition profile in moderately alkaline propylene glycol. Alkaline titration in aqueous medium, by ultraviolet and circular dichroism spectroscopy, showed two clearly defined transitions, the second of which was fully cooperative. The accompanying changes in sedimentation constants were consistent with a structure for poly(isoG) of a fourstranded helix, like neutral poly(G). In acid medium, spectral and potentiometric titrations demonstrated the existence of more than one transition in the pH range 6-12, with accompanying protonation of the isoguanosine residues. In neutral medium the polymer formed no complexes with other potentially complementary homopolymers. In acid medium, on the other hand, the protonated form of poly(isoG) did form a triple-stranded complex with poly(I), viz. 2poly(I) . poly(isoG)+. Possible structures are formulated for the neural and protonated forms of poly(isoG) which account for the two-step thermal transition in alkaline propylene glycol and on alkaline titration in aqueous medium. The nature of the protonated form, and its complex with poly(I) is also discussed.


Metabolism of phosphatidylglycerol in cell-free extracts of Escherichia coli. Cell-free extracts from Escherichia coli strain number 9, lacking among other enzymes glycerol kinase, are able to incorporate [2-3H] glycerol into phospholipids. The characteristics of this incorporation indicate that it is not taking place through the regular glycerol phosphate pathway of phospholipid synthesis which occurs when this compound is used as a precursor or even when extracts of E. coli strain 7, having a functional glycerol kinase, are incubated with [2-3H] glycerol. In E. coli strain 9 extracts glycerol is exclusively incorporated into the distal position of phosphatidyl-glycerol while in the other strains the middle position glycerol is partially labelled.


Isolation and characterization of four related peptides exhibiting alpha factor activity from Saccharomyces cerevisiae. The molecular structure of alpha factor, a mating hormone produced by alpha mating type cells of Saccharomyces cerevisiae has been investigated. From culture filtrates of alpha cells four oligopeptides exhibiting alpha factor activity have been isolated. These peptides, designated as alpha1, alpha2, alpha3, and alpha4 are structurally closely related, being composed of thirteen (alpha1 and alpha3) and twelve (alpha2 and alpha4) amino acids, respectively. The peptides were found to be composed of the following amino acids: 2 glutamic acid or glutamine, 2 proline, 1 glycine, 1 methionine or methionine sulfoxide, 2 leucine, 1 tyrosine, 1 lysine, 1 histidine, 1 or 2 tryptophan. The tridekapeptides differ from the dodekapeptides by an additional NH2-terminal tryptophan residue. Tyrosine was identified as the C-terminal amino acid in all four peptides. alpha3 and alpha4 are oxidation products containing an internal methionine sulfoxide instead of methionine. The mechanisms which could introduce the observed heterogeneity of the peptides are discussed.


On the interaction of seryl-tRNA synthetase with tRNA Ser. A contribution to the problem of synthetase-tRNA recognition. By following the tryptophan fluorescence of yeast seryl-tRNA synthetase on addition of tRNA Ser it was observed that the number of binding sites for tRNA decreases from two to one with increasing temperature, ATP or KCl concentration. Concomitantly a considerable decrease of the apparent binding constant was observed. The variation in the number of binding sites is explained by the presence of at least one temperature and ionic strength sensitive binding site and one temperature and ionic strength independent binding site. Relaxation kinetic experiments revealed two binding processes: a fast one depending on tRNA concentration and ionic strength and a slow one, which appeared to be independent of tRNA concentration and ionic strength. Enzyme kinetic studies showed that the activity of seryl-tRNA synthetase strongly depends on the KCl concentration and exhibits a maximum at 0.2 M KCl. Based on the data from relaxation and enzyme kinetic experiments a model is suggested for the recognition process involving a first unspecific step where all tRNAs, cognate and non-cognate, are bound to the synthetase (scanning step). The identification of the cognate tRNA is then performed at the recognition site by a conformational transition of the tRNA . synthetase complex (identification step).


Effect of theophylline on monoamine metabolism in the rat brain. The effect of theophylline on brain monoamine metabolism was studied in rats. Single doses of theophylline caused a striking and dose-related increase in the levels of 3-methoxy-4-hydroxyphenylethylene glycol sulfate (MOPEG-SO4) and 5-hydroxyindoleacetic acid (5-HIAA) in the brain. The level of brain homovanillic acid was only slightly affected. No appreciable change occurred, however, in the levels of brain norepinephrine, serotonin and dopamine. The increased level of brain MOPEG-SO4 or 5-HIAA after theophylline does not appear to result from its interference with the transport system for the acids in the brain since the rate of decline of the acid levels following pargyline was not affected. Under the conditions of brain monoamine oxidase inhibition, theophylline enhanced the increase in brain normetanephrine level without causing any change in 3-methoxytyramine level. The enhancement of brain normetanephrine level by theophylline became more pronounced when rats were pretreated with imipramine in addition to pargyline. These results suggest that, in the brain, theophylline may cause a release of serotonin leading to its increased turnover. The results also confirm the previous conclusion that the methylxanthine causes a release of norepinephrine and a concomitant increase in its turnover in the brain.


Octopamine, dopamine and noradrenaline content of the brain of the locust, Schistocerca gregaria. The octopamine, dopamine and noradrenaline content of the brain of the locust, Schistocerca gregaria has been determined using sensitive radiochemical-enzymatic assays. Octopamine and dopamine are present in high concentration but the noradrenaline content is only 1/25 that is octopamine. Both reserpine and fusaric acid (a dopamine-beta-hydroxylase inhibitor) produced a significant depletion of the octopamine stores.


The effects of tryptophol on immune responses and it implications toward trypanosome-induced immunosuppression. Daily administration of tryptophol to laboratory rodents resulted in significantly depressed antibody production of heterologous red blood cell challenge, did not alter cellular-mediated responses to oxazalone. These results suggest that trypanosome-produced tryptophol may account for the immunodepression observed during trypanosomiasis.


[Experimental study of 2d order tuberculostatic preparations in Mycobacterium bovis resistance to isoniazid or streptomycin (based on the data from a morphological study)]. A comparative study on the therapeutic value of the II line drugs (ethoxide, prothionamide, cycloserin) and bacteriostatics (ethambutol anf rifampicin), with their action upon experimental tuberculosis produced by streptomycin- or isoniazid-resistant mycobacteria of the bovine type, was carried out. This showed rifampicin and ethambutol to be mose effective. Ethoxide and prothionamide proved more efficient when acting upon isoniazid-resistant strains. Cycloserin turned out to be virtually inactive with regard to both streptomycin- and isoniazid-resistant mycobacteria.


[Production of phenobarbital antibodies and a study of their immunological specificity]. A conjugated phenobarbital-protein antigen has been synthetized. Immunization of rabbits with this antigen led to the appearance of antibodies specifically reacting with phenobarbital and some other barbiturates in the serum of their blood. Introduction of free phenobarbital to such animals does not produce any somniferous effect.


[Effect of thiamine bromide on cerebral circulation and arterial pressure (an experimental and clinical study)]. The effect of thiamine-bromide (5-7 mg/kg, intravenously) on the cerebral blood flow circulation rate; total arterial pressure, oxygen tension in the brain tissues (measured polarographically) and pressure in the venous vessels of the brain was investigated on anesthetized cats with controlled respiration. In patients with hypertensive disease (essential hypertension) the action of the agent (1 ml of a 6% solution, intramuscularly) on the rheoencephalographic readings and overall arterial pressure was studied. Thiamine-bromide is shown to lower the tonicity of intra- and extracranial vessels and to increase pulsed variations in the volume of blood filling of the cranial vessels. The hypotensive effect of the drug is pronounced but mildly. The oxygen tension in cerebral vessels depended mainly on changes in the cerebral circulation rate.


Nanosecond pulse fluorometry in polarized light of dansyl-L-cysteine linked to a unique SH group of F-actin; the influence of regulatory proteins and myosin moiety. The order of magnitude of the correlation time, which characterizes the dansyl cysteine residue linked to F-actin is ten times greater than the correlation time of the G-actin monomer [1]. Still it is much smaller than the correlation times of the F-actin polymer as a whole. The dansyl chromophore reveals that the C terminal end of the actin peptide chain, is mobile. As Ebashi and his co-workers have shown (13), Ca2+ triggers muscular contraction by acting on F-actin through the mediation of the regulatory proteins troponin and tropomyosin. By using spin label technique, Tonomura et al. [14] found that Ca2+ induces a conformational change on the troponin, tropomyosin actin complex. The quasi elastic scattering of laser light measurement of Fujime and Ishiwata [15] showed that troponin-tropomyosin F-actin has a rotational correlation time in the millisecond range which characterizes the flexibility of this complex; Ca2+ induces an increase of this flexibility. The present pulse fluorometry study shows an increase of mobility of the fluorescent probe induced by Ca2+. It seems difficult to correlate the results of the two kinds of measurements as long as we do not know the exact nature of the fluorescent kinetics unit.


The control of breathing in birds with particular reference to the initiation and maintenance of diving apnea. Avian respiratory pacemaker activity is dependent on some form of peripheral input but it may be inhibited by both central and peripheral stimulation. At present the results of central nervous stimulation are difficult to interpret but, aside from cortical influences, diving apnea appears to be maintained, in the face of increasing chemoreceptor input, by noxious stimulation of the upper respiratory tract and depression of the output of medullary respiratory neurons by advancing hypercapnia. If this is so, an obvious problem for future research is what initiates the prompt onset of hyperpnea when the animal surfaces and breathes. It is known that post-dive hyperpnea is little affected by either carotid body or pulmonary denervation, so peripheral chemoreceptors are unlikely to play a major role in this response.


Neural control and proprioceptive load matching in reflex respiratory movements of fishes. A brief anatomical description of topographical arrangements of respiratory neurons in the fish respiratory medulla introduces a discussion centered on neural controls of reflex breathing movements during rhythmic and ram gill ventilation, especially the role of proprioception in "load matching." Paralysis experiments show that proprioceptive signals are processed in medullary respiratory areas of the brain. Some neurons depend completely on proprioceptive input for their activity, while others "mix" proprioceptive and rhythmic information. The existence of direct proprioceptive control loops is revealed by fast compensating changes in electrical activity of respiratory muscles following mechanical interference with respiratory movements. Responses of respiratory neurons to short artificial twitches of respiratory muscles show that some neurons process proprioceptive information at higher levels of integration, often with a long latency and after effect. Other neurons react immediately to the stimulus and often show properties of elements in proprioceptive control systems in which length receptors facilitate and tension receptors inhibit motor neuron activity. Stimulus-response averaging applied to neuron activity and respiratory electromyograms, and to electrical stimuli and neurograms, leads to the conclusion that motor neurons and neurons mediating sensory signals from the respiratory muscles are situated close together in the medullary respiratory areas. Neuroanatomical studies are discussed which indicate that the sensory projection fields of some trigeminal fibers terminate adjacent to the cranial motor nuclei. These presumably project onto motor neurons forming fast disynaptic reflex arcs relaying sensory trigeminal information.


Respiration and the generation of rhythmic outputs in insects. In insects gas exchange may be: 1) entirely passive, when metabolic rate is low; 2) enhanced automatically by muscle contractions that produce movements, e.g., wing movements in flight; or 3) produced by ventilatory movements, particularly of the abdomen. In terrestrial insects such as locusts and cockroaches ventilatory movements are governed by a dominant oscillator in the metathoracic or anterior abdominal ganglion. The dominant oscillator overrides local oscillators in the abdominal ganglia and thus sets the rhythm for the entire abdomen, and it also controls spiracle opening and closing in several thoracic and abdominal segments. This ventilatory control mechanism appears to be different from that generating metachronal rhythms such as occur in the ventilatory and locomotory movements of aquatic arthropods. There are now several examples of rhythms, both ventilatory and locomotory, that can be generated by the central nervous system in the absence of phasic sensory feedback, but the mechanism of rhythm production is not known. Studies of ganglionic output suggest that neuronal oscillators can produce a range of frequencies and that some oscillators may be employed in more than one function or behavior. The mechanisms by which central oscillators are coupled to the output motorneurons are also not known; large phase changes suggest that in some cases different coupling interneurons are active. Intracellular recordings from identified neurons have begun to clarify the important roles of interneurons in the production of motor patterns.


Neurophysiology of the motor output pattern generator for breathing. In cats, there is a central pattern generator (CPG) capable of producing the motor output to drive breathing without any phasic sensory input. The phrenic activity driven by the CPG is, on each cycle, a long inspiratory discharge with continuously rising intensity, an abrupt turn-off, and a pause. The CPG is normally overridden by a reflex termination of inspiration. A sufficient lung inflation will terminate inspiration early, but has no other effect on the motor output. This is the Hering-Breuer inspiration inhibiting reflex. The lung volume necessary to terminate inspiration decreases during the course of an inspiration. The slowly adapting bronchial stretch receptors running in the vagus are the responsible afferents. These vagal afferents drive cells in the ventrolateral nucleus of the solitary tract. These cells also receive a rising excitation from the CPG. They sum these two inputs and thus show a falling threshold to vagal input. They probably generate the inspiration terminating trigger for the Hering-Breuer reflex. In deeply anesthetized breathing, the only active expiratory cells are in the nucleus retroambigualis. These do not make synapses with inspiratory cells. Thus breathing models based on reciprocal inhibition between inspiratory and expiratory cells cannot be correct. Inspiratory cells must themselves be capable of generating the rhythm. Invertebrates present many examples of repetitive burst generation by a single synergistic population. This usually involves cells that, in the absence of synaptic input, either fire continuous trains or rhythmical bursts of spikes. The cardiac ganglion of crustaceans may be a useful model for burst generation similar to breathing. In deeply anesthetized breathing there are two populations of medullary respiratory cells, those in the nucleus of the solitary tract (NST) and those in the nucleus retroambigualis (NRA). The NST has axons ramifying in the NRA and thus could drive it, but the reverse connections do not exist. Thus the current "best guess" is that the cells of the NST are the central pattern generator for breathing.


Plasmid DNA replication.

Plasmid DNA replication. Recent studies have provided some insight to the overall characteristics of plasmid replication in bacteria. The ColE 1 and R6K plasmids replicate via a covalently-closed circular intermediate. Replication is initiated at a fixed origin and is unidirectional in the case of ColE 1 and bidirectional for R6K. In the case of the plasmid R6K. the bidirectional replication is asymmetric and sequential, proceeding from a fixed origin to a fixed terminus located approximately 20% of the R6K genome from the origin. RNA serves as a primer for plasmid DNA replication. The base composition and sequence of the 5'-terminus of the RNA segments in ColE 1 DNA have been determined. Finally, the properties of plasmid relaxation complexes and the possible role of these complexes in plasmid DNA replication are discussed.


Structural evolution of bacterial plasmids: role of translocating genetic elements and DNA sequence insertions. Recent evidence suggests that plasmids have evolved by site-specific recombinational events involving translocation and insertion of discretely defined DNA segments. The role of translocating genetic elements and repeated DNA sequences in the formation and structural evolution of bacterial plasmids, and in the control of plasmid gene expression, is the subject of this brief review. Insertion sequence (IS) regions are discrete segments of DNA that are known to cause strongly polar mutations in the genes of Escherichia coli and several bacteriophages as a consequence of their insertion into bacterial or phage genomes. Recent investigations have identified three separate kinds of IS segments on plasmids, and have indicated that such regions may have a role in 1) site-specific reversible dissociation of antibiotic resistance plasmids into their component segments, 2) recombination of certain plasmids with the bacterial chromosome, and 3) translocation of segments of plasmid DNA onto other replicons, or onto different sites of the same replicon. In addition, such DNA sequences, which may be repeated on plasmid genomes in either direct or reverse orientation, are involved in the control of plasmid gene expression. Inverted repeats other than the genetically characterized IS segments also appear to be involved in recA-independent, recombination and translocation of plasmid DNA segments. These inverted repeats contain palindromic nucleotide sequences on each strand of DNA and are detectable as hairpin-loop structures by electron microscope heteroduplex analysis. Such palindromes resemble the recognition sites for restriction endonucleases, some of which are encoded by plasmids, suggesting that similar endonucleolytic enzymes may be involved in the translocation of plasmid DNA segments.


A restriction endonuclease analysis of the bacterial plasmid controlling the ecoRI restriction and modification of DNA. Genetic analyses of DNA restriction and modification mechanisms have been encumbered by the inability to rigorously select for mutant phenotypes associated with these systems. The application of restriction endonucleases has now proved to be a successful approach to the genetic analyses of small genomes that are recalcitrant to the more standard genetic techniques. Restriction endonucleases EcoRI and HindIII were used to analyze the structure of the plasmid genome responsible for the EcoRI restriction endonuclease and modification methylase. This plasmid in the original clinical isolate of Escherichia coli appears to be identical to the ColE 1 plasmid except for a 1.95 kilobase pair segment which contains these genes. A preliminary restriction map of this plasmid is presented.


The role of histocompatibility gene products in lymphocyte triggering and differentiation. Studies performed both in vivo and in vitro have demonstrated that genetic restrictions exist in the development of optimal cell-cell interactions in the immune system. Thus, in mice, the presence or absence of gene identities in the I region of the H-2 complex determines the capacity of lymphoid cells to interact. These cell interaction or CI genes appear to code for cell surface molecules integrally involved in regulatory cell interactions. Analysis of biologically active thymus-derived (T) cell factors capable of regulating differentiation events in other lymphoid cells indicates that such factors are around 40,000-50,000 daltons in mass, bear determinants coded for by I region genes, and consist of two covalently or noncovalently linked components--a heavy glycoprotein of around 40,000 daltons and a lighter protein of around 12,000 daltons. Preliminary evidence suggests that the latter component may be beta2-microglobulin.


Response of luteinizing hormone and follicle-stimulating hormone to different doses of D-leucine-6-LH-RH ethylamide in oligospermic patients. The rise in luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels after intramuscular administration of D-leucine-6-LH-RH ethylamide was studied in seven patients with idiopathic normogonadotropic oligospermia. Four tests with 2.5, 5.0, 10.0, and 20.0 mug of the analog, respectively, were carried out at 8 A.M. on each subject. The interval between tests was 1 week. Serum levels of LH and FSH were determined by radioimmunoassay before (-15 And 0 minutes) and 2, 4, 6, 12, and 14 hours after each injection. No differences in the basal values for either hormone were observed. D-Leucine-6-LH-RH ethylamide produced significant increases in LH and FSH levels for 6 to 12 hours after the injection. Peak values were obtained between 4 and 6 hours. A dose-response relationship for both gonadotropins was observed. The highest average levels of LH and FSH were obtained after injection of 20.0 mug of the drug. A great individual variation in magnitude of response was observed which seemed to be related to the functional reserve of the pituitary. It is suggested that doses of 10.0 to 20.0 mug of D-leucine-6-LH-RH ethlamide/day could be used for chronic treatment in male infertility.


Treatment of idiopathic normogonadotropic oligoasthenospermia with synthetic luteinizing hormone-releasing hormone. An assessment of the effectiveness of synthetic luteinizing hormone-releasing hormone (LH-RH) in the treatment of idiopathic normogonadotropic oligoasthenospermia was made in 21 subjects selected on the basis of at least three previous sperm counts; a history of long-standing infertility; normal urinary gonadotropin excretion; normal serum levels of luteinizing hormone, follicle-stimulating hormone, and testosterone; and lack of any evidence of other clinical, endocrine, vascular, infectious, and/or urologic involvement. On testicular biopsy, 14 patients showed hypospermatogenesis or arrest up to the spermatid stage (group 1), while the remaining 7 had more severe spermatogenetic disorders (group 2). Libido and/or potency were previously unaltered in all subjects. Five patients in group 1 and five patients in group 2 received intramuscular injection of 100 to 500 mug of LH-RH daily for no less than 60 days (long-term treatment); the remainder received the drug for 30-day periods, with 30-day intervals (short-term treatment). Qualitative and quantitative assessment of the results during and after treatment showed frank improvement in spermatozoal concentration, vitality, and motility in group 1 patients, particularly in those undergoing long-term treatment (three of five patients under these conditions were able to impregnate their wives). The long-term regimen also resulted in predomantly qualitative improvements in group 2 (one of five patients impregnated his wife). The short-term regimen was less effective. Improvements under either regimen usually occurred at the end of, or after, treatment. Six of seven patients in group 1 who were followed for a long period retained values above preliminary values for 90 to 150 days after the drug was discontinued. Increased libido and potency were spontaneously reported by 15 patients, 20 to 30 days after the start of administration of synthetic LH-RH.


Luteinizing hormone-releasing hormone for induction of follicular maturation and ovulation in women with infertility and amenorrhea. Five patients with primary infertility and secondary amenorrhea who did not respond to clomiphene with a gonadotropin or estrogen surge were treated with 500 mug of luteinizing hormone, follicle-stimulating hormone-releasing hormone (LH-RH), self-administered subcutaneously every 8 hours for 14 days. Of four patients who responded to this treatment, three showed follicular maturation, ovulation, and menses, although the luteal phase was deficient; in the fourth patient, follicular maturation and menses occurred without evidence of ovulation. For their second course of treatment these four patients were given LH-RH with the addition of human chorionic gonadotropin when the urinary estrogen levels indicated follicular maturation. All four patients responded with ovulation, an adequate luteal phase, and menses, without clinical indication of ovarian hyperstimulation. These results suggest that LH-RH may be a better alternative to human menopausal gonadotropin in the treatment of anovulatory infertility.


Prolonged anti-luteinizing hormone/follicle-stimulating hormone-releasing activities of some synthetic antagonists of luteinizing hormone-releasing hormone. A number of synthetic analogs of luteinizing hormone-releasing hormone (LH-RH) were screened for their in vivo antigonadotropin-releasing activities using a four-point bioassay test in immature male rats. Of the peptides tested, the most effective were those containing D-phenylalanine in position 2 of the decapeptide chain in conjunction with D-leucine or, preferably, D-phenylalanine in position 6. Several inhibitory peptides that were found to be very potent were reassayed and compared for duration of inhibition in immature male rats, whereupon the D-Phe2 analogs were found to be particularly long-acting. The most active and persistent peptide of the series, [D-Phe2, D-Phe6]-LH-RH, was able to inhibit the response to exogenous LH-RH for up to 6 hours after its injection.


A comparison of methods of screening for sperm antibodies in the serum of women with otherwise unexplained infertility. Sera from 102 women with infertility due to a variety of causes and from 40 pregnant women were studied for the presence of antisperm factors. Three techniques were used: sperm microagglutination, sperm immobilization, and an indirect immunofluorescent technique for detection of sperm-bound immuno-globulins. There was no correlation between the results obtained using these three different techniques. Of the three, only the results of sperm immobilization tests correlated with primary unexplained infertility. The sperm microagglutination test appeared to measure nonspecific factors. Methanol fixation of spermatozoa used in the indirect immunofluorescent technique apparently resulted in nonspecific binding of immunoglobulins. When fresh spermatozoa were used no binding of immunoglobulin to spermatozoa could be demonstrated. The nature and location of the antigen(s) involved remain to be determined.


[The role of the reticular formation at the hypothalamic level in regulating insulin secretion]. In cats, electric stimulation of direct microapplication of insulin to posterior hypothalamic nucleus increases the insulin content in the pancreatic venous blood independently of the concomitant changes of the blood sugar level. The insulin had no such effect in bilateral lesion of the lateral hypothalamus. Blockade of M-cholinergic system did not influence the changes of blood sugar level in these conditions. Electric stimulation of the anterior hypothalamic nucleus entailed no obvious changes of insulin and sugar level in the blood.


[The effect of single and long-term thyrocalcitonin administration on glucose tolerance and insulin secretion]. In male rats, i. m. administration of thyrocalcitonin (TCT)--2 and 10 units/kg for 1, 5, 10 and 15 days, reduced calcium level in the blood serum. In the TCT-treated rats the glucose-tolerance curves after i. p. injection of glucose became diabetoid-like. TCT inhibited the increase of insulin-like activity of the serum after glucose injection. The data suggest that TCT, lowering the calcium content of blood serum, inhibited the glucose-induced secretion of insulin.


Sensitivity of tumour and normal cells to the effect of immune lymphocytes in vitro and some properties of target cells determing this sensitivity. Cells from various spontaneous mammary tumours of C3H/He mice differ in their sensitivity to immune allogeneic lymphocytes in vitro. Two cell lines, differing in their sensitivity to allogeneic immune lymphocytes in vitro, were obtained from spontaneous mammary tumours which developed in C3H/He mice. Of these two lines, the cell line MT3 was considerably more immunoresistant than line MT5, as demonstrated by microcytotoxic tests in vitro. Furthermore, only the immunoresistant MT3 cell line was capable of growing in syngeneic hosts. During passages in vitro, cells of the line MT5 became as immuno-resistant as cells of the line MT3; concomitantly, cells of the line MT5 started to take in syngeneic hosts. Cell immunosensitivity to immune allogeneic lymphocytes in vitro correlates with that cell property which determines the penetrance rate of intact allogeneic and syngeneic lymphocytes into these cells (emperipolesis). Non-tumourous cells from different C3H/He mouse tissues were more immunoresistant than tumour cells from the two cell lines cultured.


Preservation of plaque-forming cells by means of deep freezing. Mouse plaque-forming cells were kept at--196 degrees C for different periods of time up to 37 weeks. The decrease in cell number occurring during the storage was found to be in no correlation with the length of the storage time. The cryoprotective effectiveness of dimethyl sulphoxide upon survival of cells was equal for concentrations ranging from 5-15%.


Interactions of nutrition and infection. Interactions between malnutrition and infection contribute directly to the health of individuals and communities. The relevance of this concept to the practice of clinical medicine, dentistry, and public health is supported by an imposing collection of evidence from the clinic, laboratory, and field. Malnutrition can interfere with any body mechanism that acts as a barrier to the multiplication or progress of infectious agents. This includes formation of specific antibodies, number and activity of phagocytes, integrity of skin, mucous membranes, and other tissues. Some of the less definite, nonspecific protective substances in body fluids are also affected by malnutrition. Infectious disease adversely influences the nutritional state in several indirect ways. Loss of appetite and intolerance for food result in metabolic changes. Cultural factors lead to substitution of less nutritious diets as a presumed therapeutic measure and to administration of purgatives, antibiotics, and other medicines that reduce digestion or absorption of specific nutrients. All of these may help to precipitate kwashiorkor in children subsisting on protein-deficient diets. Classical nutritional deficiencies precipitated by infection in individuals with borderline nutrient depletion include: keratomalacia due t avitaminosis-A; scurvey from lack of ascorbic acid; beriberi as a consequence of inadequate thiamin; pellagra resulting from insufficient niacin; macrocytic anemia due to folic acid or vitamin B12 deficiency; and microcytic anemia resulting from a shortage of iron. In well nourished individuals, body reserves and normal dietary intake assure that malnutrition will not result unless infection is prolonged.


Controversies, perspectives, and clinical implications of nutrition in periodontal disease. Many controversies concerning the role of nutrition in periodontal disease and problems in the design and analysis of nutrition-periodontal disease studies have been reviewed. In addition, recent advances in the interactions of nutrition, host defense, and infection have been conceptually related to the pathogenesis of periodontal disease. Finally, nutritional factors relevant to the diagnosis and clinical management of patients with periodontal disease have been discussed. The concepts developed with respect to experimental design and the host defenses approach to periodontal disease, will hopefully serve to encourage definitive studies which may delineate a more precise role for nutrition in periodontal disease.


Long-term perfusion of isolated rats islets in vitro. A perfusion system is described for long-term maintenance of isolated rat islets in vitro. This system permits the monitoring of the pattern, rate, and amount of insulin secretion following repeated, acute stimulations with glucose during the period of culture. Fibroblastic proliferation did not occur, thus making is possible to reclaim the islets for biochemical and morphologic studies at the conclusion of the experiments. Maintenance of the islets with a low concentration of glucose (1.0 mg./ml.) resulted in a marked decline in insulin secretion following acute stimulations with glucose (5.0 mg./ml.) during an eight-day interval. Stimulation with 10 mM theophylline and 5.0 mg./ml. glucose on day 9 resulted in enhanced insulin release. The decline in glucose sensitivity occurred even more rapidly when the islets were maintained in the presence of a lower concentration of glucose (0.5 mg./ml.). The pattern of insulin release was altered with an absence of a first phase of secretion. Adenylate cyclase activity of islets maintained with 0.5 mg./ml. glucose for four days was significantly decreased in comparison with islets from fed rats and islets maintained with 2.5 mg./ml. glucose. A means of maintaining the same biphasic pattern and amount of glucose-induced insulin release was achieved by using alternating levels of glucose (1.5 and 2.5 mg./ml) for maintenance of the islets during a 36-day interval.


Interaction of alloxan and anomers of D-glucose on glucose-induced insulin secretion and biosynthesis in vitro. The direct effects of alloxan on glucose-induced insulin secretion and biosynthesis and the interaction of alloxan and D-glucose anomers were studied in vitro by use of isolated islets from rat pancreas. Islets were pretreated by incubation for five minutes in media containing alloxan (0.2 mg./ml.) alone or alloxan with either the alpha or beta anomer of D-glucose (3 mg./ml.). After washing, batches of five islets were incubated in the medium supplemented with glucose (1.8 mg./ml.) for 60 minutes to observe insulin secretion and for 90 minutes to observe insulin biosynthesis. Prior exposure to alloxan alone produced marked inhibition of subsequent glucose-induced insulin secretion and biosynthesis. A significantly greater protection against these inhibitory effects of alloxan was observed by using the alpha anomer of D-glucose than the beta anomer. The anomeric preference of D-glucose for protecting islet cells from the inhibitory effect of alloxan on glucose-induced insulin secretion and biosynthesis was similar to that for triggering insulin secretion. Possible mechanisms of the inhibitory effect of alloxan and the protective effect of D-glucose anomers in connection with those of other sugars are discussed. It is suggested that a glucoreceptor, stereospecific to the alpha anomer of D-glucose, may exist for both insulin secretion and biosynthesis.


Virus-induced pancreatic disease by Venezuelan encephalitis virus. Alterations in glucose tolerance and insulin release. Viral infections have been implicated in the induction of diabetes mellitus in man and laboratory animals. Since virus-specific immunofluorescence (FA) is detectable in hamster pancreas during the acute phase of Venezuelan encephalitis (VE), experiments were designed to correlate pathologic and virologic events with metabolic studies in VE-infected hamsters. Golden Syrian hamsters were inoculated s.c. in groups of four to 12 with 100,000 plaque-forming units (PFU) of the vaccine strain (TC-83) of VE or 1,000 PFU of the virulent Trinidad strain of VE. Ultrastructurally, during Trinidad infection, mature virions were associated with the cell surfaces and within pancreatic beta cells in contrast to absence of virus-related changes in TC-83-infected hamsters. Virus-specific-FA was noted in islet cells and acinar cells of Trinidad-infected hamsters. VE growth curves demonstrated viral replication in pancreas with both strains. Although ultrastructural and FA changes were much more prominent in Trinidad-infected hamsters in contrast to TC-83-infected hamsters during the first few days of illness, the rapid lethality of the Trinidad-infected group necessitated performing all metabolic studies in TC-83-strain-infected hamsters. Accordingly, for the metabolic studies, glucose tolerance tests (GTT) using 2 mg. or 5 gm./kg. glucose i.p. were performed in groups of hamsters acutely infected two days earlier with the TC-83 vaccine strain and in 24-day and 90-day convalescent hamsters after TC-83 vaccine strain. Samples were obtained for glucose and immunoreactive insulin (IRI) determinations. Glucose intolerance occurred in hamsters in each of the infected groups given 5 gm./kg. glucose except for the 90-day convalescent TC-83 group. Severely decreased IRI responses occurred in the 24-day and 90-day convalescent TC-83 hamsters following both 2- and 5-gm./kg. glucose. Pancreatic IRI content in 24-day convalescent TC-83 hamsters was within normal limits, suggesting a defect in IRI release from the beta cells at this stage of convalescence.


The Chiari malformations of the posterior fossa and their relation to hydrocephalus. The classification of the developmental malformations of the posterior fossa associated with hydrocephalus proposed by Chiari is systematic and comprehensive. It is based upon the relative position of cerebellum and brainstem in relation to the foramen magnum and the upper cervical canal. Types I to II are degrees of a similar abnormality in which a conical deformity of the posterior midline cerebellum and the elongated brainstem lie at or below the foramen magnum. In the Type IV deformity, in contrast, the cerebellum and brainstem lie fully within the posterior fossa and their position in relation to the foramen magnum is normal. The most common example of this type is probably the Dandy-Walker malformation, in which a defect in the inferior vermis is congruent with a ventriculocele of the enlarged fourth ventricle. The Chiari malformations are generally associated with hydrocephalus. The most comprehensive and coherent theory of the origin of these malformations is that abnormal hydrostatic forces, acting principally during the second and early third trimesters of gestation, provide the mechanism of posterior fossa deformity. Morphological evidence indicates that posterior fossa structures in Types I to III are "pushed down" from above. Radio-isotope dilution studies suggest that the mechanism of hydrocephalus in Types I to III is overproduction of cerebrospinal fluid (CSF); initially there may be no obstructive component. In those cases without spina bifida, ventricular enlargement should occur early and be greatest in the forebrain, driving the tentorium and posterior fossa structures downward. Where there is a fistulous communication between the ventricular system and the amniotic cavity via a spina-bifida defect, a pressure gradient between intracranial and spinal compartments may be sufficient to account for downward displacement of posterior fossa structures. In this instance ventricular enlargement may occur only after cerebellar impaction causes frank obstruction to the flow of cerebrospinal fluid. In the Dandy-Walker malformation, the mechanism of hydrocephalus is probably obstruction to flow of cerebrospinal fluid through the roof of the fourth ventricle. Enlargement of the fourth ventricle is sufficient not only to compress the cerebellum and give rise to the inferior vermal defect, but also to offset forces above the tentorium and prevent its caudal displacement from the parietal region.


Somatosensory evoked cortical responses in children with spina bifida. The sensory pathways of 18 infants and children with myelomeningocele were studied by recording somatosensory evoked cortical responses to standardised electrical stimuli over major nerve trunks in the arms and legs. The 12 infants were tested between the ages of three days and eight months and the six children were tested between two and three years of age. In the majority of patients the results obtained by this technique correlated well with the clinical testing of sensation.


The use of sodium valproate in a case of status epilepticus. A child in status epilepticus, who did not respond to intravenous diazepam, was treated with sodium valproate by naso-gastric tube. Subsequent clinical and encephalographic improvement appeared to be related to the sodium valproate, and its value in cases of status epilepticus is discussed.


[Preparation of functioning recombinant (hybrid) DNA molecules in vitro (genetic engineering experiments)]. In this paper we describe the preparation of hybrid plasmid consisting of ColE1 DNA and DNA of R6K plasmid. ColE1 plasmid represents a circular DNA with the molecular weight of 4,2-10(6) daltons. It determines colicine synthesis E1, has relaxed control of replication and is present in the cell in several dozens copies. Transmissive plasmid B6K represents a circular DNA molecule with the molecular weight of 28-10(6) daltons. It confers the resistance to amplicillin and streptomycin and belongs to the compatibility group X. This plasmid also has relaxed control of replication (up to 30 copies per cell). Circular superhelical DNA of ColE1 was prepared after it is amplification with cloramphenicol according to Clewell by ultracentrifugation in CsCl-EtBr density gradient. The yield of superhelical ColE1 DNA in our experiments was up to 300 mg/gram cells. Circular superhelical R6K DNA was isolated from E. coli strain J53 R6K grown to stationary phase in the presence of 15 mg/ml of streptomycin. The yield of superhelical DNA was equal to about 45 mg per 1 gram cells. EcoR1 restrictase was prepared from E. coli KM182 as described by Thomas et al. From 37 grams of cells 10 ml of enzyme solution was prepared. 1 ml of this solution was able to restrict completely 2 mg of ColE1 DNA during Hu incubation for 30 degrees min at 37 degrees C. Restrictase activity was analyzed by electrophoresis of ColE1 DNA restricts in 0,7% agarose gel as described by Tanaka. DNA ligase was prepared from E. coli B. cells infected by T4 phage am81 mutant as described by Weiss. The activity of enzyme solution was equal to 90 units/ml (according to Richardson). In standard "hybridization" experiment 4 mug of ColE1 DNA and 4 mug of R6K DNA were incubated for 1 hour at 37 degrees in 0,2 ml of the solution containing 40 mM tris-HCl, pH 7,4; 10 mM MgCl2 50 mM NaCl, 10 mM mercaptoethanol and 5 mul of EcoR1 restrictase. The reaction was terminated by heating the reaction mixture to 65 degrees C for 5 min. In accordance with the results of others CoLE1 DNA gave one restrict and R6K DNA-two restricted fragments. 100 mul of the restricted mixture was chilled to 0 degrees and the following ingredients were added 10 mul of 50 mM MgCl2; 10 mul of 100 mM dithiotreitol; 10 mul of 0,5 mM ATP, 20 mul of water and 8 mul of ligase solution (90 units/mul). The mixture was incubated at 0-0,5 degrees for 53 hours, thereafter it was used for the transformation of E. coli C600 according to Tanaka et al. Several classes of hybrid molecules due to different combinations of restricted fragments of parental species were expected. These molecules had to confer the immunity to colicine E1 and to carry the defect in the gene controlling colicine synthesis since EcoR1 restrictase splits ColE1 DNA in the region of corresponding gene. In addition, certain classes of transformants with hybrid DNA molecules had to be resistant to one or both antibiotics as determined by the R6K plasmid portion...


[Effect of uvs1, uvs2 and xrs mutations on the radiosensitivity of and frequency of induced mitotic recombination in diploid yeast cells]. Effect of radiosensitivity mutations uvs1, uvs2 and xrs in homozygous state in yeast Saccharomyces cerevisiae on UV and X-ray sensitivity and on the induced mitotic recombination is studied. Diploids uvs2 uvs2 xrs xrs are much more UV-sensible than diploids UVS2 UVS2 xrs xrs or UVS2 uvs2 xrs xrs. Diploids uvs1 uvs1 uvs2 uvs2 and uvs1 uvs1 UVS2 UVS2 have the same radiosensitivity. These data show that mutations uvs1, uvs2 and xrs block different ways of the elimination of UV-induced damages. The diploid uvs2 uvs2 xrs3 xrs3 is much more X-ray sensible, than the diploid UVS2 UVS2 xrs3 xrs3. It shows that mutations uvs2 and xrs3 block different ways of the elimination of X-ray induced damages. All double mutant diploids are characterized by a higher frequency of UV-induced mitotic recombination, than the diploid UVS UVS XRS XRS and the UV-sensitive diploid uvs2 uvs2 XRS XRS. The diploid uvs2 uvs2 xrs5 xrs5 is characterized by an extraordinary high frequency of X-RAY INDUCED MITOTIC RECOMBINATION.


[Genetic study of Escherichia coli K-12 mutants resistant to 2,6-diaminopurine]. Mutants, resistant to the inhibitory effect of 2,6-diaminopurine and incapable of utilizing adenine as a purine source, are obtained from purinenucleoside phosphorylase-defective purine-dependent Escherichia coli K-12 strains. The mutations obtained (apt) disturb the uptake of adenosine and inosine only in the presence of a mutation for purinenucleoside phosphorylase (pup gene) in the genome of purine-dependent bacteria. The introduction of pup+ allele into the genome of mutants obtained (genotype purDpup apt) results in the restoration of the ability to uptake adenine and purine ribosides. Strains of purDpup+apt genotype are characterized by more short generation time under the growth in the presence of adenine as compared with purDpup apt+ strains which indicates the existance of an efficient pathway of adenine utilization in E. coli with the cooperation of purinenucleoside phosphorylase. Mutations apt have revealed a combined transfer with purE marker under the transduction frequency of approximately 5%. The gene order on E. coli K-12 chromosome is apt-purE-gal, as estimated from the data on conjugation crosses.


[A further study of the nature of phenotypical reversions in thymidine phosphorylase deletion mutants of Escherichia coli K-12]. Thymidine dependent (thy) strains of Escherichia coli carrying deletions for thymidine phosphorylase (tpp gene) formed phenotypical reversions for the ability of growth at the medium containing thymine as a source of thymidilate. The ability of the thy tpp strains to utilyze thymine for growth is due to mutations of the regulatory genes cytR and udpR, which control the uridine phosphorylase activity. This enzyme in constitutive amounts catalyzes conversion of thymine to thymidine in bacterial cells with a block of deoxyribose catabolism (deoxyribomutase-negative strains). The udpR mutant by contrast to cytR mutants reported by Munch-Petersen et al. (1972) are shown to contain low, inducible levels of cytidine deaminase and deo-enzymes. The udpR mutation is recessive with respect to wild type allele. It is supposed that the product of udpR gene is a specific repressor of uridine phosphorylase (udp) gene. The udpR mutation is closely linked (90% contransduction) to uridine phosphorylase (udp) gene, and is located approximately at 75 min, on the E. coli chromosome.


Hypothalamic-pituitary-gonadal function in men with cirrhosis of the liver. Hypothalamic-pituitary-gonadal function was studied in 37 cirrhotic males, 25 of whom were alcoholic. Irrespective of aetiology, cirrhotic patients had significantly reduced free testosterone concentrations. Despite low free testosterone concentrations and reduced or absent spermatogenesis, basal levels of luteinizing hormone (LH) and follicle stimulating hormone (FSH) were normal in nearly all patients, suggesting impaired function of the hypothalamic-pituitary-gonadal axis. In 14 cirrhotic men, seven of whom had gynaecomastia, the ability of the pituitary to secrete LH and FSH in response to exogenous gonadotrophin releasing-hormone (LH/FSH-RH) was asssessed. A normal LH response to LH/FSH-RH was obtained in patients without gynaecomastia. An exaggerated LH response was found in four of seven with gynaecomastia, suggesting Leydig cell failure. The Leydig cell response to exogenous gonadotrophin in eight consecutive cirrhotic patients was probably abnormal but difficult to interpret as all but one were within conventionally accepted limits of normality. The patients without gynaecomastia gave a normal or minimally exaggerated FSH response to LH/FSH-RH. Six of seven with gynaecomastia gave a markedly exaggerated response suggesting failure of spermatogenesis, and all tested were either azoospermic of oligospermic. The single patient with a normal FSH response had a normal sperm count. The pituitary cells can therefore respond to LH/RSH-RH and the Leydig cells of the testes show some response to exogenous gonadotrophin. Similar abnormalities in hypothalamic-pituitary-gonadal function have recently been described in patients with normal liver function on chronic oestrogen therapy.


Siphonaptera collected by Dr. M. Daniel in Nepal. During the Third Czechoslovak Expedition to the Himalayas, in the spring of 1973, Dr. M. Daniel collected 244 specimens of fleas belonging to 19 different taxa, including four hitherto undescribed ones, from mammals and birds in the Barun Khola valley at the foot of the Makalu mountain just south-east of Mt. Everest. Data and comments are given in this paper for the fleas collected and the new taxa are described.