[Presence of indoleamines in the pineal and parapineal organs of Lampetra planeri (Petromyzontidae)].

Using the Falck and Hillarp technique, the precursors of melatonin (5-HT and may be 5-HTP) have been identified in the pineal and parapineal organs of Lampetra planeri (Petromyzontidae). A particular kind of cell, the so-called "cells with residual bodies" (Collin, 1968), is responsible for this neurohormonal activity of the two organs.


[Action of Corynebacterium parvum on the activity of glycosidases and proteases of peritoneal macrophages in the mice].

Glycosidase and peptidase of non-treated Mouse peritoneal macrophages were characterized. The enzymatic activities of Corynebacterium parvum stimulated Mouse macrophages were compared to those of macrophages obtained from non-treated animals. All the enzymatic activities, but beta-C-galactosidase, were higher in stimulated Mouse macrophages.


[Mitotic activity in the rat palatal shelves during their fusion in vitro].

Rat palatal shelves explanted on gestational day 15 or 16 are cultivated with their medial-edge epithelial surface in contact. Observations of histological sections showed that, before and during the fusion of the processes, mitotic activity is always lower in the epithelium of the expected fusion zone (EFZ) than in the oral epithelium and progressively decreases during the processus of the fusion.


[Ultrastructural aspects of reconstitution of the basal membrane in associations of dental components cultured in vitro].

Separation of the enamel organ and pulp of mice tooth germs by trypsin removed the basal lamina. Within 18 hours in cultivated reassociations of enamel organ and pulp, a new lamina was deposited. When the epithelial component was cultivated alone, no basal lamina formed.


[Kinetics of decrease and apparent transformation of fluorescence spectra of benzo(a)pyrene absorbed by living cells: case of lymphocytes and macrophages].

A home made microspectrofluorimeter is used in order to follow the decrease of fluorescence intensity of Benzo(a)Pyrene after its absorption by single living cells. The kinetics look to be a first order one; fluorescent metabolite can be detected when peritoneal macrophages of Mice are used but not with human periferic lymphocytes pretreated with mitogens.


[Excretion of sialoglycopeptides in mucolipidosis I].

Mucolipidosis I involves a tremendous increase of the urinary excretion of sialoglycopeptides and sialyloligosaccharides. This enhancement is due to the excretion of O- and N-glycosidic peptides and oligosaccharides, which normal urine is devoid of as shown by the chemical composition and electrophoresis or thin-layer analysis. This finding is in agreement with the recent finding of an alpha-neuraminidase deficiency for this disease.


[Proliferation of adipose cells and their lipid accumulation in the rat at the perinatal stage; histoautoradiographic study].

After thymidine-3H injection into pregnant Rat, histoautoradiographic studies showed that adipose cell proliferation in different subcutaneous and visceral sites, was very important in Rat foetuses about 24 h before birth and that lipid accumulation in the labelled cells was very fast during perinatal 48 h.


[Effect of phthalic esters on lecithin cholesterol acyltransferase].

The in vitro effects of different esters of phthalic acid at increasing levels on the activity of LCAT have been studied using three currently known methods (Mickel and Foulds, Stokke and Norum, Alcindor). Inhibition from phthalates is high. It is proportional to the amounts used and in inverse ratio to the length of the carbon chains.


[Interference of hydrazine derivatives with the tyrosinase-tyrosine reaction].

A comparative study of the tyrosinase-tyrosine reaction, using both oxygen-consumption determination and spectrophotometric methods, shows that hydrazine derivatives inhibit melaninogenesis at different steps.


[2,4-dinitro-(pyrimidine-2-yl) thiophenol, a new drug with antimitotic properties].

A new thiopyrimidine derivative has been synthesized. It can inhibit cell multiplication in Chick embryo fibroblasts, in Mouse Ehrlich ascites tumor cells and in Rat hepatoma cells (line Rueber) cultivated in vitro.


[Suppression by ascorbic acid of neuromuscular fatigue due to sound-alcohol synergy].

The interaction between effects of ethanol ingestion and those of infrasound on neuro-muscular fatigue in the animal is abolished by ascorbic acid. The relevant mechanism is in acetaldehyde detoxification.


[Acute inhalation toxicity of ammonia in rabbits].

The aggressivity of ammonia at the pulmonary alveole level (intoxications in controlled ventilation) leads to an acute pulmonary oedema and cardio-vascular disfunctioning. Bradycardia is an early symptome (2,500 X 10(-6) but the real syndrome appears at 5,000 X 10(-6). Arterial pressure variations and blood gases modifications are significant, acidosis is systematic.


[Species specific esterase profiles in the genus Serratia].

Esterases of 62 Serratia marcescens, S. liquefaciens, S. plymuthica and S. marinorubra strains were analysed by electrophoresis in polyacrylamide-agarose gel. The comparative distribution of bands showed that the four Serratia species were characterized by distinct electrophoretic patterns of their esterases.


[Attempt at activating mouse intracisternal type A particles by iododeoxyuridine and dexamethasone].

In this study we have compared the effects of Iododeoxyridine (IUdR) and dexamethasone on different Mouse cell lines to determine any differences in their actions on intracisternal A-type particles (IAP). Among the nine cell lines studied only four responded to IUdR stimulation by a subsequent augmentation of IAP. No similar activating effect was detected with dexamethasome treatment.


[Ultrastructural localization of 3-beta-hydroxysteroid dehydrogenase activity in the glandular tissue cells of the testis of Pleurodeles waltlii Michach. (Amphibia, Urodela)].

With the potassium ferricyanide method, the localization of the sites of 3 beta-hydroxysteroid dehydrogenase activity has been investigated in the glandular tissue cells, previously fixed, of the Pleurodeles testis. An abundant copper ferrocyanide precipitate, the final reaction product, is observed in the vicinity of the external faces of the membranes of the smooth endoplasmic reticulum, or in contact with them. A very weak reaction occurs in mitochondrial cristae.


[Autoradiographic study of proliferative activity and cell migration in the adrenal cortex in fetal and neonatal rats].

On the 20th day of fetal life the cell proliferation is higher in the zona glomerulosa. The fate of marked cells in each cell compartment shows centripetal migration. Their displacement, from the 20th day of pregnancy up to five days post-partum, while at that time the adrenal growth is slowered down, suggests a real migration.


[Harmaline-induced tremor and cerebral activities: labeling with 14C-2-deoxyglucose in the rat].

The brain structures specifically involved in harmaline-induced tremor have been identified in the Rat by using the 14C-2-deoxyglucose marking Method. The results obtained in an animal treated with tremogenic doses of harmaline, but immobilized with Faxedil, have been compared with those of another animal, not treated, but submitted to the same experimental procedure. The most specifically marked structure was the inferior olive. Secondarily marked structures were the posterior part of the lateral reticular formation, the caudato-putamen area and the prefrontal and frontal cortex.


[Effect of 2 inhibitors of glycoprotein synthesis, tunicamycin and 2-deoxyglucose, on development of the sea urchin (Paracentrotus lividus) egg].

Tunicamycin, an inhibitor of glycoprotein synthesis, inhibits the development of the sea Urchin egg. Gastrulation is very sensitive to the Tunicamycin. Fertilization is not inhibited. 2-deoxy-D-glucose inhibits gastrulation and induces the hyperdevelopment of ectodermic structures.


[Influence of the age of the mouse on the effectiveness of stimulation of its defenses by electromagnetic radiation].

Biological effects of microwaves on experimental trypanosomiase in the Mouse depends on the state of development of the animal. In fact such stimulation needs a mature immune system. Nevertheless, the increasing of the molecular immune response is only one of the aspects of protection mechanisms stimulated by such microwaves.


Interference by tubing material in continuous-flow systems.

An anomalous behavior in the measurement of calcium with the smac multi-channel high-speed analyzer was eliminated by taking the diluted serum directly from the dilution loop of the instrument before it enters the stainless-steel riser.


Results with commercial radioassay kits compared with microbiological assay of folate in serum and whole-blood.

We compared results with three commercial folate radioassay kits [Bio-Rad, New England Nuclear (NEN), and RIA Products] with those by microbiological assay for more than 200 samples of human serum and whole blood. All but one kit (NEN) compared favorably with the microbiological assay for serum samples, although there were notable diagnostic discrepancies. Two kits (NEN and Bio-Rad) were tested on whole-blood samples; both yielded values significantly higher than those by microbiological assay. The frequency distributions of erythrocyte folate data differed strikingly between the two kits; the NEN method yielded a much narrower range of normal values than did either the Bio-Rad or the microbiological assay. Radioassay kits appear to be suitable diagnostic agents for serum folate, if the behavior of a particular kit is investigated thoroughly before its routine use. However, the diagnostic value of radioassays of erythrocyte folate needs to be validated.


Comparison of radioassay and microbiological assay for serum folate, with clinical assessment of discrepant results.

Folate assays by use of radiolabeled folate provide obvious practical advantages over the standard microbiological assay, but remain incompletely tested. We therefore compared results for 415 sera with a kit involving 3H-labeled folate and the Lactobacillus casei microbiological method. We examined the patients' data when there were discrepancies between the two methods. Although the correlation overall was satisfactory, results were discrepant in 25% of cases. In 74% of the latter, the radioassay result appeared to be the correct one, primarily because L. casei results were suppressed by antibiotics being taken by the patient. The radioassay occasionally gave falsely high values for patients with liver disease and falsely low ones for patients who had received isotopes for scanning purposes. Several assay kits that make use of 125I- or 75Se-labeled folate were also tested. Although these results correlated with the results of 3H-labeled folate assay, various problems appeared, including the possible need for serum-supernate control tubes in one kit. Answers to these and other questions and careful clinical correlation of results are needed for any folate radioassays before their adoption for routine clinical use.


Serum thyrotropin releasing hormone (TRH)-degrading activity: a sensitive and rapid radiochemical assay procedure.

A rapid and sensitive method has been developed to assay thyrotropin releasing hormone (TRH)-degrading activity in serum. In this assay, the formation of proline, a major serum degradation product of TRH (pGlu-His-Pro-NH2), is measured. The procedure is based on the finding that proline can be readily separated from TRH with Dowex 50 by a batchwise procedure in a test tube.


A study of pancreatic secretory and intracellular enzymes in pancreatic cancer tissue, other gastrointestinal cancers, normal pancreas and serum.

Serum and tumour tissue extracts from patients with pancreatic exocrine adenocarcinoma and a number of other gastrointestinal tumours were examined for digestive and intracellular enzymes or tumour associated variants by cellogel electrophoresis. Enzymes were identified within the gel by their specific catalytic activities. Normal serum and extracts of normal pancreas were also studied. All the pancreatic secretory enzymes were present in extracts of normal pancreas; their levels were markedly reduced in tumour extracts and no characteristic isozymes were found. Alkaline phosphatase, carbonic anhydrase and a number of carbohydrate metabolising enzymes were present in all extracts; tumour associated isozymes were not consistently identified. These findings suggest that most human pancreatic exocrine cancers do not produce pancreatic secretory enzymes or intracellular isozyme variants, and that the identification of these enzymes or their isozymes is unlikely to be of practical diagnostic value.


Serum growth hormone and ultrastructural studies of adenohypophysial tissue in bromocriptine treated acromegalic patients.

The therapeutic effectiveness of bromocriptine as well as the post-operative ultrastructural aspects of treated pituitary adenomas were investigated in five acromegalic patients. Although concentrations of GH basal decreased and the glucose tolerance test and the TSH responses were significantly improved, the release of GH induced by TRH was not prevented by the dopaminergic agonist. Adenomatous cells were densely granulated and contained a dilated endoplasmic reticulum. Misplaced exocytosis was frequently observed. These findings clearly indicate that bromocriptine inhibits the spontaneous release of GH but does not interfere with the abnormal GH response to TRH. This suggests a separate site of action. The drug seems not to block the synthesizing activity of the adenomatous cell, a finding in accordance with clinical observations that warns against its use as a single therapeutic agent.


Effects of oral testosterone undecanoate in hypogonadal male patients.

Effects on plasma T, DHT, A, FSH, LH and PRL concentrations and the pituitary responsiveness to the LHRH/TRH stimulation, as well as on libido and sexual, mental and physical activities were studied in ten hypogonadal male patients undergoing therapy with oral testosterone undecanoate (TU) for 9 weeks. The daily dose of TU needed for satisfactory clinical effect was 120 mg (6 cases) and 240 mg (4 cases). There was a significant rise in circulation androgen concentrations in all patients. Mean plasma T, DHT and A increased at 9 weeks from 1.20, 0.12 and 0.36 ng/ml to 4.34 (P less than 0.0004), 0.39 (P less than 0.0004) and 1.24 ng/ml (P less than 0.005), respectively. In hypergonadotrophic patients (N = 6) plasma FSH and LH fell progressively (P less than 0.05), while in hypogonadotrophic patients (N = 4) a marked rise in plasma FSH (P less than 0.05) was found, while LH tended to rise as well. Base-line plasma PRL remained unchanged. In three out of four patients with poor PRL response to TRH, normal responses were established after TU therapy. Increase in libido and sexual activity was reported by nine patients. An increase in mental and physical activity was found in seven and two patients, respectively. Tolerance was excellent. It was concluded that oral TU is an effective form of substitution therapy in male hypogonadal patients.


Normal prolactin response following thyrotrophin releasing hormone in patients with autonomously functioning euthyroid multinodular goitre.

Twelve female patients (mean age 60 years, range 41--74) with euthyroid multinodular goitre and a flat TRH test (median delta TSH 0.9 muIU/ml, range 0--3.5) had normal prolactin responses to TRH (median data prolactin 48 ng/ml, range 6--115). The results are comparable with those in euthyroid patients receiving full substitution doses of thyroid hormone without increasing T3 and/or T4 levels beyond the normal range.


Hormonal responses during treatment of acute diabetic ketoacidosis with constant insulin infusions.

Changes in glucagon, growth hormone (GH), cortisol, renin and aldosterone accompanying the metabolic disturbances and dehydration of severe diabetic ketoacidosis were studied over a 24 h period in eight patients treated with a constant intravenous insulin infusion. Mean steady state plasma-free insulin levels achieved were 28.6--49 mu/1 in patients receiving 2 u/h but a satisfactory rate of fall of glucose was not always obtained until the infusion dose was increased to 4 u/h or more. The total insulin dose administered was positively correlated with the level of plasma glucagon and cortisol on admission. During insulin infusion, both glucagon and cortisol fell but the rate of fall was not related to dose or plasma level of free insulin achieved. In six of eight patients studied increments in plasma GH above admission levels were observed during insulin treatment. Admission values of both plasma renin activity and plasma aldosterone were raised. The renin levels were highest in newly diagnosed diabetics, and two patients with long-established diabetes showed only small increments despite profound dehydration. Plasma renin activity, but not plasma aldosterone correlated with the fluid and sodium retention over the initial 24 h treatment period, but not with potassium requirements. The urinary excretion rates of the small molecular weight proteins GH and insulin, were considerably elevated over the treatment and convalescent periods.


Combined genetic deficiency of C6 and C7 in man.

By routine screening of sera, a subject was discovered who showed a sub-total deficiency of C6 and C7. No clinical disease was associated with this deficiency which was transmitted through the subject's family as a single genetic characteristic, the C6 deficiency being associated with a silent allele at the structural locus. The propositus was found to have low quantities of an abnormal C6 which was both antigenically deficient and smaller in size than normal C6 (110,000 daltons compared with 140,000 daltons) and small quantities of apparently normal C7. It is concluded that the most likely explanation for this defect is that the subject has a structural mutation in his C6 gene which produces hyopsynthesis not only of C6 but also of the closely linked gene for C7. These findings suggest the possibility that C6 and C7 may function as a single genetic unit and that the primary transcript copied from the genome includes information for both proteins.


Studies on the immunogenicity of protamines in humans and experimental animals by means of a micro-complement fixation test.

A complement fixation study with human, monkey and rabbit sera, using purified sperm nuclear basic proteins as antigens, led to the following conclusions. (1) Protamines, the sperm-specific basic nuclear proteins, may be immunogenic in mammalians. (2) Antibodies detected in the indirect immunofluorescence test on human swollen sperm heads in sera from infertile and vasectomized men, are directed primarily against human protamines. (3) The results obtained suggested that differences in the immunization site and/or in the configuration of the immunizing protamine, may lead to the formation of antibodies directed against different antigenic determinants. Autoimmunity to protamines, following vasectomy or in infertile men, is accompanied by the formation of antibodies cross-reacting with common antigenic determinants present in protamines of other species. Induction of immunity to protamines by means of immunization with protamines-RNA complexes (in rabbits), or protamine-insulin complexes (in humans), leads to the formation of antibodies reacting more specifically with the immunizing protamine, showing only slight cross-reaction with other protamines. (4) The histone-like fraction present in mature human spermatozoa is composed mainly of histone fraction H2B.


Post-vasectomy autoimmunity to protamines in relation to the formation of granulomas and sperm agglutinating antibodies.

The development of antibodies reacting with nuclear antigens was studied in sera from vasectomized men and monkeys (obtained at intervals of up to 2 and 4 years, respectively, after the operation), by means of a comparative fluorescence study on swollen nuclei of somatic cells, human and salmon spermatozoa. About 30% of forty-seven vasectomized men developed antibodies to protamines. Also four out of fifteen monkeys, vasectomized with or without ligation, developed antibodies reacting with protamines. In general, when antibodies to the homologous protamines reached higher levels, cross-reactions with salmon protamine could be demonstrated as well. No significant reactions could be detected with somatic nuclear antigens in the sera. Comparison between the results obtained with the immunofluorescence test, the agglutination tests and the clinical findings confirmed the association between the development of immune responses to various sperm-antigens and revealed a coherence of anti-protamine activity and granuloma formation at the site of the operation.


Immune function in ankylosing spondylitics and their relatives: influence of disease and HLA B27.

So as to distinguish the separate influences of ankylosing spondylitis (AS) and possible HLA B27 associated immune response genes on immune response patterns, a battery of immunological tests were performed on fourteen patients with AS and their first-degree relatives. Previously unrecognized AS was detected by clinical and radiological means. Individuals with ankylosing spondylitis had significantly higher serum IgG and IgA concentrations than both their B27 positive and B27 negative relatives. B27 positive relatives had significantly lower phytohaemagglutinin (PHA) lymphocyte transformations than B27 negative relatives (P less than 0.01), while there was no difference between the ankylosing spondylitic and B27 positive groups. Antibody titres to Streptokinase/Streptodornase were significantly higher in the B27 positive individuals, with or without AS, than their B27 negative relatives (P less than 0.005 and P less than 0.02 respectively). These results show that serum immunoglobulin differences were associated with disease, while differences in PHA stimulation and varidase antibody titres were associated with the B27 antigen. These findings may indicate the presence of HLA associated immune response genes including those involved with reactions to a particular antigenic component of Streptokinase/Streptodornase.


Complement activity in middle ear effusions.

Evidence for complement utilization in middle ear fluids (MEF) from patients with otitis media with effusion was sought. It was found that cleavage products of C3, C4 and Factor B could be demonstrated immunochemically in MEF, and that native C3 was present in much lower concentrations than other proteins, relative to their serum concentrations. Haemolytic assays for C1-C5 showed that early complement components are inactivated in MEF. Potential mechanisms for complement utilization in MEF are discussed.


Urinary acidic hydrolases in renal diseases in children.

Acidic hydrolases were assayed in urines of 19 normal children, 33 children with idiopathic nephrotic syndrome of childhood (INS), 21 children with glomerulonephritides (GN) and 7 children with persistent proteinuria/hematuria, and in plasma of 10 children each with INS or GN. Both plasma and urinary acidic hydrolases were studied in intermittent orthostatic proteinuria. Cbeta-galactosidase and Cbeta-N-hexosaminidase were done in normals and children with active renal disease. Significantly (P less than 0.01) elevated urinary acidic hydrolases excretion in active renal diseases, both in INS and GN, returned to a normal range with regression of the diseases. Increased postural proteinuria was associated with normal urinary acidic hydrolases. Both beta-galactosidase and beta-N-hexosaminidase excretion was higher than similar mol wt proteins in normals and increased further in active renal diseases. The data suggests that increased urinary acidic hydrolases is related to the activity of the renal disease, and not to urinary WBC, hematuria or proteinuria. The likely source of urinary acidic hydrolases thus appears to be the injured renal parenchyma itself.


Productivity and economic implication of a simulated practice using expanded duty dental assistants.

This investigation was concerned with determining the optimum team mix for a dental practice utilizing expanded duty dental assistants (EDDAs). A live, surrogate practice provided the basis for a computer-simulated private practice in which one dentist worked with various combinations of chairside assistants and expanded duty auxiliaries. This "composite dentist" was of average speed as an operator, functioning under conditions of an external quality control mechanism. Results indicated that the optimum team size, from the standpoint of both productivity and economic considerations, involved one dentist, one EDDA, and two assistants, as compared with the control team of one dentist and two assistants. Experimental teams involving two EDDA's, studied both with and without chairside assistants, were counterproductive from a financial standpoint. Various factors influencing team size are discussed, but dentist operator speed emerges as a major determinant of optimum team mix.


Cost analysis of 3 years of topical fluoride application.

The article gives additional information about the 3-year study of caries reduction after topical application of 4% NaF-PO4 in Ljubljana, Yugoslavia. The caries reduction obtained was about 20% after topical fluoride application once a year (Group 2) and about 30% after topical fluoride application twice a year (Group 3). Data on costs of treatment and dentist's working time required are given. The costs of repair and topical fluoride application during the 3-year experimental period were in Group 2 72% and in Group 3 158% higher than in the Control Group. The dentist's working time for repair and topical fluoride application during the experimental period was in Group 2 33% and in Group 3 80% higher than in the Control Group. It seems to be of special interest that the dentist's working time spent for total tooth repair per child at the beginning of the study was 3.3-4.9 times higher than the working time for repair in the years during the study.


Chemical occlusion of rhesus monkey oviducts with chloroquine.

Fertility control through chemical occlusion of the oviducts with chloroquine was investigated in rhesus monkeys (M.m.). Chloroquine was instilled retrograde through the tubal ostium after laparotomising the monkeys as a 10, 50 and 100% suspension (w/v) in aqueous carboxymethyl cellulose (CMC) solution which was used as a carrier vehicle. Complete occlusion of the Fallopian tubes at the uterotubal junction and also the intramural portions resulted in 15 days after a single instillation.


Neoplastic complications of neurofibromatosis.

Neurofibromatosis associated with secondary neoplasms is discussed. Neurosarcomas are the most common and most malignant secondary tumors in neurofibromatosis. Any sudden growth in a peripheral tumor in this disease should alert the physician to the possibility of malignant change and the need for biopsy. Only by early diagnosis and adequate excision can some of these patients be saved.


Immunocytoma effect upon circadian variation in murine urinary excretion of beta-aminoisobutyric acid, beta-alanine, phenylalanine and tyrosine.

Under the conditions of disynchronization by the manipulation of both the alternation of light and darkness and the availability and unavailability of food, circadian rhythms characterize the excretion of several amino acids by inbred LOU rats bearing an immunocytoma. Large amplitude rhythms can be demonstrated for urinary beta-aminoisobutyric acid, beta-alanine, phenylalanine and tyrosine. Under the same conditions of disynchronization, control animals excrete the same compounds also with a marked circadian variation but at an invariably lower average rate. These excretory rhythms, along with those demonstrated earlier for polyamines and light-chains, are of interest as potential markers for the chronotherapy of cancer.


Taurine and other free amino acids in milk of man and other mammals.

Taurine and other free amino acids have been determined in human milk of a number of other species. Taurine is the most abundant free amino acid in the milk of the gerbil, mouse, cat, dog and rhesus monkey. Taurine is the second most abundant amino acid in the milk of the rat, baboon, chimpanzee, sheep, Java monkey and man. Taurine is not a major constituent in the milk of the guinea pig, rabbit, cow and horse. The milk of each species has a characteristic free amino acid pattern which may be an indication of the relative nutritional importance of these compounds during early postnatal development.


Effects of calcium and magnesium ions on the interaction of corticosterone with rat brain cytosol receptor(s).

The apparent maximum corticosterone binding (B max) with rat brain cytosol and the apparent dissociation constant of this steroid-receptor binding (Kd) estimated with a Scatchard plot was 2.9 X 10(-13) moles/mg cytosol protein and 4.0 X 10(-9) M, respectively. When increasing amounts of CaCl2 or MgCl2 up to 5.0 mM were added, a specific [3H] corticosterone binding increased 4-fold by CaCl2 at concentrations of 1.0-2.0 mM and 1.5-fold by MgCl2 at concentrations of 0.5-5.0 mM. The addition of MnCl2 and KCl did not affect this binding. Binding of corticosterone with rat brain cytosol receptor(s) were decreased by increasing amounts of EGTA and complete inhibition was observed at concentrations equal to and greater than 2.5 mM. Inhibition of this binding by EDTA was less than by EGTA. Either theophylline or dibutyryl cyclic AMP had no effect on this binding.


Operant methodology in the study of learning.

A series of experiments is described in which operant methodology is used to study the effects of drugs on "learning." Emphasis is placed on the technique of repeated acquisition as a behavioral baseline for studying this type of transition state. In this technique, each subject is required to learn a new discrimination each session. Multiple-schedule procedures are also described in which acquisition is compared to a "performance" task, where the discrimination is the same each session. The learning baseline is more sensitive to the disruptive effects of a variety of drugs (e.g., cocaine, d-amphetamine, haloperidol) than is the performance baseline. This general finding obtains across procedural variations and species (pigeons and monkeys). The potential usefulness of these procedures for studying both acute and chronic behavioral toxicity is discussed.


Enterobacterial common antigen: isolation from Shigella sonnei, purification and immunochemical characterization.

In the studies presented the effective procedure of isolation and purification of enterobacterial common antigen from Shigella sonnei has been elaborated. The method is based on sonification of bacterial suspension in the presence of lysozyme and EDTA and subsequent extraction of the pellet with boiling water. The crude extract of common antigen was purified by fractionation with ethanol and chromatography on silica gel and Sephadex LH-20. The comparison of several extraction procedures of enterobacterial common antigen from Shigella sonnei proved that the method described above is most effective. The purified enterobacterial common antigen preparation obtained preserved full biological activity: antigenicity (precipitation and activity in enzyme-linked immunosorbent assay), immunogenicity in rabbits, ability to coat erythrocytes (passive hemagglutination) and inhibitory activity in passive hemagglutination. The pure enterobacterial common antigen was identified to 90% as a polymer of N-acetyl-D-mannosaminuronic acid and N-acetyl-D-glucosamine (2:1, molar ratio), O-acetylated and containing 3.2% fatty acids (C16:0 and C18:1, not oleic). It contains 5.3% nitrogen, less than 4% protein, less than 0.5% phosphorus and less than 1.6% neutral sugar; glycerol and RNA were not found in the preparation.


Compensation of dietary induced reduction of tetracycline absorption by simultaneous administration of EDTA.

The absorption of tetracycline in man under the influence of concomitantly administered EDTA, milk and a combination of EDTA and milk has been investigated. Urinary excretion of the drug was measured for 30 h. The inhibitory effect of milk could be counteracted by simultaneous ingestion of EDTA, which resulted in almost equivalent urinary excretion of tetracycline compared to experiments done in the fasting state. Administration of EDTA alone, in a neutral dosage form, did not significantly change absorption of the drug, which contradicted previous findings. The possible use of EDTA during tetracycline therapy is discussed.


Proteolytic cleavage of human IgG molecules by neutral proteases of polymorphonuclear leukocytes.

The effect on human IgG of the elastase-like (ELP) and chymotrypsin-like (CLP) neutral proteases derived from human polymorphonuclear leukocytes was studied. By incubating ELP with monoclonal IgG proteins, two immunochemically and electrophoretically distinct components were formed which were similar, but not identical, to the Fc and Fab fragments produced by papain digestion. When an IgG protein was incubated under similar conditions with CLP enzyme, no proteolysis was observed. IgG proteins differed in their susceptibility to proteolysis by ELP. These differences were related to the subclasses IgG1-IgG4. The IgG1 and IgG3 proteins were readily cleaved by ELP, but the IgG2 and IgG4 proteins were more resistant. Although free light chains differ in susceptibility to proteolysis by ELP, our studies showed that neither the type (kappa or lambda) nor the subgroup of light chain affected the susceptibility of complete IgG molecules to cleavage by this enzyme.


Maternal allotype dominance and allelic exclusion in the B lineage cells of the newborn rabbit.

Newborn rabbits from parents which differ at the b allotype locus (kappa chain), show a strong maternal allotype dominance, especially in the bone marrow. This concerns not only the membrane but also the cytoplasm, and it cannot be simply due to a passive uptake of maternal immunoglobulins. The spleen precedes the bone marrow for a high level of expression of the paternal allotype. In the thymus, cells which contain cytoplasmic immunoglobulin are found at low frequency, but their absolute number represent a substantial contribution to the B lineage cell pool. A small proportion of such thymus cells do not show allelic exclusion of kappa chain allotypes.


Potent depletion of 5HT from monkey whole blood by a new 5HT uptake inhibitor, paroxetine (FG 7051).

The potent 5-hydroxytryptamine (5HT) uptake inhibitor FG 7051 (paroxetine, INN) was administered to rhesus monkeys in doses of 1.0, 2.5 or 7.5 mg/kg by oral gavage once daily for 13 weeks. Blood samples for analysis of 5HT in whole blood and paroxetine in plasma were taken prior to and after 1, 4 and 13 weeks of treatment. The lowest dose 1 mg/kg caused 30% depletion of 5HT in whole blood with a level of paroxetine in plasma below 2 ng/ml. Doses of 2.5 mg/kg produced an 85% depletion of 5HT and a steady state plasma concentration of about 5 ng paroxetine/ml, while 7.5 mg/kg caused a 93% depletion of 5HT and a steady state plasma concentration of 100--450 ng paroxetine/ml. There was no concentration-dependent 5HT reduction with the highest dose level suggesting that maximal depletion was produced by concentrations below 100 ng/ml. The results showed that paroxetine is a strong depletor of 5HT from whole blood of monkeys conceivably because it inhibits 5HT uptake inhibition. The effect of the drug reached its maximum within 1 week and no tolerance developed during 13 weeks.


Bone growth aud development of secondary ossification centers of extremities in the cynomolgus monkey (Macaca fascicularis).

The development of so-called long bones in the extremity has been studied roentgenographically in forty-seven males and fifty-one females cynomolgus monkeys bred and reared at the National Institute of Health. The age of the females ranged from five months to eight years and nine months, and that of the males was from four months to seven years. In addition, the fetuses of six to twenty weeks of gestation age were examined for the time of appearance of ossification centers. As the biological parameters concerning body growth, the body weight and the bone length were measured and the secondary ossification centers were scrutinized and assessed the maturity process on the basis of the criteria that divided the state into eleven stages. Also the allometric analyses of body weight against bone length was conducted. Most of the secondary ossification centers except the proximal fibulal epiphysis appeared during the period from the prenatal stage (15-20 weeks of gestationage) to the postnatal one (several months of age). From four to five months of age, many ossification centers had developed to some extent. But, the appearance of proximal fibulal epiphysis was delayed and often lacking until 10 months of age in female and one year and three months of age in male. The earliest epiphyseal fusion was observed at the distal humeral epiphysis in both sexes. The latest epiphyseal fusion was observed at the distal ulnal epiphysis in both sexes and  at the distal ulnal and radial epiphyses in female. From this study, the time of fusion was at five and three guarters years of age in females and at six and a half years of age in males. As a result, it is suggested that the estimation of animal's age might be put to practical use by introducing the assessing method that the score was given from the observation of the secondary ossification center.


N,N-diethyl-4-aminoazobenzene (DEAB): acute actions with respect to possible carcinogenicity as well as the role of solvents. Morphological and pharmacological investigations.

The acute action of the azo dye DEAB was investigated in Sprague-Dawley (SD) and Wistar (Wi) rats. The substance was dissolved both in DMSO and sunflower oil and was administered once by stomach tube. Cytochrome P-450-DEPENDENT N-demethylation of ethylmorphine and dimethylnitrosamine are differentially altered depending on the solvent used. The excretion of DEAB as well as of N,N-dimethyl-4-amino-azobenzene (DAB) is delayed and diminished if the substances are dissolved in DMSO. Beside these effects the mitotic number in the adrenal cortex is significantly elevated in both strains of rats. But, in SD rats only DMSO-solution of DEAB is effective. In Wi rats both are effective, the oily solution more than that in DMSO. In this respect DEAB resembles DAB and various other carcinogens which are efficient in stimulating adrenocortical cell division. Considering the positive short-term assay after three other substances which revealed carcinogenic properties in long-term experiments we conclude that also DEAB may be carcinogenic in adequate long-term examination.


Digitonin induced alterations of the erythrocyte membrane as visible by freeze-fracturing.

Treatment of erythrocytes by low concentrations of digitonin results in the formation of elongated, bulged membrane areas free from particles and intramembraneous tubular structures emerged from these domains. At higher concentrations the tubular structures are present also outside of the membrane. A second, not bulged type of particle-free areas is more or less rounded, but mostly of rectangular shape. The rate of this type of domains increases with the concentration of digitonin. Breaks in these areas lead to a formation of sheets which results in a breakdown of the membrane structure finally into a brittle mass of many small sheets lying irregularly one upon the other. Already during the first steps of this membrane breakdown hemolysis takes place. Cross-linking of the proteins with glutaraldehyde does not entirely block up the membrane alterations. In fixed erythrocytes the formation fo elongated domains is restricted, no tubular structures are present and no dislocations of particles can be observed. Nevertheless smooth sheets localized also outside of the membrane are formed. However the investigated ghosts are not stabilized by glutaraldehyde against the effects of digitonin. Particle dislocations as well as all the other membrane alterations are present. The implications of the obtained results are: 1. The elongated domains and the tubular structures are probably not digitonin-cholesterol-complexes. 2. The formation of crystalline regions by digitonin-cholesterol-complexes destroys the membrane structure.


Acetic acid pretreatment of initiated epidermis inhibits tumour promotion by a phorbol ester.

Mouse skin initiated with 7,12-dimethylbenz(a)anthracene and then exposed to multiple treatments of acetic acid, shows a decreased papilloma yield on subsequent promotion with croton oil.


Autoradiographic localization of the uptake of 3H-beta-alanine in rat nervous tissue cultures.

Autoradiographic studies on the uptake of 3H-beta-alanine have shown that, in spinal cord and brain stem cultures, both neurones and glial cells have accumulated the amino acid. In contrast, in cultures of cerebellum and dorsal root ganglia, 3H-beta-alanine was only t aken up by glial elements.


High-affinity binding of beta-alanine to cerebral synaptosomes might involve glycine-receptors.

High-affinity, Na+-independent binding of beta-alanine to a synaptosomal fraction of rat brain was potently inhibited by glycine and by some other alpha-amino acids, but not by taurine or GABA. This binding mechanism, which was also sensitive to both bicuculline and strychnine, might involve synaptic receptors for both beta-alanine and glycine.


Organization of the mammalian red nucleus and its interconnections with the cerebellum.

The red nucleus in monkeys and rats consists of a magnocellular, rubrospinal portion which receives its cerebellar information from the nucleus interpositus, and a parvocellular, rubroolivary portion which receives cerebellar afferents from the nucleus lateralis. Distinct interpositorubrospinal and dentatorubroolivary projections are therefore common to these 2 species.


Estimation of plasma thyroxine concentration in ducks in relation to different environmental and experimental conditions.

Plasma thyroxine concentration was measured in ducks by the thyroxine-binding globulin technique. The assay allowed us to detect annual variations in thyroid activity as well as significant changes after starvation or cold exposure. No detectable thyroxine was formed in surgically thyroidectomized ducks.


Topographic localisation of insulinogenic and insulinoprival areas in the hypothalamus.

Electrical stimulation of hypothalamic areas through stereotaxically implanted electrodes were carried out in conscious male rhesus monkeys. There was a significant increase in immunoreactive insulin (IRI) following laterla hypothalamus (LHA) stimulation. An opposite response was obtained from ventromedial hypothalamus (VMH). Thus, insulinogenic and insulinoprival responses were obtained from feeding and satiety, suggesting a significant role of insulin in the regulation of food intake.


A method to collect cervical smears from small breeds of monkeys.

A simple method is described to collect cervical smears and to have a clear view of cervix for small breed of monkeys. This method was found useful to collect adequate cytology smears with good preservation of cellular morphology.


[Clinical evaluation of thyroid functions in chronic hemodialysis patients (author's transl)].

In order to obtain more precise information on thyroid functions in renal failure, attempts were made to analyze the Hypothalamo-hypophyseal axis in the regulation of thyroid function. TRH levels and other thyroidal function tests were carried out on sera and urine from patients with renal failure and thyroid diseases. Analysis of TRH in serum and urine was performed by mass fragmentography using a GC-MS combined method. Fifteen out of 28 patients had enlarged thyroid glands, goiter in high prevalence. Thyroid function studies showed low serum thyroxine and triiodothyronine, and high serum thyrotropic hormone levels. Serum and urinary TRH in chronic renal failure were strikingly higher than the other thyroid diseases. There were negative correlations between RIA-T3, Resomat-T4 and serum and urinary TRH levels respectively. There was a negative correlation between serum TSH and serum TRH, but no significant correlation was noticed between serum TSH and urinary TRH levels. There was a high positive correlation between serum TRH and urinary TRH levels. From these findings, it is thought that serum and urinary TRH level values are a more reliable index for the clinical evaluation of thyroid functions in renal failure than the other sample under laboratory data. In patients with chronic hemodialysis, the prevalence of goiter is relatively high, and their thyroid functions are abnormal. However, the cause of the abnormalities is not clear. It is postulated that the possibility of a breakdown of compensation in the Hypothalamo-hypophyseal axis for the regulation of the thyroid function may be seen in chronic renal failure.


[A new method for the determination of serum human placental lactogen (HPL) by single radial immunodiffusion (author's transl)].

The general radioimmunoassay (RIA) currently employed for the determination of serum HPL requires a special apparatus and a complicated technique. Therefore, in order to simplify the assay we tried to develop a new method using single radial immunodiffusion (SRID). Basic examinations were made to establish a simplified HPL assay method by SRID. The optimal conditions finally decided for SRID were as follows: the thickness of the agar plate, 1.5 mm; the diameter of the well, 3 mm; the amount of antigen added, 10 microliter; antibody concentration, 1.0% for the measurement range from 1.0 microgram/ml to 16 microgram/ml and 0.5% for the measurement range from 0.25 microgram/ml to 1.0 microgram/ml. Under these conditions serum HPL was detectable after the 9th week of gestation, and antigen was measurable over 0.25 microgram/ml. Serum HPL values measured by RIA and SRID from the 9th to the 42nd week of gestation in 138 normal pregnant women were identical, and the correlation was highly significant (r = 0.842, p less than 0.001). This SRID method was as reliable and sensitive as the RIA and made it easier than the RIA to measure the serum HPL of pregnant women. Therefore, with regard to placental function its clinical application should prove to be certainly beneficial.


Antibody binding of insulin in diabetic ketoacidosis.

This study investigates the effects of insulin antibody binding on free insulin levels measured in patients with acute diabetic ketoacidosis receiving insulin by constant infusion. In spite of antibody binding ranging from 10 to 90 per cent of the total circulating insulin, the steady state concentrations of free insulin were similar to those observed in individuals on identical infusion rates but without insulin-binding antibodies. However, the levels of free insulin in two patients were substantially lower than expected for the rate of insulin infusion, even though levels of bound insulin were not greatly elevated. An infusion rate of at least 4 U. per hour produced satisfactory rate of fall of plasma glucose, whereas lower dose regimens (2 U. per hour)--producing steady state free insulin concentrations ranging from 28 to 49 mU. per liter in different subjects--were unreliable in controlling the metabolic abnormalities of diabetic ketoacidosis.


Microtubules and microfilaments as major components of a phagocytic apparatus: the cytopharyngeal basket of the ciliate Pseudomicrothorax dubius.

The cytopharyngeal basket of Pseudomicrothorax dubius, through which filamentous blue-green algae are ingested, consists of 22 (+/- 3) nemadesmata and nemadesmal lamellae, in the form of a tube. A cytostome, delimited by the cell membrane and surrounded by 22 (+/- 3) major and minor cortical corrugations, covers the end of the basket where the latter is attached to the cell cortex. Each nemadesm, at its greatest diameter, consists of about 200 microtubules which are joined together by sheet-like cross-bridges. The cross-bridges appear to be responsible for the high structural resilience of the nemadesmata. Each nemadesmal lamella is a ribbon of 20--30 microtubules, with two arm-like structures associated with one side of each microtubule. The arms are partially embedded in a fine filamentous layer. Except for a perforated zone, the wall of the basket is completely closed due to the presence of a filamentous sheath which extends between adjacent nemadesmata. Absence of the sheath allows movement of vesicles between the cytoplasm and the lumen of the basket in the perforated zone. The sheath is capable of elastic stretching during food uptake.


[Amplification of the riboflavin operon genes of Bacillus subtilis in Escherichia coli cells].

Amplification of Bacillus subtilis DNA fragments was performed in Escherichia coli using plasmid RSF2124. The main principle of isolation and cloning hybrid plasmids was described using genes of riboflavin operon as a model. Bac. subtilis DNA was treated with restriction endonuclease EcoR; followed by the agarose gel electrophoretic separation of the resulting fragments. Gels were sliced, DNA was eluted from the corresponding slices and used to transform Bac. subtilis auxotrophs rib A72, rib S110 and rib D107. DNA fraction with the molecular weight 7 . 10(6) daltons restored prototrophy of these mutants. DNA of this fraction was ligated with EcoRI treated plasmid RSF2124 DNA and used for transformation of E. coli rk-mk+. Ampicillin resistant transformants which had lost the colicin production ability, were selected. The presence of riboflavin genes within the hybrid plasmids was detected by transformation of B. subtilis auxotrophs. Three hybrid plasmids (pPR1, pPR2 and pPR3), containing a fragment of Bac. subtilis DNA with the molecular weight 6.8 . 10(-6) daltons including riboflavin operon, were selected. The analysis of the transformation activity of Bac. subtilis DNA and plasmid pPR1 DNA revealed, that there was no restriction activity of Bac. subtilis cells against plasmid DNA amplified in E. coli. Heteroduplex analysis has shown that plasmids pPR1 and pPR2 differ in the orientation of Bac. subtilis DNA fragment. DNA of these plasmids restored prototrophy of the several studied E. coli riboflavin auxotrophs.


[Effect of Bacillus subtilis A-50 "streptomycin" mutations on the formation of molecular forms of subtilisin, an extracellular alkaline proteinase].

The patterns of subtilisin molecular forms of streptomycin-resistant (Strr) and streptomycin-dependent (Strd) mutants of Bacillus subtilis A-50, as well as the revertants of Strd to streptomycin-independence (Str1) were studied. Strr mutants had different quantitative pattern of the same subtilisin molecular forms as compared with the initial strain A-50 (the forms with Rf 0.08, 0.16 and 0.3). In comparison with the initial strain A-50, Strd mutants and Str1 revertants revealed three additional forms of the active enzyme with Rf 0.02, 0.5 and 0.7 and the molecular weights less than 35,000, 28,000 and 20,000 respectively. It was suggested that the rate and character of the enzyme secretion of the degree of its post-translational modifications might result in the different pattern of subtilisin molecular forms produced by these streptomycin mutants.


[Genetic limitations of erythrocyte lysis by bacteria of the genus Salmonella].

Lytic effect of Salmonella bacteria on the erythrocates of 1301 humans (including 150 twins), 1059 hens, 600 mice, 33 guinea pigs, 47 rabbits, 22 horses, 16 sheeps, 7 dogs, 2 cats and 2 monkeys was investigated. Erythrocytes of all horses, guinea pigs and rabbits tested appeared to be sensible. The same cells of humans, sheeps, hens, dogs, monkeys and cats turned to be either sensible, or stable. Human erythrocytes were the most stable. Erythrocytes of humans having had typhus or other Salmonella infections appeared to be more sensible. Homozygous twins developed complete two by two concordance of the sign of sensibility in all details investigated. Relation of erythrocytes to Salmonella hemolysins was not influences with time and specific immunization, did not correlate with blood groups and factors ABO, MN, P and rhesis systems, with age, sex and nationality of people tested. It also did not correlate with the presence of anti-salmonella agglutinins, with different osmotic and acid stability of erythrocytes. The display of hemolytic activity of Salmonella is limited by genetically predestinated species and individual properties of erythrocytes.


[UV-light induction of "true" revertants to adenine independence in Bacillus subtilis cells].

The UV-irradiation of Bacillus subtilis Mu5u8u16 (met5 leu8 purA) induces with relatively high frequency the revertants to adenine independence (Ade+) which form the rapidly growing morphologically uniform colonies on the solid selective medium. The genetic analysis of a portion of UV-induced Ade+ revertants (crosses in transformation system) has cleared up that their DNA does not contain the original mutation ade16. This means that they arise as the result of "true" reversions. This reversion in purA gene can serve as a good model for the study of UV-induced mutagenesis in a proximal structural locus of Bac. subtilis chromosome.


[Riboflavin biosynthesis operon of Bacillus subtilis. XIV. Operator-constitutive mutants].

Numerous operator-constitutive mutants of riboflavin biosynthesis were selected. All of them map in a short region of the Bacillus subtilis chromosome. The yield of riboflavin synthetase from this mutant is different, but in most cases much lower than the maximal yield from a repressor minus strain. Our tentative explanation is a partial overlap of the sites for the adsorption of repressor and RNA-polymerase. Therefore the affinity to the transcribing enzyme is diminished in the operator constitutive strains. The affinity of the repressor-effector complex to the operator depends on the effector structure.


Social organization and social behavior in two subspecies of squirrel monkeys (Saimiri sciureus).

Social organization and social behavior were examined in two subspecies of squirrel monkeys which differ markedly in the degree of sexual dimorphism. The Bolivian squirrel monkeys, the subspecies with greater sexual dimorphism, manifested a sexually segregated form of social organization, while the social organization of the Guyanese monkeys was sexually integrated. Dominance relationships were found to reflect these patterns of sexual segregation or integration; in the Bolivian social groups separate linear dominance hierarchies were established within each sex while the Guyanese monkeys established a single linear hierarchy which included both males and females. Relationships between males and females in the two subspecies appear to be regulated by two distinct mechanisms, dominance in the Guyanese monkeys and sexual segregation in the Bolivians.


Food transfer by wild titi monkeys (Callicebus torquatus torquatus).

The behaviours and food types associated with wild titi monkey food transfer are described and analyzed. Food transfer is not considered altruistic, and possible benefits are explored by examining the context and consequences of food transfer.


Karyotype abnormalities in two primate species, Pygathrix nemaeus and Lemur coronatus.

The karyotypes of 7 douc langurs (Pygathrix nemaeus) and 3 crowned lemurs (Lemur coronatus) were examined. Abnormalities in 23.5% of the karyotypes of 1 male douc langur were associated with a history of fathering stillborns and abortuses (38%). Karyotype analysis of an apparently normal female lemur revealed three differing karyotypes, one normal and two abnormal.


The orbital mosaic in prosimians and the use of variable traits in systematics.

Study of 579 skulls, representing all 33 extant prosimian species and several anthropoids, shows that an orbital exposure of the ethmoid occurs in most Malagasy genera. A palatine-lacrimal contact is normal only in Lemur; the zygomatic-lacrimal contact occurs in most lorisiform species. Lepilemur shows possibly significant resemblances to lorisiforms. The vomer appears in the orbit of some ceboids. Cebids are distinguished by a canal running from the orbital apex through the basicranium. It is argued that skull shape and jaw musculature affect the size and shape of certain orbital-mosaic components. Frequencies of traits, rather than typologically 'normal' traits, may be taken as synpomorphies linking different species cladistically.


Changes through the day in the food choice of wild gibbons.

Several studies of primates have found that they tend to eat different foods at different times of day. A detailed study of a small sample of siamang and lar gibbons shows that they select figs in the morning, young leaves in the afternoon and figs again for the last feed of the day. Various explanations of these choices are offered, centering on how the gibbons can best meet their energy demands overnight and at dawn.


The birth and early development of a captive sifaka, Propithecus verreauxi coquereli.

We report here our observations on parturition and infant development up to five months in a captive sifaka, Propithecus verreauxi coquereli. Comparisons are made with other reports of Propithecus. In describing the acquisition of developmental landmarks, a distinction is made between the first sighting of a behavior pattern and its regular occurrence in the infant's repertoire. We describe the development of locomotor abilities, grooming, feeding, play, and social interactions. In general, maturation of Propithecus is slow relative to species of the genus Lemur.


Some effects of living conditions upon the pattern of growth in the stumptail macaque (Macaca arctoides).

Somatometric and weight measurements of stumptail macaques (Macaca arctoides) bred in various environmental conditions are presented. Environmental parameters may influence the pattern of growth in slowing down the 'growth rate' of monkeys bred in restricted laboratory environment as compared with monkeys bred in 'optimal' captive conditions: their subadult and adult linear dimensions are smaller.


[Long-term coverage of defect-wounds using synthetic skin substitute].

According our experiences non-infected defect wounds can be covered with Epigard even for a long time without problems. Changing the skin substitute is not necessary. Thus the danger of bacterial contamination is reduced and a reliable prevention of infection results. Weeks or months later an ideal base for autodermic grafting is obtained. Our method is indicated particularly for treatment of open fractures with skin defects. Long-term cover of infected tissue cannot always be achieved; in this case another therapeutic way should be attempted.


Human placenta alpha-N-acetylglucosaminidase: purification, characterization and demonstration of multiple recognition forms.

alpha-N-Acetylglucosaminidase (EC 3.2.1.50) was purified from human placenta by a four-step procedure including ammonium sulfate precipitation, affinity chromatography with immobilized antibodies against urinary alpha-N-acetylglucosaminidase, gel chromatography and discontinuous gel electrophoresis with a 30% recovery and 26 300-fold purification. Immunological methods revealed the contamination with about 10% non-alpha-N-acetylglucosaminidase protein. Isoelectric focusing led to a distribution of activity between 4.3 and 6.5 with maxima at pH 5.1 and pH 5.7. After treatment with neuraminidase, alpha-N-acetylglucosaminidase activity assembled at pH 5.7. The multiple isoelectric forms were endocytosed with different rates by cultured human skin fibroblasts. Placenta alpha-N-acetylglucosaminidase has an apparent molecular weight of 304 000 and contains 23.4% carbohydrate consisting of glucose, galactose, mannose, hexosamines and neuraminic acid. Gel electrophoresis in the presence of 0.1% sodium dodecylsulfate separated placenta alpha-N-acetylglucosaminidase into subunits with molecular weights of 86 500 and 81 000. The activity towards various substrates, the kinetics of hydrolysis, the pH optimum and the stability of the catalytic activity were assayed.


It works better when problems are shared. Interview by Mark Perlberg.

In an exclusive interview, Jeannette R. Kramer, a specialist in the provision of long-term care, discusses the concept of the therapeutic community that she and her husband have instituted in the skilled nursing care facility that they own and operate. By providing an environment in which patients, their families, and the staff can communicate easily, the nursing facility can pinpoint problems and solve them early.


Criteria for internal auditing.

An effective, inclusive internal auditing endeavor should help assure hospital managements that (1) an adequate system of internal control exists to assure the safeguarding of assets and the reliability of data produced by the financial information system, (2) uneconomic operating practices are detected promptly so they can be remedied, and (3) program results and effectiveness levels are of sufficiently high quality to demonstrate managerial competence.


Explant culture of rat colon: a model system for studying metabolism of chemical carcinogens.

An explant culture system has been developed for the long-term maintenance of colonic tissue from the rat. Explants of 1 cm2 in size were placed in tissue-culture dishes to which was added 2 ml of CMRL-1066 medium supplemented with glucose, hydrocortisone, beta-retinyl acetate, and either 2.5% bovine albumin or 5% fetal bovine serum. The dishes were placed in a controlled-atmosphere chamber which was gassed with 95% O2 and 5% CO2. The chamber then was placed on a rocker platform which rocked at 10 cycles per min causing the medium to flow intermittently over the epithelial surface. The explants were incubated at 30 degrees C. The viability of the tissue was measured both by incorporation of specific precursors into cellular macromolecules and by monitoring of tissue morphology with light and electron microscopy. Cultured rat colon was able to metabolize benzo[alpha]pyrene, 7,12-dimethylbenz[alpha]anthracene, aflatoxin B1, dimethylnitrosamine, 1,2-dimethylhydrazine, and methylazoxymethanol acetate into chemical species that bind to cellular DNA and protein.


Binding of soluble immune complexes to Raji lymphocytes. Role of receptors for complement components, C1q and C3-C3b.

We have found that, although the binding of particulate antigen-antibody complement complexes such as EAC to lymphoblastoid Raji cells is mediated largely through receptors for C3b, the binding of complement-containing soluble complexes such as those prepared with aggregated human IgG (AHG) occurs also via receptors for C1q. Evidence supporting this conclusion included: (1) Binding of AHG to Raji cells takes place after incubation in EDTA serum; (2) Binding of AHG does not occur in C1q deficient EDTA serum but does take place after addition of C1q; (3) The extent of binding of AHG in EDTA serum is a function of the amount of C1q present; (4) Raji cells can bind up to 5-4 times 10(5) molecules of 125I C1q per cell which can be blocked by unlabelled C1q; (5) AHG pre-incubated with C can bind to a T-cell line MOLT, which lacks receptors for C3b but possesses receptors for C1q to the same extent as Raji cells; (6) Immunoassays for immune complexes in human sera yield similar results whether Raji cells, MOLT cells or C1q precipitation is used for assay; (7) EAC-Raji cell rosettes can be inhibited with inulin-treated, C1q deficient serum containing C3b or C3d whereas binding of AHG or immune complexes in patient samples to Raji or MOLT cells is not inhibited by this reagent. We conclude that receptors for C1q on certain B and T lymphocytes may play an important role in physiologic functions of lymphocytes depending on binding of soluble immune complexes to their surfaces.


Specificity and genetic restrictions of the guinea-pig immune response to dinitrophenyl-lysyl-alanyl octapeptides.

A series of 2,4-dinitrophenyl (DNP) octapeptides containing L-lysine and L-alanine were prepared to examine the specificity and genetic restrictions of both cellular and humoral immune responses in inbred guinea-pigs. Strains 2 and 13 guinea-pigs were therefore immunized with Lys4-Ala3-Lys(DNP), Lys3Ala4-Lys(DNP), Lys2-Ala5-Lys(DNP) and Lys-Ala6-Lys(DNP). Only Lys4-Ala3-Lys(DNP) was under Ir gene control and could induce both antibody and T-cell responses in strain 2 guinea-pigs. In contrast, Lys4-Ala3-Lys(DNP) injected in strain 13 guinea-pigs and the other DNP-octapeptides injected in strain 2 or 13 guinea-pigs elicited only antibody formation and no specific T-cell mediated response. Antibody formed in the absence of specific T-cell responsiveness in either strain 2 or 13 was hapten specific and lacked the capacity to discriminate the immunizing antigen from closely related DNP-peptides. Antibody produced by animals with specific T-cell responses, on the other hand, was exquisitely specific for the immunizing peptide and could discriminate it from closely related peptides.


[Clinical studies on the concentration of free amino acids in plasma and urine during post-aggression metabolism. II].

Our first report concerned the changes in the concentration of free plasma amino acids within the framework of postaggression metabolism, resulting from surgical intervention and parenteral nutrition. This study with 23 operated patients is intended to show whether an amino acid mixture is suited for parenteral nutrition if the particular metabolic action mentioned above is taken into consideration in the composition of the solution. We have shown that the change in the concentration of free plasma amino acids is very small during continuous infusion for 4 postoperative days in steady state and insignificant in comparison to the normal pattern of healthy subjects, except for phenylalanine. With a quota of 2.75% of the supply, the renal loss of free amino acids is clinically unimportant. We believe that an amino acid solution of such composition is suited for infusion in the postoperative and posttraumatic phase whereby our recommendation is explicitly limited to this indication.


[Aimed substitution of zinc, copper, magnesium and inorganic phosphates during postoperative parenteral feeding].

In 20 patients, the serum level and urinary output of zinc, magnesium, copper, calcium and anorganic phosphate were examined during postoperative parenteral feeding. The parenteral feeding was standardized. In addition, the above mentioned bioelements were systematically substituted. Effects of this substitution therapy on serum level and urinary output are discussed.


[Effect of postoperative infusion and transfusion therapy on the serum level and renal losses of zinc, magnesium, copper, calcium and phosphates].

In 52 adult patients, serum levels and renal loss of zinc, magnesium, calcium, copper and anorganic phosphate were examined during the period of parenteral postoperative nutrition. Simultaneously performed analyses of applied infusion and protein solutions, as well as of conserved blood, yielded a markedly differing content of these bioelements. Particularly pronounced are the differences in zinc concentrations of individual infusion solutions. A random substitution of zinc and anorganic phosphate is insufficient since it does not correspond with the demand. For this reason, anorganic phosphate and zinc should only be purposively substituted.


[Quality and quantity of the amino acid pattern in protracted total parenteral hyperalimentation of newborns after surgery].

The determination of plasma amino acids is of utmost importance in the newborn, since there is little information about cumulation or chronic deficiency in long-term parenteral nutrition, in particular in the operated neonate. Two patients were investigated, the first of which was subject to total parenteral nutrition for 115, the second for 56 consecutive days. The plasma levels of amino acids are discussed in relation to clinical findings and nitrogen-balances. The importance of an adequate administration of trace minerals is stressed.


Surgical treatment of nasopalatine duct cysts. A follow-up study.

In a clinical-radiological study, 43 operated and histopathologically verified incisive canal cysts were subjected to follow-up investigation. The clinical investigation revealed no subjective symptoms. Abnormalities of the sensibility to the anterior part of the palate were noted in four cases, hyposensibility in three cases and hypersensibility in the fourth. The radiological investigation showed that bone regeneration occurred 1 year postoperatively at the earliest. After 3 years, two-thirds of the patients showed complete bone regeneration. Large cysts (diameter greater than 10 mm) showed the same tendency for bone regeneration as small cysts. The five patients in whom no bone regeneration had occurred were subjected to reoperation. The histologic investigation of these patients revealed scar tissue.


Evaluation of autotransplantations of completely developed maxillary canines.

Transplantation of 28 maxillary canines was followed up in 22 patients with an average age of 25.2 s.d. 9.9 years. The root was fully developed. The mean immobilization time was 6.6 weeks and the mean follow-up period 17.8 months. Results seemed to be better in the age group of 13--20 years than in 21--30 and 31--47 years. Statistically significant better results were found in the youngest than in the older groups in the return of vitality, vertical bone resorption, periodontal space in the lamina dura. Four of the 28 teeth were extracted because of great mobility caused by poor bone regeneration and vertical resorption of the alveolar bone: three in the middle age group and one in the oldest. Pulp extirpation was performed on nine teeth: six in the oldest group and three in the middle group. Vitality returned in only three teeth, all of which belonged to the youngest group. Root resorption was observed in all age groups; in 76% of the cases it occurred in the apical third, and was generally inflammatory. The causes of the failure of transplantation were considered to include damage of the transplant during removal from deep palatal malposition, poor regeneration of the bone around the transplant and chronic periodontal infection. The prognosis for transplantation was found to be fairly good for patients under 20 years of age but poorer for older age groups.


Concentrations of azidocillin, erythromycin, doxycycline and clindamycin in human mandibular bone.

Postoperative complications after surgery in the mandible are still a clinical problem. Levels of four antibiotics--azidocillin, erythromycin, doxycycline and clindamycin--were measured in serum, dental alveolar serum, saliva and mandibular bone in 24 patients undergoing surgical removal of mandibular third molars. The concentration in mandibular bone for azidocillin was 0.8 microgram/g +/- 0.4 microgram/g, erythromycin 0.2 microgram/g +/- 0.1 microgram/g, doxycycline 2.6 microgram/g +/- 2 microgram/g and clindamycin 0.6 microgram/g +/- 0.4 microgram/g. No saliva levels were achieved with azidocillin and erythromycin, whereas doxycycline and clindamycin gave measurable saliva levels.


Prevention of postextraction bacteremia by local prophylaxis.

Four local prophylactic methods were studied in the prevention of postextraction bacteremia: 1) mouth rinsing with 1% iodine solution, postoperative bacteremia frequency 55%, 2) operative field isolation with cotton rolls and saliva ejector, bacteremia frequency 34%, 3) operative field isolation and disinfection with 10% iodine solution, bacteremia frequency 32%, 4) operative field isolation and disinfection with 0.5% chlorhexidine solution, bacteremia frequency 13%. The operative field isolation together with local disinfection with chlorhexidine was recommended for routine practice. However, when dealing with known or suspected risk group patients, a combined systemic and local prophylactic method was strongly recommended, as this was found in previous investigation to be the most effective method for reducing the frequency of bacteremia.


Melanoma of the face and mouth in Nigeria. A review and comment on three cases.

The absolute incidence of melanoma at all sites in blacks is higher than is commonly credited. There is a shift in site incidence to less pigmented areas including the mucosae but oral melanoma is still rarely reported in Africans. An unusual case of facial melanoma arising in a childhood naevus, and two oral melanomas, all in Nigerians, are described. The salient clinical features and the treatment of these cases are discussed, and the possible relationship between pigmented patches and melanomas in the mouths of blacks is examined.


Progeston therapy for menstrually related aphthae.

Ten female patients with a history of recurrent aphthous lesions for at least 5 years with the lesions occurring regularly in the latter half at the menstrual cycle were treated with progestogen in the form of an intramuscular depot injection. Medroxyprogesterone acetate and gestronol hexanoate was used. All 10 patients were satisfied with the treatment; the side effects were few.


Ultrastructure of tissue in intrabony and subperiosteal porous ceramic implants.

Porous ceramic (Al2O3) implants with pore size of 100--750 micron were placed intrabony in the maxilla and subperiosteally in the mandible of three adult monkeys. After observation periods of 10, 11 and 12 months, the animals were perfused with 1.7% glutaraldehyde and the jaws processed for electron microscopy. Tissue ingrowth into the pores was observed in all the specimens. The implant pores facing the host bone contained bone tissue to a depth of approximately 1 mm, while only unmineralized tissue was found in the implant part adjacent to the periosteum. The unmineralized tissue was of a fibrous nature with collagen fibers arranged in a lamellar fashion. Cells, often with extremely long and slender cellular processes, were seen between the lamellae. The cells had the cytoplasmic features typical of protein-producing cells. The mineralized tissue exhibited ordinary bone tissue structure with plate-like crystallites in a collagenous matrix. Some lacunae and canaliculi with osteocytes were observed, though they were scarce.


Alveolar atrophy and decreased skeletal mass of the radius.

Using absorptiometry, the bone calcium mass (BCM) at two different sites of the radius was measured in 12 male patients referred for vestibuloplasty of the mandible. The most distal site contained both cortical and cancellous bone while the other site contained cortical bone only. The BCM values were compared with those for normal patients of the same age. The patients with mandibular atrophy all had less bone in the radius than their age-matched controls (p less than 0.01). This is indicative of a systemic factor or factors operating both in the mandible and in the radius. The examination prior to oral surgery should therefore include an evaluation for diseases causing general bone loss.


Use of a miniaturized detector in facial bone scanning.

A high resolution cadmium telluride probe was used in conjunction with standard bone imaging methods in two cases of facial asymmetry. The probe improved localization of the abnormalities and led to rational treatment.


Disphosphonate dynamic imaging of experimental bone grafts and soft tissue injury. Radionuclide bone imaging.

Bone imaging provides two significant advantages over previously available methods for assessing bone processes. First, as scanning is non-invasive, it offers the operator the ability to sequentially follow any area of bone without altering it as would a biopsy. The second advantage is the ability of bone imaging to provide current information about the bone being scanned rather than having to deal with the delay that is inherent in conventional radiography. Recent developments in radiopharmaceuticals and instrumentation have made bone imaging a potentially practical clinical tool for the detection and assessment of benign as well as malignant processes affecting bone. This paper presents the results of the use of technetium 99m diphosphonate bone imaging in experimental canine mandibular autografts and surgical soft tissue injury. The implication of these results for the clinical situation will be discussed.


Intraosseous neurofibroma of the jaws.

Two cases of intraosseous neurofibromas of the mandible are presented. Only 25 cases of this tumor have been published previously. Of the present two cases, one was located at the upper border of the mandible, in the left second molar area where the tooth had been extracted 2 years previously. In the second case, the tumor presented itself as a 10 x 2 cm large destruction, mainly following the course of the alveolar nerve from the mandibular foramen to the mental foramen in the right side of the jaw. The mandibular first right molar was missing but the relationship between the extraction of the tooth and the development of the tumor could not be demonstrated. Histologically, the two tumors differed in the way that case 1 showed a conspicuous amount of delicate, wavy fibrils, some of which showed concentric lamellations resembling the pacinian corpuscle but without neurites containing nerve fiber bundles and also demonstrated a slight pleomorphism.


Giant ranula causing mandibular prognathism.

This is a case report of a 20-year-old man with ranula, the size of an orange, in the floor of the mouth causing mandibular prognathism with fan-shaped mandibular teeth anterior to the premolars. The tumor was extirpated. The pathogenesis, differential diagnosis and treatment of ranulas are discussed.


Transformation of ameloblastic fibroma to fibrosarcoma.

The direct transformation of an ameloblastic fibroma into a fibrosarcoma in a 16-year-old Caucasian male is reported. Although no ameloblastic epithelium was found in the recurrent tumor, the odontogenic origin of the fibrosarcoma was evident. The ameloblastic fibrosarcoma and the fibrosarcoma of identical odontogenic origin represent an entity which should be distinguished from conventional fibrosarcoma as these tumors demonstrate different clinical behaviors.


Chloroquine retinopathy in the rhesus monkey.

Chloroquine was administered intramuscularly 5 days a week to rhesus monkeys for as long as 4 1/2 years. No clinical, fluorescein angiographic, or electrophysiological evidence of retinal damage was observed. Yet chloroquine/chloroquine byproduct analysis of the ocular tissues revealed an enormous binding capacity of the pigmented tissues of the eye (choroid plus RPE, ciliary body, and iris) with eventual accumulation observed in the retina. Despite the normal ophthalmic appearance and function, extensive pathological changes occurred in the retinas and choroids of these experimental monkeys. The chloroquine caused an initial dramatic effect on the ganglion cells, with the photoreceptors affected shortly thereafter. Patching degeneration of the ganglion cells and photoreceptors then progressed over several years, with the choroid and pigment epithelium ultimately deteriorating as well.


[Antibiotic prophylaxis: definition and documentation].

Controlled, randomized and prospective studies were reviewed to evaluate the value of antibiotic prophylaxis in colonic and biliary tract surgery as well as in the treatment of open fractures. --Colorectal surgery: 7 studies using oral and 3 using parenteral antibiotics in addition to extensive bowel preparation have demonstrated the importance of a short term prophylaxis, instituted before surgery and discontinued soon thereafter. Different antibiotics with aerobic or anaerobic spectra reduced the incidence of wound infections significantly; further improvement could be expected from combinations covering both spectra. --Biliary surgery: cefazolin, gentamycin and rifamycin--used in four controlled studies--were effective in reducing infectious complications. No distinction was made between patients of different infectious risks. In low risk gallbladder surgery the real value of antimicrobial prophylaxis is unknown. Short term prophylaxis is recommendable in all high risk patients undergoing surgery and in the low risk case submitted to extensive interventions. --Open fractures: only three authors have submitted their patients to controlled studies. All protocols suffered from some bias. No definite conclusions can be made. The authors' personal recommendations are: no prophylaxis in first degree injuries; early preoperative administration of penicillinase-resistant drugs in second and third degree lesions.


Aqueous aldehyde (Faglu) methods for the fluorescence histochemical localization of catecholamines and for ultrastructural studies of central nervous tissue.

Aqueous solutions combining a high concentration of formaldehyde (4%) with low concentrations of glutaraldehyde (0.5--01%) have been used to simultaneously localize amines by the formation of fluorescent products and to fix central nervous tissue for electron microscopy. The fluorescence reaction is produced by the aldehyde mixture at room temperature and the fluorescence is stable when the tissue is maintained in aqueous solution. This means that nerve cell bodies and terminal fields which contain catecholamines can be located accurately in vibratome sections at the light microscope level and, after further processing, can be examined under the electron microscope. With 1% glutaraldehyde in the aldehyde mixture, ultrastructural details are well preserved; there is no significant distortion of any component of the tissue. If vibratome or cryostat sections are dried against glass slides, the intensity of the fluorescence reaction is enhanced and the sections can be permanently mounted.


Fine structure of dense-cored vesicles in glomus cells of rat carotid body after fixation with permanganate or glutaraldehyde.

Glomus (Type I) cells of the carotid body of adult rats were studied electron microscopically after fixation with potassium permanganate or with glutaraldehyde and osmium tetroxide. Two permanganate fixation methods (using Krebs-Ringer-glucose, pH 7.0, or acetate buffer, pH 5.0) were compared. Numerous dense-cored vesicles were observed only in about one tenth of the glomus cells when neutral permanganate was used for fixation, although all glomus cells showed such vesicles after fixation with glutaraldehyde and osmium tetroxide. Numerous vesicles with a dense core were observed in about one third of the cells after fixation with acid potassium permanganate. With this fixation, small dense-cored vesicles similar to those in adrenergic nerve terminals were occasionally seen in the cytoplasm of glomus cells. It is tentatively concluded that the amine-storing vesicles of the carotid body are different from those in the small intensely fluorescent (SIF) cells and those in adrenergic nerve terminals.


[Late results of surgery of the pharyngeal pouch (author's transl)].

Surgery of the posterior diverticulum of the hypopharynx is almost exclusively carried out today from an external approach. 19 patients, of whom 15 were surgically treated by invagination of the pouch, are presented and over a ten year period none have recurred. The indications and advantages of this surgical procedure are described and discussed.


The ultrastructural organization of the contractile peripheral protein layer of the human erythroycte membrane.

Erythroycte ghosts fixed in glutaraldehyde were dehydrated in (a) alcohol or acetone, (b) propylene glycol followed by Epon and embedded in an epoxy resin. A water-soluble urea/glutaraldehyde mixture was also used. The aim was to study the structure of the peripheral protein layer, which contains spectrin and actin, in the absence of OsO4 induced denaturation changes. Ghost membranes prepared in this way had an asymmetrical quadrilaminar structure. A layer of amorphous peripheral protein +/- 18 nm in width covered the entire inner face of the membrane in the form of a coarse meshwork in both Wash I (haemoglobin-containing) and haemoglobin-free ghosts. Cations (Mg2+ or Ca2+, or Mg2+ plus ATP) had no apparent effect on its fine structure. In contrast, the corresponding layer in OsO4-fixed membranes was represented by scanty, fuzzy material attached to the unit membrane only at irregular intervals. The results demonstrate the superior ability of glutaraldehyde to preserve the peripheral protein layer in thin sections, and afford further support for the view that much of this protein normally exists in an unpolymerized state.


The distribution of the chorda tympani in the middle ear area in man and two other primates.

A serial section study of the distribution of the chorda tympani in the middle ear area was carried out in man, baboon and monkey. The tissues innervated by the chorda tympani could be related to a branchiomeric pattern. The early branches distributed post-trematic facial nerve fibres to hyoid arch tissues, where they were joined by elements from glossopharyngeal and vagus nerves. The rest of the distribution was to structures derived from mandibular arch tissue where branches of the auriculotemporal nerve were also present. Contributions to perivascular plexuses were noted as well as a connexion with the otic ganglion.


Clavulanic acid inhibition of beta-lactamase I from Bacillus cereus 569/H.

Inactivation of beta-lactamase I by clavulanic acid was investigated. Clavulanic acid induced inhibition of the enzyme was found to be progressive with time. Benzylpenicillin provided protection against the adverse effects of the inhibitor initially, however, the enzyme was irreversibly inhibited in a progressive manner even in the presence of substrate. Reaction of beta-lactamase I with clavulanic acid, in the presence of ampicillin, led to a very rapid inactivation of the enzyme.


In vitro activity of HR 756, a new cephalosporin compound.

The in vitro activity of HR 756, a new cephalosporin, has been determined against recent clinical isolates and compared with that of other beta-lactam antibiotics. The geometric means of the minimum inhibitory concentrations (MIC) for different isolates of Escherichia coli (100 isolates), Klebsiella pneumoniae (84), Pseudomonas aeruginosa (121), Proteus mirabilis (52), indole-positive Proteus species (9), Salmonella species (19), Staphylococcus aureus penicillin-sensitive (29) and penicillin-resistant (39) were: 0.095, 0.124, 11.1, 0.095, 0.0107, 0.078, 1 and 0.95 mcg/ml, respectively. Its activity was affected by rise in inoculum against S. aureus and P. aeruginosa but not against K. pneumoniae and E. coli. Bactericidal activity was determined by membrane filtration method. HR 756 was found to be bactericidal to E. coli, K. pneumoniae, P. aeruginosa and Proteus species. Although the MICs of the tested S. aureus strains were 1 mcg/ml, 5 mcg/ml of HR 756 failed to kill 99% of the inoculum within 24 hours.


Incorporation of 32Pi into nucleotides, polyphosphates, and other acid-soluble compounds by Myxococcus xanthus during myxospore formation.

When glycerol was used to induce myxospore formation in Myxococcu xanthus in the presence of 32Pi, the label was incorporated into a variety of acid-soluble compounds. Incorporation into ribonucleotides was approximately fivefold greater than in vegetative cells or noninducible mutants grown in glycerol. The label was also incorporated into some unknown compounds and material tentatively identified as guanosine tetraphosphate. Marked accumulation into polyphosphates, which were present mainly in culture supernatants, occurred relatively late during myxospore formation. The kinetics of accumulation of some of these compounds and their distribution into acid-soluble cell extracts and culture supernatants are described and compared with those in vegetative cells and noninducible mutants.


Heat-modifiable outer membrane proteins of Neisseria meningitidis and their organization within the membrane.

Neisseria meningitidis group B serotype 2 strain M986 contains two predominant outer membrane proteins, with apparent molecular weights of 41,000 (protein b) and 28,000 (protein e). Heating of outer membrane vesicles at 56 degrees C for 20 min caused much of b** to disaggregate and denature into b (41,000 daltons). In contrast, protein e could be rapidly solubilized by SDS at room temperature into its monomeric state (e*), but it was not converted to its final higher apparent molecular weight of 28,000 (e) unless heated at 100 degrees C for 2 min. We propose that protein b exists in the membrane as trimers or tetramers in a transmembrane configuration and that protein e exists as subunits on the exterior surface of the outer membrane and has a highly ordered tertiary structure.


An explanation for the apparent host specificity of Pseudomonas plasmid R91 expression.

Pseudomonas aeruginosa strain 9169 has been reported to contain a plasmid that expresses resistance to carbenicillin (Cb), kanamycin (Km), and tetracycline (Tc) in Escherichia coli but resistance only to Cb in certain Pseudomonas recipients. The triply resistant plasmid in E. coli belonged to incompatibility (Inc) group P or P-1, whereas the singly resistant plasmid in P. aeruginosa was compatible with IncP-1 plasmids and other plasmids of established Inc specificity but incompatible with plasmid pSR1 that is here used to define a new Pseudomonas Inc group P-10. Additional physical and genetic studies showed that strain 9169 contained not one but two plasmids: IncP-1 plasmid R91a, determining the Cb Km Tc phenotype, and IncP-10 plasmid R91, determining Cb that differed in molecular weight and in EcoRI and BamHI restriction endonuclease recognition sites. Plasmid multiplicity rather than host effects on plasmid gene expression can account for differences in the phenotype of strain 9169 transconjugants to E. coli and P. aeruginosa.


Expression of Escherichia coli K-12 arginine genes in Pseudomonas fluorescens.

Escherichia coli argE and argH gene products were detected in Pseudomonas fluorescens argH122 carrying the E. coli F110 plasmid.


Differential effect of hydroxyurea on the replication of plasmid and chromosomal DNA in Bacillus subtilis.

The replication in Bacillus subtilis of the staphylococcal R plasmids pE194, pBD15, pUB110, pSA0501, and pSA2100 has been studied in the presence of hydroxyurea. In all cases, an enrichment for covalently closed circular DNA compared with chromosomal DNA was observed. In this respect, hydroxyurea mimics the effect previously observed with pUB110, using strains carrying the conditional mutation dnaA13. This mutation has been reported to affect ribonucleotide reductase (G. W. Bazill and D. Karamata, Mol. Gen. Genet. 117:19-29, 1972). An explanation for these effects is offered, together with some supporting evidence.


Alterations in Bacillus subtilis transforming DNA induced by beta-propiolactone and 1,3-propane sultone, two mutagenic and carcinogenic alkylating agents.

Transforming DNA was exposed to either beta-propiolactone or 1,3-propane sultone and then used for transformation of competent bacteria to nutritional independence from tyrosine and tryptophan (linked markers) and leucine (an unlinked marker). The ability to transform was progressively lost by the DNA during incubation with either of these two chemicals. For all three markers the inactivation curve was biphasic, with a short period of rapid inactivation followed by one characterized by a much slower rate. The overall rate of inactivation was different for all three markers and presumably was related to the size of the marker. The decrease in the transforming activity was in part due to the slower rate of penetration of alkylated DNA through the cellular membrane and its inability to enter the recipient bacteria. This decrease in the rate of cellular uptake, even for DNA eventually destined to enter the cell, began almost immediately after its exposure to the chemical and ended up with an almost complete lack of recognition of the heavily alkylated DNA by the specific surface receptors of competent cells. Such DNA attached to sites on the surface of competent bacteria which were different from receptors specific for the untreated nucleic acid. This attachment was not followed by uptake of the altered DNA. Presence of albumin during the incubation with a carcinogen further increased the degree of inactivation, indicating that the artificial nucleoproteins produced under such conditions were less efficient in the transformation assay than was the naked DNA. Cotransfomration of close markers progressively decreased, beginning immediately after the start of incubation of DNA with the chemicals. Extensively alkylated DNA fractionated by sedimentation through sucrose density gradients showed a peculiar distribution of cotransforming activity for such markers; namely, molecules larger than the bulk of DNA ("megamolecules") showed less ability to transform the second marker than did some of the apparently smaller molecules which sedimented more slowly through the gradient. An increase in cotransformation of distant markers was evident in DNA molecules after a short exposure to an alkylating agent, but cotransformation of such markers was absent in DNA treated for longer periods. The observed changes in the transforming and cotransforming activities of the alkylated DNA can be explained by what is known about the physicochemistry of such DNA and in particular about the propensity of the alkylated and broken molecules to form complexes with themselves and with other macromolecules.


Suppression of temperature-sensitive sporulation of a Bacillus subtilis elongation factor G mutant by RNA polymerase mutations.

A class of rifampin-resistant (rfm) mutations of Bacillus subtilis suppresses the temperature-sensitive sporulation of a fusidic acid-resistant mutant. FUS426, which has an altered elongation factor G. The rfm mutation suppressed only the sporulation defect caused by the elongation factor G mutation, but could not suppress other types of induced sporulation defects. Genetic and biochemical analyses showed that the sporulation suppression by the rfm mutation was caused by a single mutation in RNA polymerase. After the early sporulation phase, the apparent rate of RNA synthesis of FUS426, measured by [3H]uracil or [3H]uridine incorporation into RNA, became lower than that of the wild-type strain, and this decrease was reversed by the rfm mutation. However, when the total rate of RNA synthesis of FUS426 was calculated by measuring the specific activity of [3H]UTP and [3H]CTP, it was higher than that of the rfm mutant, RIF122FUS426. The possible mechanism of the functional interaction between elongation factor G and RNA polymerase during sporulation is discussed.


Cell wall assembly in Bacillus subtilis: location of wall material incorporated during pulsed release of phosphate limitation, its accessibility to bacteriophages and concanavalin A, and its susceptibility to turnover.

Addition of a pulse of phosphate to a phosphate-limited chemostat culture of Bacillus subtilis W23 led to the synthesis of teichoic acid and the consequent development by the bacteria of the ability to bind phage SP50. In cultures growing at different rates, phage-binding properties became maximal approximately one generation time after addition of the pulse. Removal of the incorporated teichoic acid by turnover also reached its maximum rate after a similar interval. After pulsed release of phosphate limitation in B. subtilis NCTC 3610, the alpha-glucosyl residues of the incorporated teichoic acid, detected by their interaction with concanavalin A, became maximally exposed at the same time that phage binding was maximum. At that time the bacteria bound phage all over the cylindrical part of the surface and at about one-third of the polar caps. That fraction of the receptor material that is exposed soon after its incorporation was distributed along the cylindrical length of most of the bacteria, but few phages bound to the polar caps, except in the case of short bacteria; these bound phages in a markedly asymmetric manner at one pole and along their length. The significance of these results is discussed in relation to the mode of assembly of the cell wall.


Kinetic specificities of BPN' and Carlsberg subtilisins. Mapping the aromatic binding site.

The kinetic specificities of BPN' and Carlsberg subtilisins [EC 3.4.21.14] were examined with various nucleus-substituted derivatives of Nalpha-acetylated aromatic amino acid methyl esters for mapping their hydrophobic binding sites in comparison with that of alpha-chymotrypsin. The Carlsberg enzyme was generally much more reactive than the BPN' enzyme due to the larger kcat value. The fact that the two sutilisins hydrolyzed Ac-Tyr(PABz)-OMe, which is a derivative of tyrosine bearing a planar trans-p-phenylazobenzoyl group at the OH-function, with the smallest Km value showed that these enzymes possess a more extended aromatic binding site than has so far been demonstrated. Ac-Phe(4-NO2)-OMe was remarkable in being hydrolyzed with a particularly large kcat value (5,500 +/- 700 s-1 at pH 7.8 for Carlsberg subtilisin). Ac-Phe(4-NO2)-OMe and Ac-Tyr-OMe were distinguished by Carlsberg subtilisin in terms of kcat but not by BPN' subtilisin, suggesting that the specificity site of the former is more sensitive to a small change in size of substituent than that of the latter. Ac-Trp(NCps)-OMe and Ac-Trp(NCps)-OH were bound to the enzyme's active site but in a competitive manner. A difference in the standard free energies of binding between the two enzymes may indicate that the hydrophobic cleft of Carlsberg subtilisin is somewhat deeper and/or narrower than that of BPN' subtilisin.


Fluorometric measurement of urinary alpha-L-iduronidase activity.

A fluorogenic substrate for alpha-L-iduronidase, 4-methylumbelliferyl alpha-L-iduronide, has been newly synthesized and the enzyme activity has been measured in urine samples obtained from normal persons and patients suffering from mucopolysaccharidosis. Urine samples derived from a patient with Scheie syndrome showed greatly reduced activity compared with a normal adult at a similar age. This patient exhibited a high level of urinary excretion of dermatan sulfate and heparan sulfate, which could be interpreted in terms of her low alpha-L-iduronidase activity. The use of the fluorogenic substrate has some advantages over existing methods because of the high sensitivity and the relative ease of handling, and it should be useful not only for diagnosis but also for following the purification process of the enzyme.