Antibodies reacting with salmon and human protamines in sera from infertile men and from vasectomized men and monkeys. A micro-complement fixation test was modified for the detection of low-titred antibodies to protamines. Specific immunological reactions with salmon protamine were found with some sera obtained from infertile men and vasectomized men and monkeys. The possibilities that allergic reactions, observed in patients receiving salmon protamine, result from immunity to the fish protamine and from immunological cross-reaction between human and salmon protamines (in cases where auto-immunity to human protamine has developed prior to the administration of fish protamine) are discussed.


An immunoglobulin heavy chain variable region (VH) marker associated with cross-reactive idiotypes in man. The VH specificities of two rabbit antisera raised against the H chains of two human IgM cold agglutinins were studied with the aid of proteins on which the VH sequence data are available. They were found to recognize a new VH antigen (VHMar), which may represent a subgroup of VHI. This antigen is found on anti-Ii antibodies which have cross-reactive idiotypes. It is moderately well-expressed in normal gamma globulin and strongly expressed on 8% of unselected myeloma and macroglobulinaemia proteins. There is evidence to suggest that a proportion of the cross-reactive idiotypes among the anti-Ii antibodies involve this VH antigen.


Cell-mediated immunity in Chagas' disease: Alterations induced by treatment with a trypanocidal drug (nifurtimox). Peripheral leucocyte migration inhibition (LMI) with Trypanosoma cruzi-specific antigens, measured as a migration index (MI), was studied in chronic Chagas' disease patients. The MI of untreated patients with polymerized antigens from culture forms (epimastigotes) of T. cruzi was significantly lower than that of controls. In contrast, when chronic Chagas' patients were treated with nifurtimox, 10 mg/kg/day for 2 months, the MI was not different from control values. Treated and untreated patients had normal T- and B-lymphocyte markers, measured by the ability to form rosettes either with sheep erythrocytes (E-RFC) or with sheep erythrocytes--antibody--complement (EAC-RFC). In addition, the number of lymphocytes bearing surface membrane Ig (SMIg) was the same as that of controls. Non-specific functional assays, such as PHA-induced blastogenesis and antibody-dependent cell-mediated cytotoxicity (ADCC) to sensitized chicken erythrocytes were also normal, both in treated and untreated patients. Thus, nifurtimox produced a particularly effect on cell-mediated immunity, specially detectable using LMI.


Kidney lesions in baboons infected with Schistosoma mansoni. Glomerular lesions in baboons (Papio anubis) infected with different dosage regimes of Schistosoma mansoni were studied by immunofluorescence and light microscopy on kidney sections and by countercurrent immunoelectrophoresis on kidney homogenates and tissue eluates. Mild lesions, characterized by focal and segmental deposits of immune complexes, developed in sixty-two out of 103 baboons, irrespective of the intensity and duration of the infection. Severe, diffuse lesions developed in six baboons after prolonged and heavy infections. Adult worm and soluble egg antigens, together with IgM, IgG and C3, were detected in most of the severe lesions and in some of the mild lesions. In some animals, antigens were detected in most of the severe lesions and in some of the mild lesions. In some animals, antigens were detected in acid homogenates and eluates of kidneys which showed no deposits of immunoglobulins or complement. These observations indicate that renal lesions in S. mansoni infections may be attributable to the deposition of immune complexes pre-formed in the circulation. However, the demonstration of antigens alone in some animals may suggest an alternative possibility, namely that antigens are deposited first with a subsequent binding of antibody and complement.


Structure and biological functions of human IgD. XII. Anti-IgD enhancement of PHA responsiveness in patients with chronic lymphocytic leukaemia. Specifically purifed anti-human delta stimulated the in vitro incorporation of [3H]thymidine by human peripheral lymphocytes from patients with chronic lymphocytic leukaemia (CLL). The peak response varied between individuals; those with 5--52% IgD-bearing lymphocytes exhibited maximum stimulation at 3 days, whereas a patient with only 1% IgD-bearing cells showed optimal activation at 6 days. In agreement with others, our data indicated that, in most instances, lymphocytes from patients with CLL respond poorly to PHA. One of the most important findings in this study is the enhancement of PHA responsiveness by anti-delta. Lymphocytes that exhibited reduced responsiveness to PHA alone, when pre-treated with anti-delta, showed transformation greater than the sum of the anti-delta plus PHA responses.


Nifurtimox-induced alterations in the cell-mediated immune response to PPD tin guinea-pigs. Positive skin reactions to PPD in guinea-pigs immunized with Freund's complete adjuvant (FCA) were reversed after treatment with 10 mg/kg/day nifurtimox for 12 days. The in vitro migration of peripheral blood leucocytes from FCA-immunized guinea-pigs was inhibited with PPD, but it returned to normal values after nifurtimox treatment. Furthermore, the cell-free supernatant from PPD-stimulated lymphocytes from FCA-immunized nifurtimox-treated guinea-pigs did not inhibit the migration of normal cells. Thus the administration of nifurtimox impaired the specific cell-mediated immune response to PPD both in vivo and in vitro.


Fibrodysplasia (myositis) ossificans progressiva treated with disodium etidronate. Two patients with fibrodysplasia ossificans progressiva (FOP) were treated with disodium etidronate, a disodium salt of ethane-1-hydroxy-1, 1-diphosphonic acid (EHPD) for two years; both had multiple ectopic ossifications of connective tissue. Regular follow-up showed no improvement in joint movement whilst laboratory examination revealed an increase in the serum phosphorus level. Serum calcium and urinary phosphorus excretion remained within normal limits. The urinary calcium excretion varied with the dose of EHDP. X-ray examination showed significant reduction in size and density of the ectopic ossifications, while the density of bones during and after EHDP therapy remained normal. Development of new ossification was inhibited in the active phase of the disease. Suspected side-effects of the drug were occult blood in stools, skin petechiae, and amenorrhoea. Previous reports on the EHDP treatment of disorders with ectopic ossification are summarised.


Visuo-spatial performance following posterior parietal and lateral frontal lesions in stumptail macaques. Three groups of four monkeys were trained to negotiate a small hand ("stylus") maze, and to use a "pointer" to guide response in a two-choice position discrimination task. One group was given bilateral lesions of posterior parietal cortex, and the second bilateral lesions of lateral frontal cortex. Postoperative impairment on the maze was evident in three of the four parietal animals, and both operated groups showed impairment on the pointer task. Since maze performance was positively associated with speed of stylus recovery in a control version of the task but was not correlated with pointer task performance, it is suggested that the parietal deficit resulted primarily from a sensory-motor dyscoordination rather than a spatial perceptual disorder. The latter could however be the cause of parietal impairment on the pointer task. Error analysis suggests an interpretation of the frontal deficit on the pointer task in terms of perseveration on position.


Form perception in stumptail macaques following posterior parietal and lateral frontal lesions. Monkeys with bilateral posterior parietal and lateral frontal ablations were compared with normal animals on a series of four tests for their ability to relate elements of a visual pattern on the basis of the Gestalt principle of proximity. The operated animals showed no clear impairment on these tests of perceptual grouping. The three groups of monkeys were then trained to discriminate two faces and tested for generalisation to different views of the same faces. The pariental-operated monkeys showed less generalisation than the other animals to lateral mirror-images of the training stimuli, but more generalisation to inverted mirror-images. It is concluded that any deficit in "gestalt" perception following such lesions must be at a relatively high level.


Reaction time variability in epileptic and brain-damaged patients. Median reaction times and intra-individual variability were studied in epileptic (N = 63), brain-damaged (non-epileptic) (N = 25) and control patients (N = 25) using a six and one half minute visual, continuous reaction time task. Epileptic and brain-damaged groups were significantly slower than control patients on median reaction times at the 10th, 50th, and 90th percentiles and on the differences between the 10th and 90th percentiles. Thus both general slowing and greater intra-individual variability were found in the epileptic and brain-damaged patients. Reaction times were not related to presence, type and severity of EEG abnormality or to age of onset of epilepsy. Grand mal patients did have significantly greater variability than other types of seizure patients. Epileptic and brain-damaged patients did not differ significantly on any reaction time variables. Both groups were discriminated significantly from the controls on all reaction time measures, especially on the intra-individual variability measure.


The chromosomal location of rDNA in selected lower primates. Hybridization in stiu was used to identify the chromosomes that carry rDNA in representative lower primates, including the baboons, Papio cynocephalus and Papio hamadryas; the colobus monkey, Colobus polykomos; the tree shrew, Tupaia glis; the lemur, Lemur fulvis; the saki, Pithecia pithecia; the marmoset, Saguinus nigricollis, and the spider monkey, Ateles geoffroyi. The marker chromosome, common to the Cercopithecines studied to date, carries the rDNA in the baboons. Another marker chromosome carries rDNA in a South American species, the spider monkey. A multichromosomal distribution of rDNA was demonstrated in the tree shrew, lemur, saki, and marmoset. None of the rDNA-containing chromosomes in the prosimians and New World monkeys show homology to the chromosomes that carry rDNA in the Hominids, Pongids, or Old World monkeys.


RNA polymerase B (or II) in heat induced puffs of Drosophila polytene chromosomes. Using indirect immunofluorescence visualization techniques we investigated the distribution of RNA polymerase B (or II) and histone H1 at heat shock puff loci in Drosophila melanogaster polytene chromosomes at different times during and after heat shock. After heat treatments of from 5 to 45 min, the heat shock puff displayed intense fluorescence when stained for RNA polymerase B, but relatively little fluorescence when stained for histone H1. Returning heat shocked larvae to room temperature resulted in the appearance of a distinctive pattern of RNA polymerase-associated fluorescence in the heat shock puff at 87C, presumably reflecting events associated with the inactivation and regression of this puff. Large differences observed in the apparent RNA polymerase B content of puffs of similar size suggest that the interaction of RNA polymerase B with chromosomal loci does not depend on simply the state of condensation or decondensation of the chromatin.


Effects of thyroxine and cold exposure on hypothalamic TRH levels in rats with various pituitary-thyroid states. The hypothalamic content and concentration of thyrotropin-releasing hormone (TRH) were determined by radioimmunoassay in normal, thyroidectomized, hypophysectomized and cold-exposed rats with or without thyroxine. In normal animals, the single administration of thyroxine (1,5 and 20 microgram/100 g B.W.) altered neither the content nor the concentration of TRH in the hypothalamus. However, seven days' administration of this hormone resulted in the dose-dependent increase in the hypothalamic TRH levels. In thyroidectomized rats the hypothalamic TRH levels were slightly reduced in spite of the marked increase of plasma TSH levels and decrease of pituitary TSH levels. In the animals given thyroxine (10 microgram/100 g B.W.) for 7 days in addition to thyroidectomy, however, the TRH levels exceeded that in the animals which underwent throidectomy alone. The hypothalamic TRH levels were markedly reduced in hypophysectomized rats. Conversely, in hypophysectomized rats given 7 days' thyroxine (1 and 5 microgram/100 g B.W.), the levels were increased dose-dependently. In cold-exposed rats, the plasma TSH levels roughly doubled, but the TRH levels remained unchanged. These findings strongly suggest that the feedback site of thyroxine extends not only to the pituitary gland but also to the hypothalamus, and that thyroxine has an increasing effect of the hypothalamic TRH level, though the mechanism(s) remain to be clarified.


Effect of magnesium on epileptic foci.

Effect of magnesium on epileptic foci. Parenteral magnesium sulfate has been used to control acute convulsions of diverse etiologies and is currently a mainstay in controlling eclamptic convulsions. However, the site of this anticonvulsant action has remained controversial and not well studied in experimental epileptic models. Therefore we determined the effects of intravenously infused magnesium sulfate on the epileptic neural activity induced by topical application of penicillin G to motor cortex in anesthetized cats and dogs and in awake, undrugged primates. Magnesium sulfate was able to directly suppress neuronal burst firing and interictal EEG spike generation at serum levels below those producing paralysis. The degree of suppression corresponded to the serum magnesium concentration and was reversible. These results corroborate the clinical observations in patients that magnesium can produce an anticonvulsant effect apart from neuromuscular blockade and suggest it may have clinical applicability in treating a wider range of acute convulsions.


[Isatin-enzyme interactions. VI. Inhibition of rat kidney alkaline phosphatase]. Interaction of isatin with rat kidney alkaline phosphatase has been studied. Mode of attachment of isatin with the enzyme protein is most likely through amino group(s), which is also imperative for catalysis. Sulphydryl group(s) do not seem to be involved in enzyme action. Zinc is also needed for enzyme activity. Use of sulphydryl compounds suggests that isatin inhibition of the enzyme is through attachment at the metal site. However, this inhibition may not only be due to simple chelation of the metal by isatin.


Homoserine dehydrogenase of Rhodospirillum rubrum. Physical and chemical characterization. A detailed physicochemical characterization of purified homoserine dehydrogenase of Rhodospirillum rubrum is presented. The enzyme has a molecular weight of 110000 and consists of two subunits of identical molecular weight of 55000. Depending on the ionic strength and protein concentration it is possible for the native enzyme to dimerize to produce an enzymatically active species of molecular weight 220000. Titrations of the native and detergent-treated enzyme with a variety of sulfhydryl reagents show 2 mol free--SH groups per 110000 g, one of which is buried in the protein interior. L-Threonine and/or high concentrations of salt can expose the buried--SH group, and this--SH group is essential for the catalytic activity of the enzyme. Two independent lines of evidence show that extensive polymerization of the enzyme caused by L-threonine and/or high concentrations of salt does not involve the formation of intermolecular disulfide bonds.


Forssman glycolipid variants of dog gastric mucosa. Structure of a branched ceramide octasaccharide. A fucose-containing ceramide octasaccharide exhibiting Forssman antigenic activity, and reacting in human H anti-H and anti-A systems, was isolated from water-soluble glycolipids of dog gastric mucosa. Defucosylation of the glycolipid resulted in the loss of H-activity, but had no effect on its Forssman nor blood-group A antigenic activity. The branched structure of glycolipid was identified by partial acid hydrolysis, sequential degradation with specific glycosidases and comparison of the permethylation products of the native and enzyme-degraded compound. The structure of this glycolipid is proposed to be: formula.


Purification and characterization of alanine carrier isolated from H-proteins of Bacillus subtilis. Alanine transport carrier was isolated and purified from H-proteins of Bacillus subtilis. The purified carrier preparation was homogeneous in migration on polyacrylamide gels containing urea or sodium dodecyl sulfate. Electrophoresis on polyacrylamide gels containing dodecyl sulfate showed a single band of molecular weight of about 7500. 1 mol alanine was bound/mol carrier protein with a dissociation constant of 0.2 micron. The binding was inhibited by p-chloromercuribenzoate and the inhibition was reversed by dithiothreitol.


A naphthoquinone adsorbent for affinity chromatography of human dihydropteridine reductase. 1. A 1,2-naphthoquinone adsorbent is described which allows simple purification of dihydropteridine reductase directly from crude extract. 2. The native molecular weight indicates that a tetramer structure of the species is isolated by this method; this is unusual and possibly reflects the capacity of the procedure to preserve the native state of the enzyme.


Complement in cystic fibrosis.

Complement in cystic fibrosis. Complement components C3, C4, and C3A were estimated in 30 patients with cystic fibrosis aged 1 to 21 years (M:F = 16:14) and were compared with results in 40 healthy, age-matched subjects. The influences of the clinical score, sputum microbiology, and the patients' sex were also investigated. In contrast to most previous communications, this paper shows that, compared to the control group, a significant decrease of C3 (P less than 0.001) and C4 (P less than 0.02) was observed whereas C3A levels were not altered. There were no increases in complement. Shwachman-scores above or below 70 did not influence the complement levels, nor did exacerbations of the disease change the levels. No influence of the patients' sex could be shown. Pseudomonas aer. in the sputum was clearly associated with complement defects (14/18). Alternative-pathway involvement of complement activation could be demonstrated in 32%. The results make complement activation due to pulmonary infection most likely. The defects observed probably represent secondary changes.


Local synthesis in the central nervous system of diclonal IgM-kappa and homogeneous free kappa light chain proteins in a case of chronic meningoencephalitis. A 60-year-old man with chronic meningoencephalitis and monuclear pleocytosis of the CSF was observed for 22 months. About one half of the CSF cells were concluded to represent IgM-synthesizing lymphocytes and plasma cells. Electrophoretic analysis disclosed two abnormal protein bands in the CSF. The bands were shown to be composed of a diclonal IgMk protein and a homogeneous k free light chain protein. No abnormal immunoglobulins were detected in serum or urine. The pleocytosis and immunoglobulin abnormalities of the CSF persisted during the observation period. Specific antibody activity of the diclonal IgM of the CSF was not demonstrated and the cause of the illness remains unknown.


Multiple transplantation of islets of Langerhans. In 6 diabetic rats about 1,000 isolated islets of Langerhans were transplanted into the liver with little effect on the uninsulinism and hyperglycemia. Subsequently, a second transplantation resulted in a decrease of blood sugar to normal and in a reversal of the loss in body weight. Finally, in animals who had reversed to diabetes again, a third islet transplantation was performed. These rats with a total of 3,100 islets showed a decrease in the glucose levels from 250-305 to 110 +/- 27 (X +/- SEM) mg/100 ml. The insulin level after glucose stimulations was measured at 27 less than X less than 35 micromicron/ml.


Metabolic response to isologous transplantation of small numbers of isolated islets of Langerhans in the rat. Pancreatic islets from adult donors were transplanted intraportally into 32 inbred, adult AGUS rats with streptozotocin diabetes. The islets were isolated with collagenase and counted individually. A relationship between the number of islets transplanted and the metabolic response could be demonstrated. Transplantation of less than 200 islets did not change the diabetic state. Rats receiving 200-220 improved, while recipients of 240-800 islets all exhibited normal values of blood glucose, plasma insulin, urine volume and urine glucose. The glucose tolerance, however, remained abnormal in all animals.


Intraportal and intrasplenic autotransplantation of pancreatic islets in the dog. Autotransplantation of pancreatic microfragments into the liver or the spleen of totally pancreatectomized dogs is described. Both modes of transplantation resulted in restoration of normal fasting blood glucose levels. A delayed response to high glucose loads was however observed in both groups. Serum amylase levels indicated a rapid decline of exocrine activity. On the basis of postoperative levels of GOT and GPT in the serum of the dogs with intraportal transplants, permanent proteolytic or ischemic damage to the liver appeared unlikely.


Induction of microsomal drug-metabolizing enzymes caused by hexobarbital. Hexobarbital was given to anaesthetized mice for a period of 7 h by repeated i. p. injection, first of 100 mg/kg,then several times of 50 mg/kg. A high level of hexobarbital was maintained in the liver. The activity of microsomal drug-metabolizing enzymes was induced by this treatment with hexobarbital. 30 min after a single i. p. injection of 100 mg/kg of hexobarbital, there was a significant inhibition of aminopyrine N-demethylase but none of cytochrome c and neotetrazolium reductases. Hexobarbital in vitro inhibits aminopyrine N-demethylase but not cytochrome c reductase.


Quantitative studies on in vitro transformation of hamster chondrocytes. Hamster chondrocytes could be transformed in a quantitative assay system which used X-irradiated feeder layer cells. Morphological transformation occurred on addition of, 4NQO, but not in control cultures. Differentiation was classified into 3 types (good, poor and none); normal and transformed colonies contained similar proportions of the 3 types.


New lectin receptors in carcinoembryonic antigen (CEA). The glycoprotein CEA (carcinoembryonic antigen) carries carbohydrate groups, which react with the plant lectins from Agaricus bisporus, Arachis hypogaea (peanut), with Tridacnin from invertebrate clams and with the anti-A lectins from snails. Accordingly, it has cryptantigenic structures, which correspond to the T or T-like antigen, the Tridacnin receptor and to the so called A-like antigen.


Somatostatin inhibits the mitogenic effect of thyroliberin. The effect of somatostatin and of thyroliberin on the mitotic incidence in the organ-cultured anterior pituitary lobe of the rat was investigated, using the colchicine metaphase-arrest technique. It was found that somatostatin added to the culture medium, together with thyroliberin, blocked the mitogenic effect of the latter.


Dopamine receptors in the central nervous system. Dopamine receptors in the central nervous system can be studied by measuring the specific binding of [3H]dopamine, [3H]haloperidol, d-[3H]LSD, [3H]dihydroergocryptine or [3H]apomorphine. The receptors are stereoselectively blocked by +)-butaclamol, a neuroleptic. All neuroleptics inhibit the specific binding of [3H]haloperidol in relation to their clinical potencies. The radioligand that desorbs most slowly from the receptor is [3H]apomorphine, thus making it a reliable ligand for dopamine receptors. Dopamine agonists that compete for [3H]apomorphine binding do so at concentrations that correlate with their potency in stimulating striatal adenylate cyclase. Structure-activity analysis, using [3H]apomorphine, confirms that the active dopamine-mimetic conformation is the beta rotamer of dopamine. Prolonged exposure in vitro of caudate homogenate to high concentrations of dopamine leads to increased binding of [3H]apomorphine or [3H]haloperidol, suggesting receptor "sensitization." Chronic haloperidol treatment of rats leads to an increased number of dopamine/neuroleptic receptors in the striatum, but a decrease in the pituitary.


Skin lesions in acquired zinc deficiency due to parenteral nutrition. The skin lesions seen in 10 patients who received parenteral nutrition during treatment of chronic enteropathy are described. All of these patients had a lowered serum zinc concentration. The skin lesions were similar to those seen in acrodermatitis enteropathica. After supplementation with zinc sulphate, the skin lesions disappeared completely. A decrease in the serum alkaline phosphatase level can be regarded as a sign of an impending zinc deficiency. Parenteral nutrition formulae should contain a sufficient amount of zinc.


Diffuse varioliform gastritis.

Diffuse varioliform gastritis. During 8 years experience, 90 cases of diffuse varioliform gastritis were observed (0.3% of examinations). Symptoms suggest either the presence of a gastroduodenal ulcer or of a digestive carcinoma when weight loss is prevalent. The disease proceeds by subacute evolutive bouts with remissions and further relapses. At endoscopy, lesions associate large folds in the fundus and erosive mucosal bulgings disseminated in the fundus and antrum. Histological alterations correspond to a superficial hyperplasic gastritis. Cellular infiltration of the lamina propria includes increased immunocytes population with an abnormal distribution of immunoglobulin classes. An increased percentage of IgE cells characterizes the disease as compared to inflammation in atrophic gastritis. Therapy is based upon agent inhibiting mastocytes degranulation and histamine antagonists. Diffuse varioliform gastritis must be separated from antral varioliform gastritis, a lesion with a higher frequency but no symptomatic individuality.


Results of the single-radial-diffusion test with formaldehyde-treated influenza virus. We found reduced values for formaldehyde-treated influenza virus in the single-radial-diffusion test by comparison with non-treated virus. The reduction correlates with the formaldehyde concentration. Hemagglutination and neuraminidase activities were not affected by the same treatment. We therefore suggest that this reduction is not based on antigen destruction.


Relevance of the immunoprecipitation assay to human immunogenicity of influenza vaccines. Influenza vaccines representing each of the four U.S. manufacturers' output for the 1975-76 respiratory season were characterized clinically and assayed by immunoprecipitation. All vaccines contained 350 CCA units/dose each of the A/Port Chalmers/1/73 (H3N2) and A/Scotland/840/74 (H3N2) viruses plus 550 CCA units/dose of B/HK/5/72 virus. Two of the vaccines were whole virus while the other two were subunit products; one made by extraction with ethyl ether, the second by detergent treatment. The vaccines were compared for serologic efficacy in children naturally primed to the (H3N2) family of viruses and by immunoprecipitation techniques against monospecific goat antiserum to the viral hemagglutinin prepared at the Bureau of Biologics of the U.S. Food & Drug Administration. The subunit vaccines had significantly greater specific activity (human immunogenicity/unit mass of type-specific precipitable antigen) than the whole virus products. It is concluded that clinical immunogenicity is as much a function of antigen form (subunit vs whole virus) as it is of mass and that setting a level for precipitable antigen content alone is an insufficient criterion for potency standardization. Since antigen form, as well as mass, must be considered, immunoprecipitation may be useful for standardization of human immunogenicity only if candidate lots are compared by this technique to an homologous, reference vaccine of identical manufacture and form which is tested for potency in humans.


Quantitation of influenza vaccine hemagglutinin by immunoelectrophoresis. A technique for the quantitation of hemagglutinin (HA) in influenza vaccines by immunoelectrophoresis (IEP) is described. This method gives accurate, and reproducible results and requires approximately 9 h to complete. The HA content of four manufacturers' vaccines correlated with the chick cell agglutination (CCA) values for each manufacturers' vaccine. However, when comparisons were made between vaccines produced by different manufacturers, up to a four-fold difference in HA content was observed. Comparison with single radial immunodiffusion (SRID) gave comparable results for vaccines produced by three of four manufacturers; the fourth manufacturers' vaccine gave 1.6-fold higher results by SRID. Human seroconversion rates in seronegative individuals correlated better with IEP determined HA content than the current assay of vaccine potency using CCA.


Single-radial-complement-fixation: a new immunodiffusion technique. 2. Assay of the antibody response to the internal antigens (MP and NP) of influenza A virus in human sera after vaccination and infection. This new immunodiffusion method involves the incorporation of antigen and complement in a primary agarose gel. Heat-inactivated serum samples are filled into wells and allowed to diffuse radially overnight at 4 degrees C. A secondary gel, containing antibody-coated sheep erythrocytes, is layered on top of the first gel and the system is incubated for 45 min at 37 degrees C. Where complement is fixed, i.e. around wells with positive serum samples, zones of unlysed indicator cells appear. Otherwise, the red bloods cells are lysed. This sensitive technique is employed in a study of human sera for the measurement of the antibody response to the internal antigens (MP and NP) of influenza A virus after infection and vaccination. It was found that none of the vaccinees developed antibodies to the MP antigen, whereas 35% of the post-infection group showed a significant rise. The response to the NP antigen was less frequent and of lower order of magnitude after vaccination than after infection.


[The olfactor-genital syndrome (author's transl)]. 1. Only FSH was increased after intravenous administration of 0.025 mg of GnRH in 2 female patients suffering from olfacto-genital syndrome. LH-serum-concentrations increased significantly, only when 0.1 mg GnRH was applicated intravenously twice with an interval of one hour.2. The response of hPRL-secretion to 0.2 mg TRH was found normal in both patients. However, when 25 mg chlorpromacine were administered intramuscularly, only one patient responded with an adequate rise of serum-hPRL. 3. One patient became pregnant during treatment with HMG/HCG. The clinical course of pregnancy was quite normal. HPL- and HCG-levels were in the normal, hPRL in the low normal range during pregnancy.


Application of an improved single-radial-immunodiffusion technique for the assay of haemagglutinin antigen content of whole virus and subunit influenza vaccines. An improved single-radial-diffusion technique for the assay of influenza haemagglutinin antigen is described. The modified method enables the results of assays of antigen to be obtained more rapidly and with greater precision than previously. The use of immunoplates containing varied, preselected concentrations of antihaemagglutinin antibody allows accurate assays to be performed over a wide range of antigen concentration. In tests on "whole virus" antigen preparations it was found that the ratio between haemagglutination titre (iu/ml) and haemagglutinin antigen activity (microgram/ml) determined by single-radial-diffusion was relatively constant for antigens containing a given strain but showed variation between strains (range 16.5 to 26.8 iu/microgram HA activity). For the subunit vaccines examined this ratio showed a large degree of variation (range 1.4 to 16.6 iu/microgram HA activity) and in general was considerably lower than for whole virus antigens. It was concluded that single-radial-diffusion may be of value for assays of the haemagglutinin concentration of both "whole virus" vaccines and subunit vaccines. The data presented suggest that, in the event of antigenic drift, it may be possible to use the existing reference anti-haemagglutinin serum to assay a variant strain of influenza virus.


The meiotic effect of a deficiency in Drosophila melanogaster with a model for the effects of enzyme deficiency on recombination. The meiotic effects of heterozygosity for a deficiency of the zeste-white region of the X chromosome include reduced recombination and increased non-disjunction of the entire chromosome complement. Reduced dosage of a gene or genes in the zeste-white interval, rather than structural heterozygosity, is responsible for the meiotic effect. A model for the recombination effects of reduced enzyme concentration has been developed, and its consequences are comparable with the results obtained for deficiency heterozygosity. Thus, all of the observations can be accounted for by imagining a dosage-sensitive locus in the zeste-white region that codes for an enzyme involved in the recombination process. The interaction of the interchromosomal effect of heterozygous inversions with the deficiency has been examined, and the possibility of using the model for the analysis of other meiotic phenomena is considered.


Chromosomal analysis of DDT-resistance in a long-term selected population of Drosophila melanogaster. The genetic basis of DDT-resistance was studied in a population of Drosophila melanogaster. This population was unique in that it had been continually selected for DDT-resistance since 1952 and had achieved a very high level of resistance. The genetic basis of resistance was studied by means of a chromosomal analysis. Fifteen combinations of resistant and control chromosomes were tested using a time-based DDT test. The analysis of the data showed that resistance was multifactorial with each of the three major chromosomes involved. Dominant and recessive second and third chromosome effects were found to be much more important than those of the first chromosome, which had no detectable recessive effects. Second and third chromosome resistance genes showed incomplete dominance. The average dominance of the second chromosome was much less than that of the third chromosome. These large-scale differences between chromosomes' effects and average dominance may indicate that a small number of resistance genes are involved. Two significant interactions between chromosomes were found. Scaling difficulties make the interactions difficult to interpret without further data. It seems possible that positive interactions between resistance have been developed by the long-term directional selection in this population.


Fitness effects of EMS-induced mutations on the X chromosome of Drosophila melanogaster. I. Viability effects and heterozygous fitness effects. Drosophila melanogaster X chromosomes were mutagenized by feeding males sucrose solutions containing ethyl methanesulfonate (EMS); the concentrations of EMS in the food were 2.5 mM, 5.0 mM, and 10.0 mM. Chromosomes were exposed to the mutagen up to three times by treating males in succeeding generations. After treatment, the effective exposures were 2.5, 5.0, 7.5, 10.0, 15.0, and 30.0 mM EMS. X chromosomes treated in this manner were tested for effects on fitness in both hemizygous and heterozygous conditions, and for effects on viability in hemizygous and homozygous conditions. In addition, untreated X chromosomes were available for study. The viability and heterozygous fitness effects are presented in this paper, and the hemizygous fitness effects are discussed in the accompanying one (MITCHELL and SIMMONS 1977). Hemizygous and homozygous viability effects were measured by segregation tests in vial cultures. For hemizygous males, viability was reduced 0.5 percent per mM EMS treatment; for homozygous females, it was reduced 0.7% per mM treatment. The decline in viability appeared to be a linear function of EMS dose. The viabilities of males and females were strongly correlated. Heterozygous fitness effects were measured by monitoring changes in the frequencies of treated and untreated X chromosomes in discrete generation populations which, through the use of an X-Y translocation, maintained them only in heterozygous condition. Flies that were heterozygous for a treated chromosome were found to be 0.4% less fit per mM EMS than flies heterozygous for an untreated one.


[Bacillus subtilis mutant with altered ability for heterologous transformation]. Mutant strain of Bacillus subtilis, which produced in certain conditions significantly reduced quantity of trnasformants during transformation by homologous DNA, as compared with transformation by heterologous DNA from Bac. aterrimus, is isolated. The ability to transfection by phage SPO1 DNA and the efficiency of infection of the mutant by this phage are also decreased. The causes of such alterated properties are discussed.


[Induction of auxotrophy in the brucellosis microbe under the effect of N-methyl-N'-nitro-N-nitrosoguannidine]. Mutagenic activity of N-methyl-N'-nitro-N-nitrosoguanidine with respect to Brucella melitensis was studied. In the course of this study the method of obtaining auxotrophic brucella mutants was elaborated. Numerous assortiment of auxotrophic mutants of different types was obtained. In a number of cases the acquisition of auxotrophy was accompanied by profound changes of the strain, such as the changes in morphology of colonies, in the biochemical activity, in the capacity of agglutination and in the virulence.


[Mutagenic effect of chemical compounds on laboratory mice]. Mutagenic effect of thioTEPA applied at a dose of 1.25 mg/kg was studied in late spermatids of C57BL/L male mice. The mutagen induced dominant lethal mutations in germ cells (39%) and symmetric translocations in 33.5% of F1 male offspring. The common frequency of sperms with chromosome mutations was 60%, that is ten times as much as the mutagenic effect in bone marrow cells. 39% of embryos at 3.5 days of development died or delayed their development at 2--22 blastomers stages. Structure chromosome aberrations were found in the cells of such embryos. The scheme of genetical screening of chemical compounds in laboratory mice, based on the data obtained early and in the present experiment, is proposed.


[Contibution of the X chromosme to the increased resistance of inbred Drosophila melanogaster hybrids to physical effects]. Effects of high sub-lethal temperature and UV-irradiation on surviving of inbred lines of Drosophila melanogaster and its F1 hybrids derived from reciprocal crosses between these lines are investigated. High resistance of F1 hybrids to these factors was observed as compared with that of parental inbred lines. D. melanogaster females in inbred lines and F1 hybrids were more resistant than males. Differences in the resistance between females and males were more pronounced in hybrids.


Another type of iatrogenic problem.

Another type of iatrogenic problem. Physicians' lack of careful attention to transfer forms for elderly patients being moved from acute-care hospitals to facilities for long-term care can create serious--even life-threatening--problems.


Appraisal of the 14C-glycocholate acid test with special reference to the measurement of faecal 14C excretion. The (14)C-glycocholate test, including the measurement of marker corrected faecal (14)C, has been assessed in the following groups of subjects: normal controls (18), patients with diarrhoea not attributable to altered bile acid metabolism (21), patients with diverticula of the small intestine (12), patients with previous resection of ileum and often proximal colon (34), and established ileostomists (10). Patients with diverticular disease had increased breath (14)CO(2) excretion, but normal faecal excretion of (14)C, and this test was more frequently abnormal than the Schilling test. Ileostomists excreted increased amounts of faecal (14)C, even when the ileum was intact and apparently normal. The pattern after resection was complex. Breath (14)C output was normal if the ileal resection was less than 25 cm in length, although some of these patients had increased faecal (14)C excretion if, in addition, at least 15 cm of proximal colon had been resected or by-passed. Longer ileal resections were associated with increased breath and/or faecal (14)C excretion, depending in part on the length of colon resected or by-passed and the 24 hour faecal volume. Fewer than half these patients had both increased breath and faecal excretion of isotope and faecal (14)C alone was occasionally normal with an ileal resection of 50 cm of more. The (14)C-glycocholate test was more frequently abnormal than the Schilling test in this group. The use of faecal marker correction had only a minor impact on the results. These data suggest that, in patients with ileal resection, faecal (14)C, like faecal weight, is determined by the extent of colonic resection as well as by the amount of ileum resected.


Influence of antimicrobial agents on contamination and chlortetracycline production. The possibility of shortening the thermal sterilization time for cultivating media was demonstrated in chlortetracycline fermentation with an industrial strain of Streptomyces aureofaciens. The medium was artificially contaminated with a mixture of eight strains of G+ and G- bacteria isolated from contaminated industrial fermentors, and the following chemical agents, either alone or in combination, were added: formaldehyde, phenol. dimethylformamide, p-aminosalicylic acid and nitrofurazone. Dimethylformamide was inhibitory even at 0.08%.  formaldehyde concentrations higher than 0.05%, Nitrofurazone stimulated chlortetracycline production. The best combination was 0.01% formaldehyde added before, and 2.10-3% nitrofurazone added after short sterilization at 120 degrees C.


Aspects of reproduction in ruffed lemurs (Lemur variegatus). Results of breeding of ruffed lemurs (Lemur variegatus) at the Duke Primate Facility provide information concerning estrous cycle, sexual behavior, age of first conception, gestation period, parturition, litter size. L. variegatus differs from other Lemur species in these respects.


Urinary excretion of biologically active chorionic gonadotrophin by the pregnant marmoset (Callithrix jacchus jacchus). No significant correlation exists between the amount of biologically active marmoset chorionic gonadotrophin (mCG) in urine and results obtained with an immunological pregnancy test. The pregnant marmoset excretes large amounts of oestrogenic steroids, which must be removed, to prevent the enhancement of the response of the bioassay for mCG. More than 99% of these unconjugated and conjugated urinary oestrogens can be removed by extraction with acetone and ether. mCG is excreted throughout pregnancy, maximum levels occurring between the 8th and 9th week of gestation. There is a considerable within- and between-animal variation in the amount of mCG excreted. However, the pattern of gonadotrophin excretion by the pregnant marmoset is similar to that of man and the apes but unlike that of baboons and macaques.


[Obstetrical risk factors in perinatally deceased boys and girls]. Statistical analysis reveals that there is a correlation between fetal sex and kind and incidence, respectively, of intrauterine and perinatal manifestation of risk factors. Male fetuses are more likely to be affected by risk factors than females. These observations correspond to the higher mortality rate of male versus female fetuses.


Trihexosyldiacylglycerol and acyltrihexosyldiacylglycerol as lipid anchors of the lipoteichoic acid of Lactobacillus casei DSM 20021. The lipoteichoic acid of Lactobacillus casei DSM 20021 was isolated. It consists of glycolipid and a poly(glycerophosphate), 37-44 units in length. The lipid portions can be released by treatment with hydrofluoric acid or acetic acid. They were determined to be 1,2-di-O-acyl-3-O-[O-beta-D-glucopyranosyl-(1 leads to 6)-O-alpha-D-galacto-pyranosyl-(1 leads to 2)-alpha-D-glucopyranosyl]glycerol and 1,2-di-O-acyl-3-O-[O-beta-D-glucopyranosyl-(1 leads to 6)-O-alpha-D-galactopyranosyl-(1 leads to 2)-(6-O-acyl-alpha-D-glucopyranosyl)]glycerol. Lipoteichoic acid with a third carbohydrate-linked fatty acid in the glycolipid portion represents a novel subclass.


A new variety of spondyloepiphyseal dysplasia characterized by punctate corneal dystrophy and abnormal dermal collagen fibrils. Several individuals from one family are described with a unique form of spondyloepiphyseal dysplasia. Characteristic features include short-trunked short stature, punctate corneal dystrophy and marked disorganization of dermal collagen fibrils when examined by transmission electron microscopy. Inheritance is compatible with either dominance and a variable expression or X-linkage. Although the basic defect has not been determined, the tissue distribution is consistent with a defect in a non-collagenous component that affects collagen fibril formation or stability.


[Immunological aspects of inflammatory bowel diseases (author's transl)]. The gastrointestinal tract is replete with immunocompetent tissue represented by appendix, Peyer's patches and nonaggregated lymphocytes in the intestinal epithelium. This local immune system plays a vital role in the physiologic immune response and in the control of food, drug, microbial or viral antigens. Under certain situations this lymphoid tissue may contribute to the immunological reactions underlying the pathogenesis of various diseases. Generalised deficiencies of the immune apparatus frequently give rise to disturbances of gastrointestinal function. Furthermore plenty of immune reactions occurring in connection with human inflammatory bowel disease have been described. Some immunologic features are reported as typical for a disease, the majority seems to indicate only nonspecific autoimmune reactions. The relevance of immunological mechanisms for the pathogenesis of these diseases is still not full evaluated.


Induction of hapten-specific cell-mediated immunity in rhesus monkeys treated with adsorbent purified antilymphocyte antibody. Rhesus monkeys were treated with sufficient amounts of rabbit antilymphocyte sera and immunoadsorbent purified antilymphocyte antibody to retain full-thickness skin grafts 23 and 29 days respectively. During this immunosuppressed period hapten-specific cell-mediated immunity was induced in six of seven rhesus monkeys immunized with p-azophenylarsono-N-acetyltyrosine (R-tyr) in complete Freund's adjuvant. These observations suggest that in rhesus monkeys different populations of T cells may be responsible for cutaneous sensitization to antigen and rejection of skin grafts.


Monoclonal gammopathies in rheumatoid arthritis. A total of 2560 sera from patients with and without rheumatoid diseases were examined for the incidence of monoclonal proteins. The incidence of monoclonal protein was higher (3.3%) in patients with rheumatoid arthritis than with other non-rheumatoid diseases of connective tissue (0.7%). Of 16 monoclonal proteins found in rheumatoid arthritis, 15 were IgG and 1 IgA; 7 sera belonged to the kappa and 9 to lambda type.


The binding of adjuvant-active mycobacterial peptidoglycolipids and glycopeptides to mammalian membranes and their effect on artificial lipid bilayers. The nature of the binding of adjuvant-active amphiphilic mycobacterial peptidoglycolipids and glycopeptides to mammalian plasma membranes was investigated together with their effect on the stability of artificial lipid bilayers. The importance of these interactions between mycobacterial components and membranes is discussed in relation to immunopotentiation, mitogenicity and tuberculous infection.


Antibody mediated mechanisms of immunity to malaria induced by vaccination with Plasmodium knowlesi merozoites. Rhesus monkeys vaccinated with merozoites in FCA are protected against challenge with several strains and variants of Plasmodium knowlesi. Vaccination induces sterilizing immunity which is species specific. Merozoite-blocking (inhibitory) antibody usually correlates with clinical immunity and protection can be passively transferred with immune sera provided these contain high levels of inhibitory antibody. However, vaccination using adjuvants other than FCA may induce inhibitory antibody without clinical protection. In addition, vaccinated animals may become susceptible to challenge 4-5 weeks after splenectomy, although inhibitory antibody levels are not reduced. These observations indicate that immunity induced by merozoite vaccination involves: (i) merozoite blocking (inhibitory) antibody, (ii) specific antibody or immune complexes acting synergistically with cytotoxic splenic cells stimulated by FCA.


Allograft cytotoxicity co-operation between alloimmune T cells and macrophages. T cells from the spleens of C57BL 10 (H-2b) mice 7-12 days after immunization with P815Y (H-2d) mastocytoma cells have been shown to co-operate synergistically with an adherent component of non-immune starch induced peritoneal cells in the cytostasis of target cells. Although significant values for synergy could be obtained using the (125I) UdR incorporation assay to measure cytostasis, normal peritoneal cells were incapable of co-operating with T cells in cytolysis as measured by 51Cr release from pre-labelled target cells. Initially, the synergistic interaction was immunologically specific, but non-specific activity could be induced by challenge with specific antigen.


Specificity of salivary-bacterial interactions: role of terminal sialic acid residues in the interaction of salivary glycoproteins with Streptococcus sanguis and Streptococcus mutans. Four highly purified salivary glycoproteins were used to study salivary-bacterial interactions. One pair of glycoproteins was mucin-like in composition, whereas the second pair was not. By an agglutination assay, it was found that only the mucin-glycoproteins agglutinated Streptococcus sanguis and S. mutans. Removal of sialic acid from these molecules resulted in a loss of agglutination of S. sanguis but not of S. mutans. The agglutination phenomenon was shown to require a salivary macromolecule of at least 150,000 daltons.


Acrylamide gel electrophoresis of proteins of Neisseria gonorrhoeae as an epidemiological tool. The electrophoresis patterns of gonococcal cell proteins were assessed as an aid to gonococcal epidemiology. Lyophilized gonococci were extracted in heated lauryl sulfate with 2-mercaptoethanol. The solubilized gonococcal cell proteins were subjected to alkaline sodium dodecyl sulfate gel electrophoresis, and the resulting patterns were scanned with a spectrophotometer. The electrophoresis patterns were judged to be alike by superimposition of the gel scans. Gonococci thus compared were also auxotyped and tested for sensitivity to penicillin, ampicillin, tetracycline, and spectinomycin. The electrophoresis patterns were reproducible and not affected by serial subculture of the isolates. Patterns from nongonococcal Neisseria species did not resemble those of gonococci. To test the utility of the electrophoresis patterns, duplicate isolates were made from 12 cultures of Neisseria gonorrhoeae. After randomization and blind coding of the 24 isolates, all were successfully matched by their electrophoresis patterns. Cervical-rectal paired isolates from 45 patients were studied by means of their electrophoresis patterns, auxotypes, and results of antibiotic sensitivity tests. Results indicated that the electrophoresis patterns are useful in characterizing paired isolates of gonococci.


Effect of metabolic inhibitors on the formation of antibody to sheep erythrocytes, on development of delayed hypersensitivity, and on the immune response to infection with Mycobacterium tuberculosis in mice. The effect of a number of metabolic inhibitors was determined on: (i) the production of cellular immunity to infection with Mycobacterium tuberculosis in mice by vaccination with mycobacterial ribonucleic acid (RNA), (Ii) the production of cellular immunity to infection with M. tuberculosis in mice with viable H37Ra cells, (iii) the induction of antibody formation to sheep erythrocytes, and (iv) the induction of delayed hypersensitivity in mice to purified protein derivative. The pattern of inhibition produced by metabolic inhibitors on cellular immunity to infection with M. tuberculosis produced by mycobacterial RNA was entirely different from the pattern of inhibition produced by the same metabolic inhibitors on antibody formation to sheep erythrocytes. The effect of the metabolic inhibitors on the induction of delayed hypersensitivity to purified protein derivative did not correlate with the pattern of inhibition produced by the same compounds on antibody formation or on the development of immunity produced by mycobacterial RNA. Cellular immunity to infection produced in mice by viable H37Ra cells was not reduced by any of the metabolic inhibitors except actinomycin D. The possible reasons for the lack of activity of the metabolic inhibitors on the immune response to viable H37Ra cells and the lack of correlation with the pattern of inhibition found in mice vaccinated with mycobacterial RNA is discussed.


Mechanism of action of Pseudomonas aeruginosa exotoxin A in experimental mouse infections: adenosine diphosphate ribosylation of elongation factor 2. The data presented indicate that one of the primary actions of Pseudomonas aeruginosa exotoxin during experimental infection is the inactivation of elongation factor 2 (EF-2) in various mouse organs. Organs from mice infected with the toxigenic P. aeruginosa strain PA103 contained considerably less EF-2 activity than did organs from uninfected controls. Whereas EF-2 activity was reduced in all organs examined from PA103-infected animals, the largest decrease was observed in the liver, where the active EF-2 levels were reduced by 70 to 90%. In addition, consistent inhibition of protein synthesis in livers but not in other organs was observed in mice infected with the toxigenic PA103 strain. Treatment of mice with antitoxin before infection with strain PA103 prevented inactivation of EF-2. When mice were infected with lethal doses of the nontoxigenic P. aeruginosa WR5 strain, tissue EF-2 levels were not markedly reduced below those derived from uninfected control animals.


Activation of the alternate complement pathway by peptidoglycan from streptococcal cell wall. Activation of the alternate complement pathway in human serum by several bacterial components was compared. Peptidoglycan from group A streptococcal cell walls was the most active material, on a weight basis, followed by cell walls, protoplast membranes, and whole cells. The group-specific carbohydrate was inactive. Treatment of peptidoglycan with low concentrations of lysozyme or short periods of sonic treatment enhanced complement activation. High concentrations of lysozyme or extended sonic treatment of peptidoglycan destroyed or greatly reduced the capacity to activate complement. Lysozyme treatment of group A streptococcal cell walls or lipopolysaccharide had no measurable effect. Activation of the alternate complement pathway by group D streptococcal cell walls was destroyed by lysozyme. Activity of peptidoglycan was not inhibited by N-acetyl glucosamine, N-acetyl muramic acid, or D-alanine-D-alanine. Conversion of C3 and factored B by peptidoglycan was shown to occur by immunoelectrophoresis and crossed immunoelectrophoresis.


Studies on gonococcus infection. XII. Colony color and opacity varienats of gonococci. Colonies with markedly differing color and opacity characteristics were found for Neisseria gonorrhoeae propagated on translucent, solid medium. These optical properties of gonococcal colonies were independent of pilation and appeared to be related to the degree of aggregation among the gonococci comprising the colonies. Dark, opaque colonies contained highly aggregated gonococci which were more susceptible to killing and to solubilization of their 125-labeled surface components than were organisms comprising light, transparent colony forms.


Studies on gonococcus infection. XIII. Occurrence of color/opacity colonial variants in clinical cultures. Colonial variants of Neisseria gonorrhoeae differing in color and opacity characteristics are distributed differently between male urethral and female cervical cultures. Colonial characteristics of cultures isolated from female cervices differ with time of isolation within the menstrual cycle and use of oral contraceptives. These differences may reflect selective forces in the ecology of the cervix, particularly proteolytic enzymes in cervical mucus and menstrual blood. Cycle changes in the characteristics of gonococci isolated from females may have implications for pathogenesis of gonococcal infections and immune response to the gonococcus.


Purification of Pseudomonas aeruginosa exotoxin by affinity chromatography. Pseudomonas aeruginosa exotoxin A was purified by affinity chromatography from culture supernatants by elution of toxin from antitoxin immunoglobulin G-Sepharose 4B with 3 M NaSCN. The purity, toxicity, and enzymatic activity of exotoxin obtained were comparable to those of toxin purified by previously reported multiple-step procedures.


Induction of cell-mediated immunity to Mycobacterium leprae in mice. The immune response of mice to armadillo-derived, irradiation-killed Mycobacterium leprae (I-ML) was investigated. Following injection of 100 microgram of I-ML into the left hind footpads of mice, a state of cell-mediated immunity (CMI) was engendered to antigens of M. leprae. The evidence for CMI was as follows: (i) development of delayed-type hypersensitivity to both human tuberculin purified protein derivative and soluble M. leprae antigens; (ii) T-lymphocyte-dependent macrophage activation at the inoculation site; (iii) specific systemic resistance to the cross-reactive species M. tuberculosis; and (iv) immunopotentiation of the delayed-type hypersensitivity response to an unrelated antigen. The CMI induced by I-ML in aqueous suspension was greater than that obtained with the same antigen in water-in-oil emulsion, even though the latter generated a more severe reaction at the site of immunization. I-ML also induced a stronger CMI response than the corresponding dose of heat-killed BCG.


Chemical transformation of human revertant cells induced by murine sarcoma virus. A human revertant cell line, derived from non-producer human osteosarcoma cells (NP/KHOS) induced by Kirsten murine sarcoma virus, was treated in vitro with various levels of polycyclic aromatic hydrocarbons (3-methylcholanthrene, 7,12-dimethylbenz(alpha)anthracene, and benzo(alpha)pyrene [BP] or dimethyl sulfoxide (control). Cells treated only with 3-methylcholanthrene and 7,12-dimethylbenz(alpha)anthracene underwent morphological alteration in vitro, and produced tumors when injected into NIH nude mice. These human revertant cells may be a useful in vitro tool in screening for the potential chemical carcinogens in human cell systems.


Ultrastructure of the condylar articular surface in severe mandibular pain-dysfunction syndrome. Specimens from articular surfaces of normal human mandibular condyles are compared with very small biopsies from articular surfaces of condyles taken at conservative operations in severe pain-dysfunction syndrome (P.D.S.). Immediate fixation was followed by examination using transmission electron-micrography. Normal surfaces exhibit a nearly structureless layer about 2 micron thick, which corresponds with the lamina splendens described in other diarthrodial joints. This layer surmounts a dense main structure of wavy interlacing bundles of collagen interspersed with fibrocytes. Occasional straight elastic fibres were found. Surfaces of all condyles from P.D.S. patients showed loss of lamina splendens, alteration of collagen fibre size, and tendency to dissociation of both collagen and its surrounding ground substance. Depper levels showed aggregations of bizarre structures which the author terms "vermiform bodies", and which appear to be collections of abnormal amounts and types of elastic tissue. It is suggested that the appearances are those of stress elastosis. Such profound ultrastructural changes may affect the joint sliding properties, and also its mechanical integrity under stress. Examination at this degree of magnification suggests a direct relationship between long-standing pain-dysfunction syndrome and the onset of degenerative disease.


Periapical condition of anterior teeth after surgical correction of mandibular prognathism. A radiographic study. Extrusion and root resorption of the mandibular anterior teeth have been reported after surgical correction of mandibular prognathism by horizontal osteotomy of the rami. The aim of the present study was to find out whether oblique sliding osteotomy also has such side effects. Intraoral radiographs of the maxillary and mandibular anterior teeth of 61 patients were studied. Enlargement of the periodontal space and signs of root resorption were estimated postoperatively and at a later review 1 1/2-5 years after the operation. Enlargement of periodontal space occurred postoperatively in 16.2% of the maxillary incisors and 52.2% of the mandibular incisors. The enlargements were mostly reversible and at the final review only 1.1 and 8.5% of the maxillary and mandibular incisors, respectively, showed such enlargements. Root resorption was rarely found in radiographs from the postoperative control. At the final review, however, there were signs of root resorption of 1.6 and 20.8% of the maxillary and mandibular incisors, respectively. It appears warranted to conclude that the anterior teeth, especially the mandibular incisors, are exposed to strong forces also after oblique sliding osteotomy of the mandibular rami.


Frenum surgery. I. A comparison of three surgical methods. The present study was undertaken to evaluate and compare three surgical methods to relocate labial frena to a more apical position. Thirty patients with frenum insertion at the gingival margin were selected for the study. All had been peridontally treated prior to frenum surgery. Stone casts from rubber base impressions in individual trays were used to measure the differences in frenum insertion before surgery and 2, 12, 26 and 52 weeks afterwards. The surgical methods, each carried out in 10 cases, consisted of frenum excision, vestibular extension of Z-plasty. The results showed that a significant reduction of the frenum was obtained with all three methods used, although after 52 weeks, the difference between the pre- and postoperative status were most marked where Z-plasty had been carried out and least so after frenum excision. Post-surgical symptoms were most frequent after vestibular sulcus extension and fewest after frenum excision.


Lingual exposure during mandibular third molar surgery. The necessity for surgical exposure of the lingual aspect of mandibular third molars will vary according to the severity of the impaction and the surgical technique adopted. A retractor is described for use in those cases when major lingual retraction is required. The retractor provides optimum access to the mandibular third molar area with adequate protection for the lingual nerve, tongue retraction and enhanced illumination by light reflection. It can be used under both local and general anaesthesia.


Leukokeratosis nicotina glossi-smokers' tongue. "Leukokeratosis nicotina glossi" or "smokers' tongue" is a homogeneous leukoplakia with evenly distributed pin-point hemispherical depressions. Histologically, there is a loss of glossal papillae, hyperkeratosis, acanthosis and the formation of large drop-shaped rete pegs with central clefting and occasional parakeratotic plugging. Mitotic activity and atypia are not marked and there is no evidence of Candida species infection. In some respects the lesion histologically resembles verrucous carcinoma but, unlike that condition, papillomatosis is not clinically noticeable and an invasive "leading edge" is not apparent. All but one of the subjects in which the lesion was seen were men, all had concurrent leukokeratosis nicotina palati and two gave histories of laryngeal carcinoma.


Mammalian cones: disc shedding, phagocytosis, and renewal. During the past several years we have examined a variety of different mammalian retinas for ultrastructural evidence of cone disc shedding and RPE phagocytosis. In this paper we review our previously published evidence from squirrel and human retinas as well as present new evidence of cone disc shedding in rhesus monkey and cat. All these species show definitive evidence for the shedding of discs from cone outer segments and the phagocytosis of shed discs by apical processes of the RPE; both of these events closely resemble those described for mammalian rods. The occurrence of cone disc shedding leads directly to the conclusion that new membrane must be added to the cone outer segment in order to maintain its length. The successive evaginations, which are observed at the bases of cone outer segments, we consider to be indirect evidence for the addition of new discs. Finally, we propose a model for the structural organization of mammalian cone outer segments.


Phototoxicity to the newborn primate retina. Newborn stump-tail monkeys were continuously exposed to 400 f-c of cool, white, fluorescent light for periods varying from 12 hr to 7 days. The right eye of each monkey was occluded by a patch of black velour material to serve as a control. The protected eyes retained normal ultrastructure; the exposed eyes showed progressive damage to the retina from the 12 hr to the 7-day exposure periods. Early changes were evident in the outer nuclear layer with darkly staining pyknotic nuclei and electrondense cytoplasmic processes that could be traced to their synaptic terminals. Late changes included marked distortion, vacuolization, and fragmentation of the rod and cone outer segments. The potential for phototoxicity to be additive to the normal aging of the retina is proposed, and we conclude that there is a sound basis for the current practice of patching the eyes of infants undergoing phototherapy.


Accuracy of aqueous humor flow determination by fluorophotometry. After corneal iontophoresis of fluorescein, freshly enucleated rhesus monkey eyes were perfused at a known rate with glutathione-bicarbonate-Ringer's solution. Total fluorescein mass and aqueous fluorescein concentration were measured with a fluorophotometer every 15 min for 6 to 8 hr. The aqueous flow was calculated from the fluorophotometry data by the second method of Jones and Maurice. This flow was then compared to the known perfusion rate. The curve for total fluorescence decay was not precisely parallel to the curve for aqueous fluorescence decay. This affected the value of the ratio of total:aqueous fluorescence. The most accurate and consistent flow values were obtained with the decay constant from the aqueous fluorescence decay curve combined with the ratio of total:aqueous fluorescence extrapolated back to zero time. Under these two conditions, the calculated flow in five experiments was 5.4% +/- 3.3 (+/- S.D.) lower than the known flow. The 99% confidence interval of the true mean percent difference between the known and calculated flows is--12% to +1%. A small effect of corneal fluorescein concentration on measured aqueous fluorescence was observed.


Can polymorphism be maintained by selection favouring an intermediate optimum phenotype? Populations of Drosophila melanogaster containing the special chromosome, M-5, were subjected to artificial selection to canalise scutellar bristle phenotype at four bristles. At the end of the experiment allele frequencies at three enzyme loci were measured. In the selected population, these frequencies were found to have diverged from their starting values less than would have been predicted on the basis of random genetic drift. This was not found in control populations or in populations selected for low bristle number.


Chromosome breakage: a possible mechanism for diverse genetic events in outbred populations. Evidence is summarised to support the hypothesis that genetic characteristics frequently assayed in natural populations, including mutator activity, sterility, male recombination, and distortion of segregation, may often be expressions of the same general event--chromosome breakage in outbred populations.


Genetical variation for enzyme activity in a population of Drosophila melanogaster. IV. Analysis of alcohol dehydrogenase activity in chromosome substitution lines. Chromosome substitution lines derived from two inbred strains of Drosophila melanogaster homozygous for the AdhS allele of alcohol dehydrogenase but differing significantly in ADH activity have been analysed. Variation in activity can be attributed to all three major chromosomes. The effect of the second chromosome, where the ADH structural gene is located, can be modified significantly by the genotype of both the first and the third chromosomes. The most substantial single effect results from homozygous differences between the third chromosomes. In contrast, differences between the X chromosomes are revealed only when the second or second and third chromosomes are heterozygous.


New apparatus for exercising a monkey seated in a primate chair. A new apparatus and method for exercising a monkey seated in a primate chair are described. The apparatus consists of a set of hand and foot bars mounted at either end of a rocker arm that pivots in a vertical plane. This arm is connected by a steel cable to a wooden box that houses lead weights. By simultaneously pushing the foot bar and pulling the hand bar, the monkey lifts a weight and triggers a microswitch which releases a banana-flavored food pellet into a well close to the animal's mouth. The amount of work performed is varied by changing the a) amount of weight to be lifted, b) distance the weight must be lifted, or c) number of times that the animal lifts the weight for a single reward. During 30 min of exercise, the heart rate of a given monkey rose 120 beats/min, respiratory rate 24/min, and colonic temperature 1.2 degrees C above base-line values. Total work performed in 30 min varied from 250 to 360 kpm. Major advantages of this apparatus over a treadmill or bicycle ergometer are that the animal 1) is restrained sufficiently to permit the delicate experimental manipulations and physiological measurements, 2) can be exercised strenuously in a quantitative fashion, and 3) is readily trained to perform the task in a short time.


The coraco-acromial ligament and projection index in man and other anthropoid primates. The coraco-acromial ligament in man is a trait shared only with other hominoids (apes) among anthropoid primates. The associated form of the coracoid process and acromion, their lateral projections and the shape of the glenoid cavity likewise distinguish the Hominoidea. These anatomical features add credence to the view that the living hominoids share an independent ancestry within the Anthropoidea associated with the development of several unique locomotor/feeding adaptations.


A teased-fibre study of the median nerves of vitamin B12-depleted baboons. Isolated nerve fibres of the median nerve of normal baboons and baboons kept on diets deficient in vitamin B12, and supplemented with potassium cyanide and potassium thiocyanate injections, were examined by the teased-fibre technique. Regression lines of internodal length on fibre diameter were obtained. Small differences between the groups were apparent but were of uncertain significance. There was occasional evidence of segmental demyelination and Wallerian degeneration but this was not characteristic of any particular group.


The enamel--dentine junction of human and Macaca irus teeth: a light and electron microscopic study. The enamel--dentin junction of human and Macaca irus teeth has been studied using light and scanning and transmission electron microscopy. There are differences between species and within the same tooth. Scalloping, when present, is more developed in the cuspal areas and approximally, and the scallops appear to be formed at the time of maturation of the hard tissues. Enamel tufts and spindles are briefly discussed. The depth of scallops was less in the human deciduous than in the permanent teeth, but no differences were noted between monkey deciduous and permanent dentitions.


Spore location patterns in sporulating doublets of Bacillus cereus and Bacillus megaterium, derived from single doublet isolates with differing sporulation geometry. The distribution of spore loci in pairs of Bacillus cereus and B. megaterium showed different degrees of polarization from random location towards location at the old end of the sporangium. When individual doublets containing both spores at either the old or new ends of the sporangia were isolated by micromanipulation, subsequent culture showed the same spore location patterns.


Genetic recombination in Streptomyces griseus. Low-frequency (10(-6)) genetic recombination was observed in a cephamycin-producing strain of Streptomyces griseus. The recombinants were predominantly heteroclones. Heteroclone analysis was performed involving four heteroclones of one cross. In 100 mutants correlation was found between the type of auxotrophy and the level of antibiotic activity. A cross of this strain with a streptomycin-producing strain of S. griesus is described.


Growth properties of Cellulomonas flavigena mutants affected in cellulose utilization. The role of cellobiose metabolism in cellulose utilization by Cellulomonas flavigena was investigated by studying mutants unable to grow on cellobiose or cellulose. The results show that the ability to utilize cellulose is strictly dependent on the ability to utilize cellobiose.


Effect of cerulenin on Streptococcus faecalis macromolecular synthesis and cell division. The antibiotic cerulenin has been used to study macromolecular synthesis and cell division in Streptococcus faecalis. The data suggest that lipid and lipoteichoic acid synthesis as well as cell number increase are affected prior to any observable effects on overall mass increase or DNA, RNA, protein, or peptidoglycan synthesis. Treatment with cerulenin of cultures growing at various rates and analysis of the subsequent cell divisions indicate that the antibiotic may block a cell cycle event that precedes the completion of chromosome replication by about 10 min.


TOL plasmid in Pseudomonas aeruginosa PAO: thermosensitivity of self-maintenance and inhibition of host cell growth. The TOL plasmid originally isolated in Pseudomonas putida (arvilla) mt-2 was transmissible to strains of the fluorescens group of Pseudomonas, i.e., P. putida, P. fluorescens, and P. aeruginosa, except for a strain of P. aeruginosa, strain PAO. The same strain, however, could accept the plasmid when its restriction and modification abilities were lost by mutations or by growing at high temperature. In addition, the transmissibility of the TOL plasmid from strain PAO to P. putida was low when the plasmid was modified by the donor. By using P. aeruginosa PAO carrying the TOL plasmid, the stability and genetic expression of the plasmid as well as its effect on the host cell growth were examined. Thus the self-maintenance of the plasmid was found to be thermosensitive. Furthermore, the TOL plasmid inhibited the growth of strain PAO at high temperature, accompanied by the formation of some filamentous cells. These thermosensitive properties of the TOL plasmid were host dependent and not exhibited in another strain of P. aeruginosa.


Comparative immunological studies on arylsulfatase in bacteria of the family Enterobacteriaceae: occurrence of latent arylsulfatase protein regulated by sulfur compounds and tyramine. The arylsulfatases of 21 strains of the family Enterobacteriaceae were compared by measuring their enzymatic activities and immunological reactivities. Enzyme formation under repressing, nonrepressing, and derepressing conditions was tested. Antiserum prepared against pure arylsulfatase from Klebsiella aerobgenes W70 was tested against the enzyme extracts from the strains using double diffusion, quantitative precipitation, and immunoelectrophoresis. No close relationship was found between arylsulfatase activity and immunological cross-reactionship was found between arylsulfatase activity and immunological cross-reactivity. The strains in the family Enterobacteriaceae could be divided into two groups on the basis of the immunological properties of their enzyme. Antisera formed a precipitin band with both active and inactive enzyme proteins from Escherichia, Citrobacter, Salmonella, Klebsiella, and Enterobacter, but not with the proteins from Serratia, Proteus, and Erwinia, even though some strains of these species had enzyme activity. It was also found that the formation of arylsulfatase proteins, irrespective of whether they had enzyme activity, were under regulation by sulfur compounds and tyramine.


Regulation of beta-1,3-glucanase synthesis in Penicillium italicum. The filamentous fungus Penicillium italicum produced a certain level of beta-1,3-glucanase during active growth in a glucose-supplemented medium; however, at a low glucose concentration (2 to 10 mM), derepression took place and the specific activity of the enzyme increased significantly. Derepressed cells (incubated in a glucose-limited medium) accumulated a capacity for the synthesis of beta-1,3-glucanase, which led to a subsequent increase in the specific activity even when the cells were transferred to a medium with an excess of glucose (180 mM). Two protein synthesis inhibitors, cycloheximide and trichodermin, immediately stopped the increase in specific activity when added to derepressed cells. On the other hand, 8-hydroxyquinoline, an RNA a synthesis inhibitor, acted differently, since it permitted the specific activity to increase for some time after being added to depressed cells. Moreover, the concentration of glucose did not affect the 8-hydroxyquinoline-insensitive synthesis of beta-1,3-glucanase. It is concluded that the glucose repression effect on beta-1,3-glucanase production must be exerted at a pretranslational level that could be either mRNA synthesis or some stage of the process involved in its maturation or stabilization.


Induction of anaerobic, photoautotrophic growth in the cyanobacterium Oscillatoria limnetica. Anaerobic photoautotrophic growth of the cyanobacterium Oscillatoria limnetica was demonstrated under nitrogen in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (5micron), a constant concentration of Na2S (2.5 mM), and constant pH (7.3). The photoanaerobic growth rate (2 days doubling time) was similar to that obtained under oxygenic photoautotrophic growth conditions. The potential of oxygenic photosynthesis is constitutive in the cells; that of anoxygenic photosynthesis is rapidly (2 h) induced in the presence of Na2S in the light in a process requiring protein synthesis. The facultative anaerobic phototrophic growth physiology exhibited by O. limnetica would seem to represent an intermediate physiological pattern between the obligate anaerobic one of photosynthetic bacteria and the oxygenic one of eucaryotic algae.


Membrane-bound nucleotidase of Bacillus cereus. A membrane-bound nucleotidase of Bacillus cereus T was solubilized by digestion with trypsin and subsequently purified more than 300-fold. The purified nucleotidase was most active on ribonucleoside 5'-monophosphates and was slightly less active (40 to 60%) on deoxyribonucleoside 5'-monophosphates and ribonucleoside 3'-monophosphates. In addition to hydrolytic activity, the nucleotidase preparation possessed phosphotransferase activity by which phosphate is transferred from a phosphate donor to the 5' position of nucleosides.


Autolytic defective mutant of Streptococcus faecalis. Properties of a variant of Streptococcus faecalis ATCC 9790 with defective cellular autolysis are described. The mutant strain was selected as a survivor from a mutagenized cell population simultaneously challenged with two antibiotics which inhibit cell wall biosynthesis, penicillin G and cycloserine. Compared to the parental strain, the mutant strain exhibited: (i) a thermosensitive pattern of cellular autolysis; (ii) an autolytic enzyme activity that had only a slightly increased thermolability when tested in solution in the absence of wall substrate; and (iii) an isolated autolysin that had hydrolytic activity on isolated S. faecalis wall substrate indistinguishable from that of the parental strain, but that was inactive when tested on walls of Micrococcus lysodeikticus as a substrate. These data indicate an alteration in the substrate specificity of the autolytic enzyme of the mutant which appears to result from the synthesis of an altered form of autolytic enzyme.


Microbial oxidation of methane and methanol: crystallization of methanol dehydrogenase and properties of holo- and apomethanol dehydrogenase from Methylomonas methanica. Procedures are described for the purification and crystallization of methanol dehydrogenase from the soluble fraction of the type I obligate methylotroph Methylomonas methanica strain S1. The crystallized enzyme is homogeneous as judged by acrylamide gel electrophoresis and ultracentrifugation. The enzyme had a high pH optimum (9.5) and required ammonium salt as an activator. In the presence of phenazine methosulfate as an electron acceptor, the enzyme catalyzed the oxidation of primary alcohols and formaldehyde. Secondary, tertiary, and aromatic alcohols were not oxidized. The molecular weight as well as subunit size of methanol dehydrogenase was 60,000, indicating that it is monomeric. The sedimentation constant (s(20,w)) was 3.1S. The amino acid composition of the crystallized enzyme is also presented. Antisera prepared against the crystalline enzyme were nonspecific; they cross-reacted with and inhibited the isofunctional enzyme from other obligate methylotrophic bacteria. The crystalline methanol dehydrogenase had an absorption peak at 350 nm in the visible region and weak fluorescence peaks at 440 and 470 nm due to the presence of a pteridine derivative as the prosthetic group. A procedure was developed for the preparation of apo-methanol dehydrogenase. The molecular weights, sedimentation constants, electrophoretic mobilities, and immunological properties of apo- and holo-methanol dehydrogenases are identical. Apo-methanol dehydrogenase lacked the absorption peak at 350 nm and the fluorescence peaks at 440 and 470 nm and was catalytically inactive. All attempts to reconstitute an active enzyme from apo-methanol dehydrogenase, using various pteridine derivatives, were unsuccessful.


Mapping of a genetic locus that affects glycerol 3-phosphate transport in Bacillus subtilis. Two types of fosfomycin-resistant mutants of Bacillus subtilis were isolated. Mutants of the first type (GlpT mutants) were resistant to at least 200 microgram of fosfomycin per ml and failed to take up exogenous glycerol 3-phosphate. Mutants of the second type were resistant to lower concentrations of fosfomycin and transported glycerol-3-phosphate as efficiently as wild-type bacteria. The glpT mutations, but not the mutations in the second type of fosfomycin-resistant mutants, map in the cysA-aroI region of the B. subtilis chromosome.


Nutrition of Myxococcus xanthus, a fruiting myxobacterium. The minimal requirements for vegetative growth of Myxococcus xanthus have been sought. Isoleucine, leucine, and valine were required, and vitamin B12 was needed for the synthesis of methionine. Pyruvate was an excellent energy source and an efficient source of cellular carbon. Acetate, aspartate, glutamate, and most tricarboxylic acid cycle intermediates could also be utilized, but were less efficient sources of carbon and energy than was pyruvate. Many mono- and disaccharides were tested, but, in agreement with earlier results, none served as carbon-energy sources. A minimal medium (A1) has been devised that includes the essential amino acids and vitamin B12, with pyruvate and aspartate as carbon-energy sources. In this medium, M. xanthus could propagate indefinitely, and on it vegetative cells formed colonies with greater than 75% efficiency; hence, it is likely that no organic cofactors other than those present in A1 are required in more than trace amounts.


Functional expression of two Bacillus subtilis chromosomal genes in Escherichia coli. EcoRI-cleaved deoxyribonucleic acid segments carrying two genes from Bacillus subtilis, pyr and leu, have been cloned in Escherichia coli by insertion into a derivative of the E. coli bacteriophage lambda. Lysogenization of pyrimidine- and leucine-requiring auxotrophs of E. coli by the hybrid phages exhibited prototrophic phenotypes, suggesting the expression of B. subtilis genes in E. coli. Upon induction, these lysogens produced lysates capable of transducing E. coli pyr and leu auxotrophs to prototrophy with high frequency. Isolated  DNAs of these bacteriophages have the ability to transform B. subtilis auxotrophs to pyr and leu independence and contain EcoRI-cleaved segments which hybridize to corresponding segments of B. subtilis.


Cellular autolytic activity in synchronized populations of Streptococcus faecium. The autolytic capacity of Streptococcus faecium (S. faecalis ATCC 9790) varied during synchronous cell division. This phenomenon was initially observed in rapidly dividing populations (TD=30 to 33 min) synchronized by a combination of induction and size selection techniques. To minimize the problems inherent in studies of cells containing overlapping chromosome cycles and possible artifacts generated by induction techniques, the autolytic capacities of slowly dividing populations (TD=60 to 110 min) synchronized by selection only were examined. Although the overall level of cellular autolytic capacity was observed to decline with decreasing growth rate, sharp, periodic fluctuations in cellular autolytic capacity were seen during synchronous growth at all growth rates examined. On the basis of similar patterns  of cyclic fluctuations in autolytic capacity of cultures synchronized by (i) selection, (ii) amino acid starvation followed by size selection, and (iii) amino acid starvation followed by inhibition of DNA synthesis, a link of such fluctuations with the cell division cycle has been postulated.


Bacteriocin and antibiotic resistance plasmids in Bacillus cereus and Bacillus subtilis. A number of plasmids have been isolated as covalently closed circular DNAs from strains of Bacillus cereus and B. subtilis. From 12 out of 15 strains of B. cereus, plasmids could be isolated. Most of the B. cereus strains contained two or more plasmids. Their molecular weights ranged from 1.6 X 10(6) to 105 X 10(6). Bacteriocin production could be attributed to a 45 X 10(6)-dalton plasmid (pBC7) from B. cereus DSM 336, and tetracycline resistance to a 2.8 X 10(6) plasmid (pBC16) from B. cereus GP7. Two streptomycin-resistant strains of B. subtilis harbored plasmids of 5.2 X 10(6) and 9 X 10(6), respectively, which were, however, not correlated with the antibiotic resistance. The plasmid carrying resistance to tetracycline, pBC16, which was originally isolated from B. cereus, could be subsequently transformed in B. subtilis, where it is stably maintained.


Induction of a biopolyester hydrolase (cutinase) by low levels of cutin monomers in Fusarium solani f.sp. pisi. Cutin hydrolysate induced the production of an extracellular cutinase by glucose-grown Fusarium solani f. sp. pisi. The rate of production depended on the amount of cutin hydrolysate added up to 80 mug/ml, and saturation was attained at this level. Glucose was found to be a repressor of cutinase production. A radial immunodiffusion assay for cutinase was developed, and the induction of cutinase by cutin hydrolysate was confirmed by this direct assay. When cutinase was induced by cutin hydrolysate, exogenous labeled phenylalanine was incorporated into cutinase, which was shown to be the major (>70%) protein in the extracellular fluid. Induction of cutinase by cutin hydrolysate was not inhibited by actinomycin D and was stimulated ( approximately 100%) by cordycepin. Addition of cycloheximide with the inducer, or up to 12 h after the addition of the inducer, resulted in a nearly immediate cessation of cutinase production. Deoxyglucose, an inhibitor of proten glycosylation, inhibited the induction of cutinase by cutin hydrolysate. omega-Hydroxy fatty acids were more effective in inducing cutinase than any of the other more polar acids of cutin. Experiments with derivatives and analogues of omega-hydroxy C(16) acid indicated that a free hydroxyl group at the omega-position was the most important factor determining the cutinase-inducing activity. n-Aliphatic primary alcohols with 14 or more carbon atoms induced cutinase, and n-C(16) was the most effective inducer. These results strongly suggest that the monomers function as the chemical signal which induces the extracellular hydrolase.


Study of pole assembly in Bacillus subtilis by computer reconstruction of septal growth zones seen in central, longitudinal thin sections of cells. The septal growth of Bacillus subtilis 168/s has been studied by making a number of observations from thin sections of cells from exponentially growing cultures. The process was initiated by the formation of a new cross wall under a preexisting layer of cylindrical wall. An annular notch appeared to cut through the overlying wall and presumably allowed the cross wall to split into two layers of peripheral wall. During this initial notching process, two raised bands of wall material were produced which resembled those previously observed in morphological studies of Streptococcus faecalis. Through an improved fixation technique, it was possible to preserve the bands seen in B. subtilis to the extent that they were used as markers to study the subsequent stages of septal growth. These stages included (i) the continued displacement of the two bands from the cross wall (as the two nascent polar surfaces enlarged and as the diameter of the cross wall decreased), (ii) the closure of the cross wall, and (iii) the final severance of the common cross wall connection between two completed poles. To study this process in a more quantitative manner, three-dimensional reconstructions of the envelope observed between pairs of the raised bands were made from axial thin sections of cells. The process of reconstruction was based on a technique by which x, y coordinates were taken from thin sections and were rotated around the cell's central axis. These reconstructions were used to estimate the surface area or volume of the reconstructed zones or their parts. A round of septal growth was then simulated by arranging 118 reconstructions in order of increasing surface area or volume. The topology of the process was studied by noting how various measurements of septal thickness, length, surface area, and volume varied as a function of increasing septal zone size. This analysis was based on several assumptions, of which three of the most important are: (i) the bands produced by the initial notching process are markers which separate septal from cylindrical wall growth; (ii) a septal zone observed between pairs of bands is made up of two nascent poles and a single cross wall; and (iii) as septal zones develop in terms of relative age they increase in size (volume or surface area) or amount of wall. The data suggested that the S. faecalis model of surface growth (in which polar growth occurs through a regulated constrictive separation and expansion of a cross wall) also seems applicable to the pattern of septal growth observed here for B. subtilis. This was indicated from measurements which showed that increases in the size of nascent polar surfaces were correlated with decreases in cross wall diameter. An explanation of these observations may be that decreases in cross wall diameter were due to a progressive splitting of the cross wall that removed surface from the outer circumference of the cross wall and converted it into new polar surface. Calculations further suggested that if the poles of B. subtilis were made by this model a sizeable and variable increase in surface area of the cross wall would also be required to convert these separating cross wall layers into two curved polar structures. Measurements of wall thickness taken from various locations within septal zones indicated that while the thickness of the polar wall of B. subtilis was constant over its surface, the width of the cross wall varied considerably during a round of synthesis. Again, one of the simplest explanations compatible with these observations and those previously made in S. faecalis is that the B. subtilis cross wall is brought to a constant thickness (possibly by remodeling or precursor addition) before or during separation. Although most observations made from the reconstruction of the septal zones of B. subtilis may fit the S. faecalis model of surface growth, differences in the pattern of septal growth were seen when the two organisms were compared. These have been discussed in terms of differences in the regulation of their respective septal growth sites and basic mechanisms of wall assembly and modification.


Genetic transformation of pilation and virulence into Neisseria gonorrhoeae T4. Genetic transformation of nonpilated strains of Neisserai gonorrhoeae to pilated forms is described. The transformants displayed phenotypic T1 and T2 colonial morphology on agar and possessed pili visualized by electron microscopy. When T1 or T2 transformant cells were injected into 11-day-old chicken embryos, they exhibited virulence characteristics only slightly less than the parental donor strains, though the parental recipient strains were avirulent. Competence was maximal in the late log phase of growth, and the frequency of transformation of clonal T4s to pilation and virulence approached 2%. DNA extracted from transformants could be used to transform other T4 cells. In the course of this work, a shift to a novel colonial type, designated T2-T3 wrinkled, was observed as a consequence of growth of T4 in presence of enzymatic digests of either DNA or RNA, nucleases or individual deoxy- or ribonucleosides. In sharp distinction to the parental T4, these novel organisms were very pilated; however, they were only minimally virulent. Various nucleic acid analogs could neither induce nor inhibit this population shift. Additionally, DNA extracted from this T2-T3 wrinkled variant could be used to transform genetically both T1 and T4 gonococci to the new morphology.


Isolation and characterization of outer and inner membranes from Pseudomonas aeruginosa and effect of EDTA on the membranes. The outer and inner cytoplasmic membranes of Pseudomonas aeruginosa were separated as small and large membranes, respectively, from the cell envelope of this organism treated with lysozyme in Tris-chloride buffer containing sucrose and MgCl2 by differential centrifugation. The small membrane fraction contained predominantly 2-keto-3-deoxyoctonate (KDO), and little cytochromes or oxidase activities. The small membrane was composed of only 9 polypeptides and showed homogeneous small vesicles electron-microscopically. On the other hand, the large membrane fraction had high cytochrome contents and oxidase activities, and little KDO. The large membrane was composed of a number of polypeptides and showed large fragments or vesicles electron-microscopically. These results indicate that the small and large membranes are the outer and inner cytoplasmic membranes of P. aeruginosa, respectively. The isolated outer membrane showed a symmetrical protein peak with a density of 1.23 on sucrose density gradient centrifugation and the isolated inner membrane showed an unusually high density, probably due to association with ribosomes and extrinsic or loosely bound proteins. EDTA lowered the density of both membranes and caused lethal damage to the outer membrane, causing disintegration with the release of lipopolysaccharide (LPS), proteins and phospholipid.


Catabolic conversion of sepiapterin to 6-(1-carboxyethoxy)pterin by Bacillus subtilis. Structural elucidation of an intensely blue fluorescent compound (A) formed from sepiapterin by Bacillus subtilis is described. The structure of the catabolite (A) was found to be 2-amino-6-(1-carboxyethoxy)-4(3H)-pteridinone (9) from both spectroscopic and degradation studies. This was confirmed by an unambiguous synthesis of 9. The stereochemical structure of the side chain at the 6-position of A was confirmed to be the L(or S) configuration, as in sepiapterin, by analysis of the lactic acid formed from A on acid hydrolysis. This suggests that the side chain is rearranged intact during the catabolic conversion of sepiapterin. A possible mechanism for the conversion is discussed.


Purification and properties of two forms of rat alpha-lactalbumin. Two species of alpha-lactalbumin, alpha-lactalbumin1 and alpha-lactalbumin2, were separated from rat milk and purified to homogeneity by gel filtration, followed by the DEAE-cellulose chromatography. alpha-Lactalbumin1 is a bigger molecule in contrast to other known alpha-lactalbumins, and has a molecular weight of 21,500 as determined by sedimentation equilibrium and sodium dodecyl sulfate-polyacrylamide gel analysis. alpha-Lactalbumin2 has a molecular weight of 16,000 measured by sedimentation analysis. alpha-Lactalbumin2, however, exhibits abnormally high molecular weight of 22,500 on sodium dodecyl sulfate polyacrylamide gels. Both alpha-lactalbumins are active in lactose synthase assay and are glycoproteins containing 7 to 9% carbohydrate. Antiserum raised against alpha-lactalbumin1 cannot discriminate between the two species in a radioimmunoassay.