The effects of total parenteral nutrition on immunodepression due to malnutrition.

An experimental study was performed in 16 dogs to investigate the effects of sub-acute malnutrition on humoral and cellular immunity and phagocytic functions and, subsequently, to investigate the ability of total parenteral nutrition (TPN) to restore abnormal immunological variables. Deficiencies of IgG, C3, primary immune response to sheep red blood cells (SRBC),lymphocyte counts, lymphocyte response to phytohemagglutinin (PHA), and neutrophi chemotaxis were found to be caused by malnutrition. Nutritional repletion by means of TPN resulted in a return to normal or supranormal serum concentrations of IgG, IgM, and C3, and the primary immune response to SRVC was prompter and higher. Moreover, TPN resulted in restoration of normal neutrophil chemotactic responses. TPN did not improve lymphocyte response to PHA in these experiments. The study demonstrates that subacute malnutrition results in broad based deficiencies of the immunological response of the type that predispose to infection and that the proper use of TPN can correct most of these abnormalities.


Metabolic effects of experimental bacteremia.

Hemodynamic and metabolic effects of a lethal 5-hour infusion of Ps. aeruginosa at a dose 10(8) organisms per ml per min were studied in 39 dogs. Blood glucose, insulin, catecholamines, body temperature, WBC, and hemodynamic parameters were measured before and at 1-hour intervals during controlled bacterial infusions. Induced bacteremia in the upper 10(4) range per ml of blood was accompanied by a decline of mean arterial blood presure from 130+/-6 mm Hg to 84+/-12 mm Hg at 4 hours, hypothermia, leukopenia, and hypoglycemia. Death within 24 hours was associated with hypoinsulinemia and increased blood catecholamines. Survival was characterized by maintenance of arterial blood pressure, only moderate decline in blood glucose levels, and normal plasma insulin concentrations with little change in plasma catecholamines. Mortality could be reduced significantly by glucose administration. This was associated with correction of hypoglycemia, rise in plasma insulin activity and increased energy production.


Acute erythroleukemia complicating prolonged chemotherapy for ovarian carcinoma.

Two patients with ovarian carcinoma developed acute erythroleukemia following prolonged chemotherapy with an alkylating agent. An analysis of our patients, together with literature review of similar cases, suggests that the duration of chemotherapy may have been the most important factor in the leukemogenesis. We suggest that the duration of chemotherapy required to achieve a cure in patients with ovarian carcinoma in complete remission can be determined only by prospective controlled trials.


Mechanisms of CO2 fixation in bacterial photosynthesis studied by the carbon isotope fractionation technique.

The carbon isotope discrimination properties of a representative of each of the three types of photosynthetic bacteria Chlorobium thiosulfatophilum, Rhodospirillum rubrum and Chromatium and of the C3-alga Chlamydomonas reinhardii were determined by measuring the ratio of 13CO2 to 12CO2 incorporated during photoautotrophic growth. 2. Chromatium and R. rubrum had isotope selection properties similar to those of C3-plants, whereas Chlorobium was significantly different. 3. The results suggest that Chromatium and R. rubrum assimilate CO2 mainly via ribulose 1,5-diphosphate carboxylase and the associated reactions of the reductive pentose phosphate cycle, whereas Chlorobium utilizes other mechanisms. Such mechanisms would include the ferredoxin-linked carboxylation enzymes and associated reactions of the reductive carboxylic acid cycle.


The cell content and secretion of water-soluble vitamins by several freshwater algae.

Three green algae, Chlamydomonas reinhardii, Chlorella vulgaris and Scenedesmus obliquus, and one blue-green alga, Anabaena cyclindrica, were grown in chemically defined media. All the algae examined contained folates, beta-carotene and vitamins C and E; several of the B-vitamins and vitamin A were found in varying amounts in some but not in all the algae examined. All the green algae secreted significant amounts of folate and biotin and all but Scenedesmus secreted pantothenate into their growth medium; Anabaena secreted folate and pantothenate.


Coupling between the initiation of DNA replication and cell division in the blue-green alga Anacystis nidulans.

The effect of mitomycin C on cell mass increase, cell division, RNA synthesis and DNA synthesis in the blue-green alga Anacystis nidulans has been examined. Data suggests that the initiation of DNA replication, rather than its termination was the necessary event for cell division to occur.


Chimpanzee livers after infection with human hepatitis viruses A and B: Ultrastructural studies.

Electron microscopical studies were carried out on coded liver biopsy specimens from chimpanzees inoculated with human hepatitis A or B virus. Hepatitis B was recognized by the presence of hepatitis B core particles in hepatocellular nuclei. Hepatitis A was characterized by unidentified large, dense, and more irregular heterochromatin-like particles in hepatocellular nuclei coincidental with peak aminotransferase activities. As type A hepatitis illness became manifest in the chimpanzees, mitochondrial cristae were curled and attenuated, and clusters of endoplasmic reticulum were tightly packed. In contrast, the livers in viral hepatitis B showed mainly hypertrophy of tubular smooth endoplasmic reticulum. This suggested different pathogenetic mechanisms in A and B chimpanzee viral hepatitis.


[The epileptic crises of the Lennox-Gastaut syndrome. I. Convulsive forms].

Clinical and electroensephalographic aspects of twenty seven (27) patients with Lennox-Gastaut syndrome were studied (20 without previous West syndrome, group A, and 7 with this antecedent, group B). The epileptic seizures were characterized through descriptions made by relatives who were in close contact with the patients, and also by direct observation by the author in the clinic. The direct observation was possible due to high frequency of the seizures. Denominations were given to the manifestations not previously mentioned in the literature, according to the quality and eventual sequence of observable phenomena, in agreement with the terminology used by the Clinical and EEG Classification of Epileptic Seizures. The incidence, prognostic, clinical and evolutive aspects of elementary forms of epileptic seizures were discussed. Great variability of the convulsive seizures was verified, permitting their division into simple, complex and mixed forms.


[Epileptic crises of the Lennox-Gastaut syndrome. II. Partial and nonconvulsive forms].

Clinical and electroencephalographic aspects of partial and non convulsive forms of epileptic seizures of twenty seven (27) patients with Lennox-Gastaut syndrome were studied (20 without previous West syndrome, Group A, and 7 with this antecedent, group B). The epileptic seizures were characterized through descriptions made by relatives who were in close contact with the patients, and also by direct observation by the author in the clinic. The direct observation was possible due to high frequency rates of the seizures. Denominations were given to the manifestations not previously mentioned in the literature, according to the quality and eventual sequence of observable phenomena, in agreement with the terminology used by the Clinical and EEG Classification of Epileptic Seizures. The incidence and clinical aspects of elementary partial and non-convulsive forms of epileptic seizures were discussed. Great variability of the non-convulsive seizures was verified permiting their division into simple, complex and mixed forms, similarly to the absences. Unilateral forms of atonic seizures were identified.


Macular cherry-red spots and beta-galactosidase deficiency in an adult. An autopsy case with progressive cerebellar ataxia, myoclonus, thrombocytopathy, and accumulation of polysaccharide in liver.

An adult patient with macular cherry-red spots, a gargoyle-like physical appearance, cerebellar ataxia, myoclonus, convulsive seizures, and pyramidal tract signs showed a profound deficiency of beta-galactosidase in liver and brain. Thrombocytopathy of undetermined etiology was evident since childhood, and the patient died of intracranial bleeding at age 22. Cerebral ganglioside pattern was normal. Hepatic mucopolysaccharides were not increased. GM1-gangliosidosis and mucopolysaccharidosis were ruled out by those analytical data. However, a large amount of amylopectin-like polysaccharide was found to be accumulated in liver. Hepatocyte contained numerous inclusion bodies with granulofibrillary structure similar to Lafora bodies, corpora amylacea, and inclusion bodies in glycogenosis type IV. This case seems to represent a new inborn metabolic disease closely related to GM1-gangliosidosis and mucopolysaccharidosis. The primary metabolic defect is not known at present.


Evoked potential in neglect.

The evoked potential was studied in Macaca speciosa before and after they received a lesion inducing neglect. Significant evoked potential changes associated with neglect were seen only in late components (N2, P3). These observations support the hypothesis of the arousal-attention mechanism of neglect.


Induction of subcapsular cataracts in cynomolgus monkeys by echothiophate.

Sixteen eyes of 13 cynomolgus monkeys were treated topically twice daily with echothiophate iodide. The dosages were increased gradually over several weeks to plateau levels ranging from 125 to 500 mug daily. The opposite eyes of the ten unilaterally treated monkeys received a control solution (DILUENT) identical save for the absence of echothiophate. All 16 echothiophate-treated eyes developed posterior subcapsular cataracts and 15 developed anterior subcapsular cataracts. The lenses in all ten DILUENT-treated eyes remained clear. The cataracts in the echothiophate-treated eyes strongly resembled those attributed to cholinesterase inhibitors in humans. They were first noted from 2 1/2 to 14 weeks after beginning echothiophate treatment. Prior total iridectomy caused a more rapid progression and/or greater severity of lens changes. Higher echothiophate dosages caused the cataracts to appear more rapidly.


Aeruginocine typing and antibiotic sensitivity of Pseudomonas aeruginosa of poultry origin.

Aeruginocine typing and an antibiotic sensitivity test were conducted on 277 strains of Pseudomonas aeruginosa isolated from dead-in-shell embryos and on water from waterers of a chick hatchery. Thirty different aeruginocine patterns were obtained using 8 Wahba indicator strains. Of the 277 strains, 129 were typable and 148 were not. The aeruginocine typing patterns of the strains isolated from dead-in-shell embryos and water were similar, suggesting that contaminated water may be the source of infection. Antibiogram revealed that all the strains were resistant to methicillin and tetracycline, and had varying degrees of resistance to other antibiotics tested.


Serologic response to Haemophilus gallinarum in artifically infected and vaccinated chickens.

Serologic responses to Haemophilus gallinarum (HG) were compared in chickens artifically inoculated with living HG and vaccinated with HG bacterin, using tube agglutination (AGG), hemagglutination-inhibition (HI), and agar-gel precipitation (AGP) tests. Infected and vaccinated chickens differed markedly, as did infection routes early after treatment. HI response was delayed and lower in chickens infected intranasally than in vaccinated chickens. Both AGG and AGP responses peaked sooner than HI. AGP response was earlier in infected chickens, but later in vaccinated chickens. A maximum of three lines were observed. All which appeared for vaccinated chickens were similar to those for inoculated ones. A residual line remaining for very long periods for inoculated and vaccinated chickens appeared near the side of well of the HG antigen. The duration of symptoms following HG infection had no correlation with HI, AGG, and AGP titers. From these results, the HI test seems not to be the only serological diagnosis of HG infection in the field.


Rapid fluorimetric assay of minocycline in plasma or serum: comparison with microbiological assay.

1. Plasma concentrations of minocycline were assayed by measuring the fluorescence emission of an aluminum-minocycline chelate in a spectrofluorimeter. 2. The assay gives reproducible results over the concentration range 0.05-5.0 mug ml-1, and compared favourably with results achieved using a conventional large plate agar diffusion microbiological assay in a double-blind comparison of the two methods. 3. Fluorimetric assay of minocycline in plasma was found to correlate closely with antibiotic activity (r = 0.95). The method is easy and economical to perform, results being obtained in 1 h.


Biochemical characteristics of different forms of protein-energy malnutrition: an experimental model using young rats.

I. In three separate experiments, four groups of five to eight young male rats were fed either (i) a high-protein diet, for which the net dietary protein:total metabolizable energy ratio (NDp:E) was 0-1 (HP diet); or (ii) a low-protein diet, for which NDp:E was 0-04 (LP diet). In both these groups, food intake was ad lib. In group (iii) the HP diet was given in an amount approximately equal to that taken by the LP group fed ad lib. (HP-restricted). In group (iv) rats were fasted for 48 h after receiving the HP diet (HP-fasted). Each experiment lasted 4 weeks. 2. In the LP and HP-restricted groups, food intake was about 50% of that of the HP rats, while body-weight, after 4 weeks on diet was about 35% and 55% of that of HP rats, for LP and HP-restricted respectively. Both groups of malnourished rats gained some weight during the experiment. 3. Measurements of oral glucose tolerance and plasma insulin levels were made in the fourth week. LP and HP-restricted rats both showed low fasting insulin levels and low insulin to glucose ratios during the glucose tolerance tests; the LP rats were more seriously affected. 4. At the end of the fourth week the rats were killed and blood, liver and gastrocnemius muscle were analysed. LP rats showed specifically and consistently low values for haemoglobin and plasma protein concentration, and low activities of hepatic glucose-6-phosphatase (EC 3-1-3-9) and of alanine aminotransferase (EC 2.6.1.2) in liver and muscle. The activity of hepatic aspartate aminotransferase (EC 2.6.1.1) was, if anything, increased. The plasma amino acid concentrations and ratios showed a specific fall in branched-chain amino acids. Liver fat concentration was consistently elevated. The HP-restricted rats had normal values for haemoglobin, plasma protein andliver fat, and near-normal values for plasma amino acids. Hepatic alanine aminotransferase showed increased activity compared with HP rats, but muscle alanine aminotransferase showed reduced activity. The HP-fasted rats had increased haemoglobin, plasma protein and liver fat concentration, and very low liver glycogen concentrations. Hepatic alanine aminotransferase activity was elevated. Plasma alanine concentration was specifically reduced. 5. The results are consistent with suppression of gluconeogenesis, liver dysfunction and essential amino acid deprivation in LP rats. These biochemical changes found in rats on a low intake of a diet of low protein and high carbohydrate value are similar to those found in kwashiorkor. An equally low intake of a diet of good protein value (HP-restricted) led to marginally better growth, accompanied by biochemical signs of increased gluconeogenesis, analogous to those reported for nutritional marasmus. This nutritional state was not biochemically identical with that of acute fasting. 6. The results are discussed in terms of the consistency of the rat model, and its contribution to understanding biochemical changes found in infant malnutrition.


Balance study of twenty trace elements during total parenteral nutrition in man.

1. Balances of twenty trace elements (silver, arsenic, gold, bromine, cadmium, cobalt, chromium, caesium, copper, iron, mercury, lanthanum, molybdenum, rubidium, antimony, scandium, selenium, samarium, tungsten and zinc) have been determined in four male patients during total parenteral nutrition incliding fat emulsion and a special solution for addition of Fe, Zn, manganese, Cu, fluorine and iodine, besides calcium and magnesium, to the infusion solutions. 2. The analyses for trace elements were made with the aid of an ion-exchange technique based on neutron activation, and combined with subsequent gamma spectrometry. 3. The intended intravenous supply of trace elements correspond approximately to the analysed supply. However, all the other trace elements determined were found to be unintentionally administered in small amounts. 4. There was a substantial retention of Fe. Other elements retained were Ag, Co, Cr, Cu, Sb, Sc, and W. 5. Particularly Br and Rb were lost by the patients, but negative balances were also found for As, Au, Cd, Cs, Mo, Se and Zn. However, Zn was retained by one patient with short bowel syndrome. 6. The serum concentrations of thirteen (Ag, Br, Co, Cs, Cu, Fe, Hg, Mo, Rb, Sc, Se, W and Zn) of the trace elements were found to have some decrease during the period of total parenteral nutrition, mostly in accordance with the corresponding balance values, Fe, in particular, was found to have the derectional change in concentration. 7. The administration of trace elements is recommended in long-term total parenteral nutrition.


[Nutritional evaluation and use of a nutritional complement in children undergoing periodical hemodialysis].

Six uremic dhildren in periodic hemodialysis with protein-calorie malnutrition were studied. Three of them were given diet supplementation with a compound constituted by carbohydrates and essential amino acids. Evaluation at ,3 and 6 months with somatometry, rutine laboratory analysis, intravenous glucose tolerance test and plasma amino acid determinations, showed that patients with diet supplementation had a slight increase in height and body weight, improved glucose in tolerance, that was initialy detected, and an abnormal pattern of plasma amino acids not modified during the study. Patients without diet supplementation showed no changes in height, body weight, glucose tolerance and plasma amino acids. These results suggest that diet supplementation with carbohydrates and amino acids is useful to improve nutrition in uremic children on hemodialysis, but it is neccesary to study more patients.


Isolation and characterization of an organic solvent soluble polypeptide component from photoreceptor complexes of Rhodospirillum rubrum.

An organic solvent soluble polypeptide has been isolated from photoreceptor complexes and chromatophores of Rhodospirillum rubrum. After extraction of the protein from lyophilized samples with 1:1 chloroform-methanol, it was purified by column chromatography. Its isoelectric point determined by isoelectric focusing was 7.10. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified polypeptide ran as a single band of an apparent molecular weight of 12 000. However, according to amino acid analysis, the minimal molecular weight based on one histidine residue per polypeptide is 19 000. The polypeptide contains no cysteine and no tyrosine. Amino acid analysis indicated that three methionines were present per histidine residue and cyanogen bromide cleavage gave four smaller peptides which were isolated by two-dimensional electrophoresis and chromatography. Spectroscopic analysis indicated the presence of three tryptophan residues per histidine and N-bromosuccinamide cleavage also gave four smaller peptides which could be isolated by two-dimensional electrophoresis and chromatography. The C-terminal amino acid was shown to be glycine by two methods, while the N-terminal amino acid appears to be blocked. The organic solvent soluble polypeptide accounts for approximately 50% of the chromatophore protein and seems to bind the antenna bacteriochlorophyll and carotenoid molecules. Using this procedure, organic solvent soluble polypeptides were isolated from several photosynthetic bacteria and were found to have substantially different amino acid contents.


Tyrosine emission in the tryptophanless azurin from Pseudomonas fluorescens.

A strain of Pseudomonas fluorescens contains an azurin with no tryptophan and two tyrosines. This protein is interesting because it allows one to study both the structure of azurin and the emission of tyrosines in proteins. Comprehensive measurements were carried out including spectrophotometric and fluorimetric titration, fluorescence quantum yield, fluorescence polarization, and I- quenching. In the copper-containing protein, almost independent of the copper ion oxidation, the fluorescence quantum yield is approximately 60% of that of the apoprotein. The latter has the remarkable property that its quantum yield is even greater than free tyrosine. The two tyrosines in the metalloprotein have different pKa's, 10.75 and 12.78, but there is only one average pKa, 10.9 in the apoprotein. The polarization of the fluorescence at 310 nm (290-nm excitation) is 0.32 for the metalloproteins and 0.34 for the apoprotein. I- hardly quenches the fluorescence. The conclusion is that the two tyrosines are inaccesible to the solvent, located in nonpolar environments, larger than or equal to 20 A apart, and not adjacent to the disulfide bridge.


Study of the triplet state properties of tyrosines and tryptophan in azuring using optically detected magnetic resonance.

Optically detected magnetic resonance (ODMR) signals and phosphorescence spectra were seen of tyrosine in the P. aeruginosa and tryptophanless P. fluorescens azurins and of tryptophan in the former. This confirmed a conclusion from other experiments that the tryptophan of P. aeruginosa cannot effectively quench the singlet energy of both tyrosines. The ODMR signals were all very narrow, additional evidence that the chromophores are buried in the interior of the protein. Accurate values of the zero-field coupling constants D and E lead to a tentative correlation of D values with the red shift of the 0 leads to 0 peak of the phosphorescence spectrum. The environment of tryptophan in P. aeruginosa is the most hydrocarbon like of any tryptophan so far observed. The experiments raise a number of unanswered questions concerning rate processes. The intensities of the 2E transition of tyrosine and the phosphorescence of both tyrosine and tryptophan are substantially reduced when the copper is oxidized. Nevertheless the phsphorescence lifetimes are unaffected. A hole cannot be burned in the ODMR resonances. The homogeneously broadened lines may conceivably be a result of low-temperature proton tunnelling.


The photoaddition of trimethylpsoralen to Drosophila melanogaster nuclei: a probe for chromatin substructure.

Derivatives of the furocoumarin, psoralen, can penetrate intact cells or nuclei and cross-link opposite strands of the chromosomal DNA under the influence of long wave-length ultraviolet light. The potential of trioxsalen (4,5',8-trimethylpsoralen) as a probe for chromatin structure has been investigated. The DNA in both embryo nuclei and tissue culture cells from Drosophila melanogaster was found to be about 90% protected from trioxsalen binding relative to purified DNA. Digestion of trioxsalen-treated nuclei by micrococcal nuclease and gel electrophoresis of the resulting DNA gave the same type of band pattern that is characteristic of native, untreated nuclei are digestion. Nuclease digestion was therefore used to examine the distribution of bound trioxsalen in the DNA. The resulting DNA fragments were analyzed both by radioactivity measurements and quantitative electron microscopy. The nuclease cleaved intact photoreacted nuclei in such a way that preferential excision of trioxsalen containing regions of the DNA occurred, but, when acting upon purified DNA that contained bount trioxsalen, it attacked the trioxsalen-free regions preferentially. It was thus concluded that trixosalen binds at the sites corresponding to the regular nuclease-sensitive regions of the chromatin in nuclei.


Transcription and translation of cloned Drosophila DNA fragments in Escherichia coli.

The expression of three unique DNA fragments from Drosophila melanogaster which have been inserted into Escherichia coli (E. coli) via the plasmid, pSC 101, was studied. The hybrid plasmid DNA molecules containing Drosophila DNA were transformed into the minicell producing strain of E. coli, X1411. Drosophila DNA-directed RNA synthesis was studied by hybridizing newly synthesized RNA isolated from the minicells with various DNA fragments which were immobilized on nitrocellulose filters. RNA was synthesized as readily from the inserted Drosophila DNA as from the original bacterial plasmid, pSC 101. In one case, transcription appeared to be initiated preferentially on one of the two strands of a Drosophila DNA fragment regardless of the orientation of that Drosophila DNA fragment with respect to the pSC 101 sequences. Two of the three Drosophila DNA fragments did not induce the synthesis of new polypeptides in minicells as detected by autoradiography of [35S]methionine-labeled polypeptides on polyacrylamide gels. The third Drosophila DNA fragment caused the synthesis of one additional polypeptide of 29 000 daltons. When an 8200 base pair portion of the third inserted Drosophila DNA (63% OF THE TOTAL Drosophila insertion) was removed by digestion with the restriction enzyme, Eco R1, this new polypeptide was no longer synthesized by minicells containing the remaining Drosophila DNA. When the 8200 base pair fragment was placed back into its parent plasmid as an inversion, the new polypeptide did not reappear. In addition, the presence of some, but not all, of the Drosophila DNA insertions affected the relative synthesis of the six polypeptides coded for by the parent plasmid, pSC 101.


Kinetic properties of crystalline enzymes. Carboxypeptidase A.

Spectrochemical probes have demonstrated that the conformations of carboxypeptidase A differ in solution and in the crystalline state. Detailed kinetic studies of carboxypeptidase A crystals and solutions now show that the physical state of the enzyme is also a critical parameter that affects this enzyme's function. Thus, for all substrates examined, crystallization of the enzyme markedly reduces catalytic efficiency, kcat, from 20- to 1000-fold. In addition, substrate inhibition, apparent in solution for some di- and depsipeptides, is abolished with crystals, while longer substrates with normal kinetics in solution may exhibit activation with the crystals. The physical state of the enzyme also affects the mode of action of known modifiers of peptidase activity of the enzyme. In solution, addition of benzoylglycine or cinnamic acid markedly increases the rate of hydrolysis of CbzGly-Phe, but, with the crystalline enzyme, their addition hardly alters the activity. This is in accord with the weakening or absence of inhibitory enzyme-substrate binding modes. Kinetic studies on crystals were carried out over a range of enzyme concentrations, substrate concentrations, and crystal sizes, and in all instances the results are in good agreement with the theory developed by Katchalski for enzymes insolubilized by other means. Importantly, these kinetic parameters are determined under conditions which obviate artifacts due to diffusion limitation of substrates or products. The differences in the kinetic behavior of carboxypeptidase crystals, on the one hand, and of their solutions, on the other hand, bear importantly on efforts to interpret the function of the enzyme in structural terms. Hypothetical modes of substrate-enzyme interaction, generated by superimposing substrate models on the crystal structure of carboxypeptidase to stimulate kinetics in solution, have failed to detect all of these changes which affect inhibitory or activating binding modes.


Heavy-chain variable-region sequence from an inulin-binding myeloma protein.

The entire variable-region sequence of the heavy chain from ABE-47N, a BALB/c inulin-binding myeloma protein, has been determined. This protein is unusual in that the third complementarity region (H3) is extremely short, consisting of at the most three and probably only one amino acid. A comparison of the heavy-chain hypervariable regions from mouse, human, and rabbit proteins shows that the variability in length of H3 is greater than that seen in the first or second hypervariable regions. This variability in H3 length suggests a specialized function for this region.


Characterization of dog small intestinal fucolipids with human blood group A activity. Differences in dog and human A-active fucolipids.

Glycolipids containing fucose (fucolipids) which carried human blood group A activity were isolated from a number of dog small intestines and analyzed. On the basis of sugar analysis, methylation, periodate oxidation, enzyme degradation, mass spectrometry, and immunologic studies, a structure is proposed for these substances. The ceramides of the dog fucolipids containing only hydroxylated fatty acids with 85% saturated and 15% monoenoic acids ranging from 16 to 25 carbon atoms. Sphingosine and phytosphingosine comprised 48% each of the long chain bases. An A-active fraction isolated from human small intestine was shown to have two components, one of which was immunologically distinct and the other identical with the dog intestinal fucolipids. The human fraction differed from the dog fucolipids in migration on thin-layer chromatography and contained two types of amino sugar substitution. It is proposed that the human fraction was composed of two fucolipids with incomplete structures.


Isolation and identification of cytokinins from Euglena gracilis transfer ribonucleic acid.

Three ribonucleosides responsible for cytokinin activity in Euglena gracilis var Bacillaris tRNA have been isolated and identified as 6-(3-methyl-2-butenylamino)-9-beta-D-ribofuranosylpurine, 6-(4-hydroxy-3-methyl-cis-2-butenylamino)-9-beta-D-ribofuranosylpurine, and 6-(4-hydroxy-3-methyl-2-butenylamino)-2-methylthio-9-beta-D-ribofuranosylpurine. The structures of these compounds were assigned on the basis of their chromatographic properties and ultraviolet and mass spectra which were identical with those of the corresponding synthetic compounds. The elution profiles of cytokinin bioassay activity and of 35S radioactivity suggest the presence of a trace amount of 6-(3-methyl-2-butenylamino)-2-methylthio-9-beta-D-ribofuranosylpurine.


Specificity of the weak binding between the phage SPO1 transcription-inhibitory protein, TF1, and SPO1 DNA.

The interaction of the phage SPO1 protein transcription factor 1 (TF1), with DNA has been analyzed by membrane filter binding and by sedimentation methods. Substantially specific binding of TF1 to helical SPO1 DNA can be demonstrated by nitrocellulose filter-binding assays at relatively low ionic strength (0.08). However, TF1-DNA complexes dissociate and reequilibrate relatively rapidly and this makes filter-binding assays unsuitable for quantitative measurements of binding equilibra. Accordingly, the sedimentation properties of TF1-DNA complexes have been explored and a short-column centrifugation assay has been elaborated for quantitative measurements. Preferential binding of TF1 to the hydroxymethyluracil-containing SPO1 DNA has also been demonstrated by short-column centrifugation. TF1 binds relatively weakly and somewhat cooperatively to SPO1 DNA at many sites; TF1-DNA complexes dissociate and reequilibrate rapidly. At 20 degrees C in 0.01 M phosphate, pH 7.5, 0.15 KC1, one molecule of TF1 can bind to approximately every 60 nucleotide pairs of SPO1 DNA.


Purification, properties, and partial structure elucidation of a high-molecular-weight glycoprotein from cervical mucus of the bonnet monkey (Macaca radiata).

A high-molecular-weight glycoprotein has been purified from the cervical mucus of the bonnet monkey (Macaca radiata). The glycoprotein was shown to be homogeneous by electrophoresis, sedimentation equilibrium, and N-terminal group determination, and to contain 19% protein, 19% D-galactose, 18% N-acetyl-D-galactosamine, 15% N-acetyl-D-glucosamine, 11% L-fucose, 10% sialic acid, and 1% sulfate groups, corresponding to about 1800 amino acid residues and 400 carbohydrate side chains of about 9 monosaccharides. The carbohydrate chains are linked to the peptide backbone through N-acetyl-D-galactosamine and serine (or threonine) residues. Reduction with dithiothreitol and alkylation with iodoacetic acid reduced the molecular mass from 1 to 0.5 X 10(6) daltons and produced subunits having the same size, charge, and N-terminal amino acid. Electrophoretic studies suggested the presence of disulfide bonds between two chains of the glycoprotein. Degradation with alkaline borohydride gave, after fractionation on Bio-Gel P-2, fractions containing L-fucose, D-galactose, N-acetyl-D-galactosaminitol, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, and sialic acid in the ratio of 1.0:3.0:1.0:1.0:1.3:1.0. Further fractionation by electrophoresis and paper chromatography gave a charged fraction representing 13% of the original glycoprotein. Enzymic degradation and methylation studies indicated the presence of the structure alpha-Gal-(1 leads to 3)-[Fuc(1 leads to 2)]-Gal-(1 leads to 4)-GlcNAc, linked to a core component containing N-acetyl-D-galactosaminitol.


Composition of a photosystem I chlorophyll protein complex from Anabaena flos-aquae.

The use of Triton X-100 to solubilize membrane fragments from Anabaena flos-aquae in conjunction with DEAE cellulose chromatography allows the separation of three green fractions. Fraction 1 is detergent-solubilized chlorophyll, and Fraction 2 contains one polypeptide in the 15 kdalton area. Fraction 3, which contains most of the chlorophyll and shows P-700 and photosystem I activity, shows by SDS gel electrophoresis varying polypeptide profiles which reflect the presence of four fundamental bands as well as varying amounts of other polypeptides which appear to be aggregates containing the 15 kdalton polypeptide. The four fundamental bands are designated Band I at 120, Band II at 52, Band III at 46, and Band IV at 15 kdaltons. Band I obtained using 0.1% SDS contains chlorophyll and P-700 associated with it. When this band is cut out and rerun, the 120 kdalton band is lost, but significant increases occur in the intensities of Bands II, III, and IV as well as other polypeptides in the 20-30 kdalton range. The use of 1% Triton X-100 coupled with sucrose density gradient centrifugation allows the separation of three green bands at 10, 25 and 40% sucrose. The 10% layer contains a major polypeptide which appears to be Band IV. The 25 and 40% layers show essentially similar polypeptide profiles, resembling Fraction 3 in this regard, except that the 40% layer shows a marked decrease in Band III. Treatment of the material layering at the 40% sucrose level with a higher (4%) concentration of Triton X-100 causes a loss (disaggregation) of the polypeptides occurring in the 60-80 kdalton region and in increase in the lower molecular weight polypeptides. Thus, aggregation of the lower molecular weight polypeptides accounts for the variability seen in the electrophoresis patterns. Possible relations of the principal polypeptides to the known photochemical functions in the original membrane are discussed.


Immobilised monomers of human liver arginase.

Human liver arginase (L-arginine amidinohydrolase, EC 3.5.3.1) was immobilised by attachment to nylon with glutaraldehyde as a crosslinking agent. Incubation of the immobilised tetrameric enzyme with EDTA followed by dialysis resulted in the dissociation of the enzyme into inactive matrix-bound and solubilised subunits. Both species recovered enzymatic activity after incubation with Mn2+, and the activity of the reactivated matrix-bound subunits was nearly 25% of that shown by the enzyme initially attached to the support in the tetrameric form. When the reactivated bound subunits were incubated with soluble subunits in the presence of Mn2+, they 'picked-up' from the solution an amount of protein and enzymatic activity almost identical to that initially lost by the immobilised tetramer after the dissociating treatment with EDTA. This occurred only in the presence of Mn2+. It is suggested that the reactivation of the subunits of arginase involves the initial formation of an active monomer, which then acquires a conformation that favours a reassociation to the tetrameric state.


Selective tissue distribution of carbonic anhydrase isozymes in the ox.

By affinity chromatography the isozymic distribution of carbonic anhydrase (carbonate hydro-lyase, EC 4.2.1.1) has been studied in extract from various bovine tissues. Carbonic anhydrase II forms isolated from erythrocyte, kidney and brain are indistinguishable by specific activity, amino acid composition, fingerprint, electrophoretic and immunological behaviour. By these criteria they differ from carbonic anhydrase I isolated from rumen epithelium.


Soluble and microsomal forms of NADH-cytochrome beta 5 reductase from human placenta. Similarity with NADH-methemoglobin reductase from human erythrocytes.

In congenital methemoglobinemia associated with mental retardation a generalized deficiency of NADH-cytochrome beta 5 reductase (NADH : ferricytochrome beta 5 oxidoreductase, EC 1.6.2.2) has been found in soluble extracts of red blood cells, as well as in deoxycholate-treated extracts of leukocytes, muscle, liver and fibroblasts (Leroux et al. (1975) Nature 258, 619-620). In the present study the relationship between the microsomal (I) and the soluble (II) NADH-cytochrome beta 5 reductase was investigated, using human placenta as a source of enzyme. Both forms were compared to the human red-cell soluble NADH-methemoglobin reductase (III) and NADH-cytochrome beta 5 reductase (IV). The four entities exhibited great immunological similarities. It is concluded that the three soluble enzymes (II, III and IV) are identical. The detergent-solubilized microsomal NADH-cytochrome beta 5 reductase (I) is immunologically very similar to the soluble enzymes, but presents distinct features possibly due to the presence of a hydrophobic part.


Inhibition of lysyl hydroxylase by catechol analogs.

Catechol analogs inhibit the activity of lysyl hydroxylase (peptidyllysine, 2-oxyglutarate: oxygen 5-oxidoreductase, EC 1.14.11.4), a microsomal enzyme which catalyzes the transformation of certain lysyl residues in collagen to hydroxylysine. Chick embryo lysyl hydroxylase activity was measured by specific tritium release as tritiated water from an L-[4,5-3H]lysine-labelled unhydroxylated collagen substrate prepared from chick calvaria. Catechol analogs did not bind irreversibly to either enzyme or substrate, as full activity was restored with dialysis. Addition of excess cofactor, Fe2+, ascorbic acid, or alpha-ketoglutarate, did not affect inhibition. Kinetic analysis revealed that with respect to collagen substrate, catechol demonstrated a noncompetitive type of inhibition with a Ki of 15 muM.


Human erythrocyte pyruvate kinase. Total purification and evidence for its antigenic identity with L-type enzyme.

Erythrocyte pyruvate kinase (ATP:pyruvate 2-0-phosphotransferase, EC 2.7.1.40) has been purified 40 000 times from human erythrocytes, according to an original method. The whole purification procedure included toluene extraction, ammonium sulphate fractionation, DEAE-Sephadex batchwise chromatography and affinity chromatography on a Dextran Blue-Sepharose column with specific elution by fructose 1,6-diphosphate. The final preparation had specific activity of 290 I.U./mg of proteins and the overall yield was about 30%. Pyruvate kinase showed only one protein band as judged by sodium dodecyl sulphate acrylamide gel electrophoresis. Pure enzyme was injected into rabbits and monospecific antiserum was obtained able to neutralize, per ml, 150 I.U. of erythocyte-type pyruvate kinase as well as of L-type enzyme. L-type and erythrocyte-type pyruvate kinases showed reactions of complete identity when tested in immunodiffusion against anti-erythrocyte type pyruvate kinase sera; in all cases a single precipitation line could be detected. L-type pyruvate kinase when mixed with anti-erythocyte pyruvate kinase serum suppressed all ability of that antiserum to react immunological with erythocyte enzyme. Finally the microcomplement fixation curves using anti-erythrocyte pyruvate kinase serum were identical for erythrocyte and L-type enzymes. From these results it appeared that no antigenic difference between L-type and erythocyte enzyme could be detected. Consequently the most likely hypothesis is that both these enzymes are coded by  the same single gene, the slight electrophoretic differences between them being due to post-synthetic tissue-specific changes.


Retinol receptors in corneal epithelium, stroma and endothelium.

Specific receptors for retinol are present in the cytosol fraction of corneal epithelium as demonstrated by sucrose density gradient centrifugation. These appear to be (1) protein in nature (2) of small molecular size (2 S) (3) specific for retinol and (4) present in several species. Assuming a receptor molecular weight of 15 000 and a single mole of retinol bound/mole of receptor protein, the association constant value is 5.26-10(7) with deltaG degrees = -8.53 kcal/mol. 2-S receptors are also observed in stroma and endothelium along with another binding species of approximately 8 S. Binding of [3H]retinol in bovine epithelial cytosol can also be demonstrated by disc gel electrophoresis and gel filtration. Immunodiffusion techniques demonstrate that monkey corneal epithelial and stromal cytosol samples do not contain contaminating serum retinol binding-protein.


The fine structure of luminescence spectra of azurin.

The spectra of azurin absorption, fluorescence, phosphorescence and fluorescence excitation have been measured in aqueous solutions at ordinary and liquid nitrogen temperatures. The fluorescence spectra of azurin even at ordinary temperatures have a well resolved fine vibrational structure. The frequency analysis reveals practically the same wave number distances between the main structure peaks in fluorescence spectra at room and low temperatures and in phosphorescence spectra. The comparison of the protein absorption and excitation spectra shows that all the energy absorbed by tyrosine residues is transferred onto indole chromophore. These data suggest an unusual tryptophan environment in this protein, which is characterized by the absence of any hydrogen bonding or other polar interaction of tryptophan with its environment. The problem of the possibility of contributions of two electronic transitions (1La in equilibrium A and 1Lb in equilibrium A) in absorption and emission spectra of azurin tryptophan arising from their mirror symmetry is discussed.


Fractionation of primary amyloid fibrils. Characterization and chemical interaction of the subunits.

Amyloid fibrils of kappa origin from a patient with primary amyloidosis are dissociated in various denaturants and fractionated into their subunit components on Sepharose 6B. Solubilization of the fibrils in 4 M guanidine-HCl followed by reduction and alkylation produced 22 000 and 17 000 dalton fractions. Without prior reduction and alkylation, these fractions exist as a high molecular weight protein which can be separated on Sepharose 6B. A high molecular weight protein can be directly dissociated from the amyloid fibril with 1% sodium dodecyl sulfate or 1 M NaCl. Reduction and alkylation of this material produces the two lower molecular weight fractions, i.e., 22 000 and 17 000. These have in the first 20 residues identical N-terminal amino acid sequences; they share immunologic identity and have similar tryptic peptide map profiles. Amino acid analysis of the 22 000 dalton fraction is identical with the intact immunoglobulin light chain isolated from the patient's serum. These data suggest that the insoluble amyloid fibril is the result of aggregation by disulfide linkages between the 22 000 and 17 000 dalton fractions.


The N-terminal activation fragment of bovine prothrombin. Immunological studies leading to a one step purification.

Some immunological studies on prothrombin fragment 1 from bovine prothrombin and its warfarin-induced precursor acarboxyprothrombin are reported. Based on the results, a rapid and simple immunoadsorption method for the isolation of prothrombin fragment 1 in good yield has been established. The method exploits the conformational change induced in the fragment by removal of Ca2+. The principle may be applicable to other gamma-carboxyglutamyl-containing proteins or fragments therof.


Temperature-dependent changes of myeloma immunoglobulin G (K) IVA, Bence-Jones protein (K-type) IVA and its fragments.

1. The temperature function of the myeloma IgG(K) IVA, Bence-Jones protein (K-type) IVA and its fragments (Fab(t), Fc'(t), VL and CL) was studied by thermal perturbation difference spectroscopy and circular dichroism. 2. The IgG and Bence-Jones protein studied were found to be capable of a fully reversible structural changes at temperatures between 25 and 35 degrees C. The changes occurring at the higher temperature are accompanied by the screening of the significant part of exposed tyrosine residues. The transition is not accompanied by an appreciable change in the main IgG secondary structure-beta-pleated sheet, according to the CD data. 3. It was found that the temperature-dependent changes of IgG occur in its Fab fragments, the changes of Bence-Jones protein occur in its variable part (VL domains). 4. The temperature changes in the interval 25-35 degrees C are explained by the flexibility of amino acid side chains composed hypervariable loops delineated the "sides" of cavity between variable domains.


Late hexokinase activity expression during Bufo bufo development.

Hexokinase activity was detected in cytosols and homogenates from different developmental stages of Bufo bufo embryos starting from stage 17. Free glucose was measured in the embryo cytosol and was detected at each stage tested. At stage 15, a large increase of glucose content of the embryo cytosol occurs. Hexokinase expression in the embryo thus occurs after the increase of cytosol glucose content occurring at stage 15. The findings rule out that glucose by itself is the hexokinase inducer in vivo. The very low glucose utilization found by many authors during early amphibian development may be related to the late hexokinase expression during Bufo bufo development.


Immunoreactive insulin in amniotic fluid from Rh-immunized women.

Immunoreactive insulin was measured in amniotic fluid from Rh-immunized women (anti-D antibody). The degree of immunization was expressed by the spectrophotometric index of the amniotic fluid and by the capillary hemoglobin concentration of the newborn. Higher levels of insulin were found in amniotic fluid from severely Rh-immunized mothers than from non-immunized, and a correlation between the degree of immunization and insulin concentration was indicated. To our knowledge this has not previously been demonstrated, and may justify some new hypotheses concerning the pathogenesis of the observed higher frequency of hyperinsulinism in babies of Rh-immunized mothers.


Developmental pattern of hepatic uridine diphosphoglucuronosyl transferase activity in growing male rats.

Hepatic uridine diphospho(UDP)glucuronosyl transferase activity was measured in growing male rats to gain the developmental activity pattern of this enzyme catalyzing conjugation of xenobiotics and endogenous substrates. The activity was at a very low level at the age of 14 days showing a 10-fold increase to the age of 72 days whereafter a slight decrease of activity was observed. Simultaneously there was about 30% increase in the hepatic postmitochondrial supernatant protein content. The highly enhanced UDPglucuronosyl transferase activity during the growth may be caused by increased synthesis of enzyme protein or by changes in the micro-environment of the enzyme.


Endocrine studies in the untreated F1 and F2 progeny of rats treated neonatally with thyroxine.

When neonatal rats are made hyperthyroid with large doses of thyroxine during the first 5 days of life, they develop hypothalamopituitary, thyroidal and gonadal abnormalities that persist through life. When female rats, which have been treated neonatally, are subsequently mated to normal males, their offspring show unexpected gonadal and thyroidal defects, such as reduced weaning weight and delayed vaginal opening and first estrus in females; males show a significant increase in relative thyroid weight and a significant decrease in ventral prostate weight. Cross-fostering was done to separate prenatal from postnatal influences. Even more surprising was the finding that when the untreated female F1 progeny were mated with normal males, the untreated F2 progeny showed more defects and to a greater degree than those present in the maternal parent.


A method using solubility to predict dry matter digestibility of cellulosic materials.

A rapid chemical procedure based on the solubility of a holocellulose sample in a system of dimethyl sulfoxide with paraformaldehyde has been developed to provide a laboratory method for predicting dry matter digestibility of cellulose containing samples. The amount of dry matter solubilized by the chemical procedure was closely correlated with anaerobic, in vitro, rumen fluid digestion and with digestibility as measured by aerobic Cellulomonas, sp. bacteria. The quantity of solvent and dissolving time had little effect on solubility over a wide range. The method is rapid, well suited for various cellulosic materials, and may be carried out with simple equipment and facilities.


The growth of L-cells and Vero cells on an autoclavable MEM-peptone medium.

The growth of L-60TM cells (a suspension culture adapted L-cell) on media composed of MEM (minimum essential medium (Eagle)) and bactopeptone autoclaved together or separately under a variety of conditions has veen determined. It has been found that MEM autoclaved with 0.5% bactopeptone at 15 psi for 20 min, cooled and then neutralized with NaHCO3, consistently supported good cell growth of L-60TM and L-929 cells. Similar results were obtained when the MEM and bactopeptone were autoclaved separately. The cells grew initially as a monolayer, subsequently becoming a stationary suspension. Some experiments were carried out with agitated suspension culture of L-60TM cells in the autoclaved MEM-bactopeptone combination with and without added methylcellulose and results were obtained which indicate that large scale suspension culture is possible in this system. Other peptones were also found to support cell growth. The autoclaved MEM-bactopeptone combination also supported the growth of Chang liver and Vero cells. The Chang liver cells rapidly dissociated from the plastic surface but the Vero cells remained sufficiently securely attached so that it was possible to grow them near to confluency in roller bottles.


Gamma heavy chain disease: clinical aspects and characterization of a deleted, noncovalently linked gamma1 heavy chain dimer (BAZ).

This report describes the clinical and immunoglobulin features of a patient with gamma heavy chain disease (HCD), who presented with a clinical picture suggestive of an underlying malignancy rather than the usual picture of lymphoma or granulomatous disease. A unique clinical feature was the nearly total replacement of the submaxillary glands by plasma cells. The patient's serum and urine contained a paraprotein, gammaHCD protein BAZ, which belongs to the gamma1 subclass and forms noncovalently linked dimers with a molecular weight of approximately 60,000 daltons. This mutant protein exhibited a deletion which encompassed most of the variable (V) region, the first constant domain (CH 1), and the hinge region. In addition, preliminary structural analyses demonstrated the replacement of alanine by glycine in position 431 of the carboxyterminal octadecapeptide. This substitution may possibly represent another allotypic marker on IgG1 proteins.


Blood viscosity in Waldenström macroglobulinemia.

Patients with Waldenström macroglobulinemia were studied for the presence or absence of the hyperviscosity syndrome, the relative serum viscosity value, and the calculated whole blood viscosity to identify a level at which symptoms occurred. The majority of symptomatic patients had whole blood viscosity values above 8.0 centipoises. There was a direct correlation between whole blood viscosity and relative serum viscosity, r = 0.75. One patient with central nervous system abnormalities was identified as having a high whole blood viscosity but a low serum viscosity. It was concluded that the vast majority of patients with the hyperviscosity syndrome will be identified by measuring the relative serum viscosity. In patients with central nervous system findings and a low serum viscosity, the whole blood viscosity should be determined either by direct measurement or by calculation.


Determination of the bacteriostatic capacity of neutrophils and monocytes in mixed cell populations.

Cline [1, 2] described a method of determining the phagocytic and bacteriostatic activity of individual types of leukocytes within mixed cell populations. We tried to improve the applicability of this method for the investigation of clinical problems.--Bacteria in the log-phase of growth were incubated in test tubes with leukocytes separated from venous blood. After a short period of phagocytosis 3H-thymidine was added to label DNA-synthesizing organisms. Smears were prepared and processed by autoradiography. The labeling indices of extracellular bacteria and of those phagocytized by neutrophils and monocytes were determined microscopically. The intracellular inhibition of DNA-synthesis was taken as indicative of the bacteriostatic activity of the leukocytes. The proposed modification of Cline's assay is suited to investigate clinical problems of phagocyte dysfunction.


The content of ward rating scales for long-stay patients.

Although ward behaviour rating scales for use with long-stay psychiatric patients by nurses have been in existence for many years, their content tends to reflect the opinions of psychiatrists and psychologists rather than those of nurses. An analysis of five commonly used rating scales illustrates the lack of agreement on what should be included. The opinions of 76 nurses from four separate hospitals were sought, using a 34-item questionnaire, to establish what areas of content were considered important in nursing practice. Apart from agreement on general content, consideration should also be given to the presentation of individual items of ward rating scales.


Treatment tolerance and early complications with extended field irradiation in gynaecological cancer.

Treatment tolerance and early complications have been analysed in patients treated with extended field irradiation and pelvic irradiation alone. At the Centre Georges-François Leclerc, 126 patients were treated for carcinoma of the uterine cervix and endometrium from May, 1972 to November, 1974. All patients received external irradiation using a 25 MV X-ray beam. Follow-up has been nearly complete in all patients with the minimum follow-up being 15 months and the average 24 months. Treatment tolerance was approximately the same for all field sizes except when the field was extended to the top of D12. In this situation the patients occasionally required a short rest during their irradiation. The age of the patient was found to contribute significantly to the tolerance, with 70% of the patients who experienced poor tolerance being older than 60 years. Complications were evenly distributed in all all catagories of field size and appeared to be affected only by age and previous surgery. Of the patients who experienced complications 68% were older than 60 years, and of the 37 patients who had previous surgery, 32% had a complication. With certain treatment modifications the treatment tolerance and early complications appear acceptable when using extended field irradiation for gynaecological cancer.


[Secretory IgA and the antibacterial defenses of the respiratory tract (author's transl)].

The secretory IgA (IgA S) in bronchial mucus is produced by cells which are derived, at least in part, from the BALT (Bronchus Associated Lymphoid Tissue). The physico-chemical characteristics of IgA S (the amino-acid structure of the alpha chain, the secretory component and the J chain) explain three properties of this antibody which enable it to play its part as the first line of defense on the surface of the mucus, namely its resistance to proteolysis, its adherence and its ability to penetrate mucus. The role of IgA S in the antibacterial defense of the respiratory tract has been in doubt for some time because it does not follow the usual processes of antibacterial action, having neither opsonising nor lytic activity. It is thought at present that IgA S exerts its action in an individual way, more directly and peculiar to the secretory immune-apparatus. It acts by 1) causing agglutination of bacteria and by inhibiting their adsorption on the mucous membranes, 2) inhibiting bacterial reproduction and 3) by inactivating bacterial toxins. The combination of these three processes produces a surface defense system which inhibits bacterial penetration of the mucus. Despite the uncertainties which still exist, it is essential that the fundamental immunological data should be included in the complex picture of the mechanisms of defense of the respiratory tract.


Immune responses to soluble antigens of treponemes.

Sonicates from five cultivable treponemes were used as antigens in delayed hypersensitivity tests and macrophage inhibition assays. Immunodiffusion analysis showed that the sonicate comprised two major antigenic components which were not distinguishable in the skin tests. The sonicate antigens elicited significant cell-mediated immunity in guinea-pigs. Treponema refringens biotype Nichols proved to induce the strongest delayed response. Delayed skin hypersensitivity to the antigens of Treponema pallidum was found in eight rabbits without orchitis, but not in six rabbits with T. pallidum orchitis. In contrast, the rabbits with syphilitic orchitis gave the strongest reactions with the non-pathogenic spirochetes. In terms of the cell-mediated immunity responses, Treponema phagedenis Reiter was found to be related to T. phagedenis Kazan 4 and Treponema denticola. Treponema scoliodontum was related to T. phagedenis Kazan 5, and T. refringens biotypes Nichols and refringens. The antigens of T. pallidum had the closest relationship to T. refringens biotypes refringens and Nichols ,T. phagedenis biotype Reiter, and T. scoliodontum. It was also demonstrated that some three of 12 human syphilitic sera reacted with the antigens of T. pallidum but not with control sera.


Scanning electron microscopical examination of gonococcal urethral exudate.

The relationship of gonococci and phagocytes in gonococcal urethral exudate was examined using a scanning electron microscope. Several stages in the phagocytic process were evident. These included pseudopodias, microvilli, and simple attachment of gonococci to phagocytic surfaces. Epithelial cells, also a part of the exudate, were examined in this study. Parallels are drawn between this and other phagocytic systems. The technique provides excellent preservation of phagocyte-bacterial interrelationships and offers a means of investigating the nature of the resistance of Neisseria gonorrhoeae to phagocytosis.


Columnar distribution of cortico-cortical fibers in the frontal association, limbic, and motor cortex of the developing rhesus monkey.

The terminal distribution of cortico-cortical connections was examined by autoradiography 7-8 days following injections of tritium labeled amino acids into the dorsal bank of the principal sulcus, the posterior part of the medial orbital gyrus, or the hand and arm area of the primary motor cortex in monkeys ranging in age from 4 days to 5.5 months. Labeled axons originating in these various regions of the frontal lobe have topographically diverse ipsilateral and contralateral destinations but virtually all of these projections share a common mode of distribution: they terminate in distinct vertically oriented columns, 200-500 mum wide, that extend across all layers of cortex and alternate in regular sequence with columns of comparable width in which grains do not exceed background. Spatial periodicity in the pattern of transported label in such regions as the prefrontal association cortex, the retrosplenial limbic cortex and the motor cortex indicates that columination in the intracortical distribution of afferent fibers is not unique to sensory specific cortex but is instead a general feature of neocortical organization. A columnar mode of distribution of cortico-cortical projections is present in monkeys at all ages investigated but is especially well delineated in the youngest of them. Thus, grain concentrations within columns are very high in monkeys injected at 4 days of age, somewhat lower in monkeys injected at 39-45 days of age, and least dense in those injected at 5.5 months. The distinctness of the spatially segregated pattern of innervation in the cortex of neonates indicates that the columnar organization of association-fiber systems in the frontal and limbic cortex is achieved before or shortly after birth.


Effect of lid suture on retinal ganglion cells in Macaca mulatta.

Previous work has established the pronounced effect of unilateral lid suture during visual immaturity on cell growth in the lateral geniculate nucleus of cats, dogs and monkeys. Most investigators have reported the retinas to be grossly normal but only a few quantitative studies are available and the results are contradictory. We have compared cell section diameters and cell density of parafoveal retinal ganglion cells in the deprived and non-deprived eye of Macaca mulatta after the lids of one eye were studied between the ages of 1-9 weeks for varying periods of time. The results show a decrease in the size and density of parafoveal retinal ganglion cells after long-term (24 months) visual deprivation. Lid suture of lesser duration (2-56 weeks) had no effect on the size of retinal ganglion cells.


Amino acid incorporation in medulla, pons, midbrain and cortex following spinal cord hemisection in the cebus monkey (Cebus apella).

The effects of spinal cord hemisection on protein labeling in the medulla, pons, midbrain, and cortex in the Cebus monkey were studied by measuring the uptake of [3H]lysine into the trichloroacetic acid (TCA) precipitable and non-precipitable fractions of brain. Animals were sacrificed 3, 6 or 13 days after hemisection. Two normal animals and a 3-day sham operate were utilized as controls. Differences included individual animals, operated vs. unoperated animals, brain areas, and variation of brain areas over all postoperative days. The overall trend in brain radioactivity over postoperative days indicates stress and non-stress related components of brain response to spinal injury. Left-right side differences (L greater than R) were noted in the uptake of [3H]lysine into specific areas of brain over postoperative time, including leg areas of motor cortex, occipital cortex, and superior colliculus. A biphasic action of leg sensory cortical areas over time was noted, where L greater than R for the first 6 postoperative days and R greater than L at day 13. The factors possibly mediating radiolabeled amino acid uptake into protein are discussed in relation to demonstrated anatomical and neurochemical regenerative processes.


Heterogeneous afferents to the inferior parietal lobule of the rhesus monkey revealed by the retrograde transport method.

The sources of afferent connections to the inferior parietal lobule (rostral part of the area 7 of Brodman; PF and rostral part of PG of von Bonin and Bailey) were examined with the retrograde transport method in infant and adult rhesus monkeys. Two to 3 days after injections of horseradish peroxidase (HRP) into the cortex, the animals were anesthetized, and the brains fixed and processed for the histochemical demonstration of the enzyme marker. Labeled neurons were found in layer III in the ipsilateral prefrontal, parietal, occipital and temporal cortices, notably in areas 5, 19, 22 and 46 of Brodmann, and in area 7 of the contralateral parietal cortex. In the thalamus, HRP-positive cells were located ipsilaterally in the medial pulvinar nucleus in the nuclei centrum medianum and parafascicularis, as well as in the rostral thalamus, lateral and medial to the mammillothalamic tract, in the nucleus ventralis anterior and nucleus paracentralis. Numerous labeled cells were also identified in the magnocellular nuclei of the basal forebrain, in the dorsal and medial raphe nuclei, and in the locus coeruleus. Most of the cells in these regions were located in the hemisphere ipsilateral to the injections, but a number of them were also found in the contralateral hemispher. In adult monkeys, brownish granules in the cytoplasm of some cells were interpreted as endogenous pigment or due to various pigment precursors. However, all 14 locations listed above were identified in the infant monkey in which endogenous pigment was not a confounding factor.


Efferent connections of the hippocampal formation in the rat.

In this investigation the projections of the hippocampal formation to the septal area and hypothalamus were studied in the rat with the combined use of 3H-amino acid radioautography and horseradish peroxidase histochemistry. The results indicate that all of the fibers which project to the hypothalamus and the majority of fibers which project to the septum arise from the subicular cortex and not from hippocampal pyramidal cells. The projection to both of these areas are topographically organized along the longitudinal axis of the hippocampal formation. Specifically, fibers from subicular cortical cells situated at the septal end of the hippocampal formation which project through the medial part of the dorsal fornix terminate in the dorsomedial quadrant of the lateral septal nucleus and in the dorsal portion of the pars posterior of the medial mammillary nucleus. Fibers from progressively more posteroventral levels of the hippocampal formation which project through more lateral portions of the dorsal fornix and fimbria terminate in progressively lateral and ventral quadrants of the lateral septal nucleus and in progressively more ventral portions of the pars posterior. Concerning the specific origin of the fornix system, fibers from only the prosubiculum and subiculum project through both the pre- and postcommissural fornix. Hippocampal pyramidal cells from all CA fields have a restricted projection through the precommissural fornix and terminate in the caudal half of the septum while the presubiculum projects solely through the postcommissural fornix. The medial corticohypothalamic tract (MCHT) was found to arise from cells located in anterior ventral levels of the subicular cortex. Fibers from this tract appeared to be distributed throughout the pericellular region of the entire ventromedial extent of the hypothalamus from the level of the suprachiasmatic nucleus through the level of the medial mammillary nucleus. In this way, the mammillary bodies receive input from the subicular cortex via two routes: the descending column of the fornix and the MCHT.


Ethane-1-hydroxy-1, 1-diphosphonate (EHDP) effects on incorporation and accumulation of osteoclast nuclei.

Male rats weighing 100 g were injected with vehicle (control group), 0.4 or 4.0 mg/kg/day of ethane-1-hydroxy-1,1-diphosphonate (EHDP). Beginning after 6 days of EHDP treatment all rats received six subcutaneous injections of 3H-thymidine at 8 h intervals. The multiple labeling index of autoradiographically defined labeled cells was determined for the osteoprogenitor cells of the primary spongiosa of the proximal tibial metaphysis. The osteoclasts in the proximal tibial metaphisis were assayed for population parameters and incorporation of labeled nuclei at sequential intervals after the administration of 3H-thymidine. After 6 days of EHDP pre-treatment the multiple labeling index of the osteoprogenitor cells was reduced with 4.0 mg/kg/day dose of EHDP. At the beginning of the experimental kinetic sampling period, after the 6 days of EHDP pre-treatment, EHDP treatment resulted in significant increases in the numbers of osteoclasts, nuclei per osteoclast profile, and total osteoclast nuclei. During the sampling period, the 4.0 mg/kg/day dose of EHDP caused further increases in the number of total osteoclasts. EHDP treatment caused a marked increase in the rate of incorporation and accumulation of 3H-thymidine-labeled nuclei in osteoclasts. Thus, EHDP causes an accelerated rate of osteoclast production from percursor cells in spite of its ability to slow bone resorption. Although the origin of osteoclast nuclei is not clear, the kinetic data from this study suggests that osteoclast nuclei are not arising from osteoprogenitor cells or osteocytes in the EHDP-treated animals.


A comparative study of peripherally inserted silicone catheters for parenteral nutrition.

One hundred patients receiving parenteral nutrition with lipids and hypertonic amino acids and glucose were divided into five groups of 20, depending on the type of intravenous catheter used for the infusion. Least satisfactory were the short Butterfly needles (average 3.3 days in place) and the long peripherally inserted polyvinyl central venous catheters (average 6.2 days in place). Subclavian catheters of polyvinyl (average 15.3 days) or silicone elastomer (average 17.5 days) were equally efficacious. A new long silicone elastomer catheter inserted peripherally was most satisfactory (average 29.5 days). Problems common with polyvinyl catheters (phlebitis, thrombosis, and sepsis) rarely occurred with either the long or short silicone elastomer catheter.


Retinal tumours in neurofibromatosis.

A 45-year-old male with von Recklinghausen's disease was noted to have hamartomatous lesions of the retinae similar to those more commonly described in Bourneville's disease and von Hippel-Lindau's disease. These findings would appear to support the concept that the retinal changes in neurofibromatosis occupy an intermediate position between those seen in the other two phakomatoses.


Discrete-trial control of morphine self-injection behaviour in monkeys: effects of injection dose and trials per session.

Responding for intravenous injections of morphine was studied using a discrete-trials procedure in squirrel monkeys. For one group, each session consisted of 10 trials at an inter-trial interval of 15 min; for the second group, each session consisted of 100 trials at an inter-trial interval of 1.5 min. When different injection doses of morphine (1-1000 mug/kg per injection), including saline as a control procedure, were substituted at random for blocks of five consecutive sessions, the frequency of morphine self-administration was found to be an inverted U-shaped function of the injection dose. This relationship was observed in each group of monkeys, despite a 10-fold difference in the total amount of the drug which was available for self-administration per session when a given injection dose was substituted for both groups. The results show that the injection dose of morphine acted as a primary determinant of response probability, even under circumstances in which trial spacing imposed a significant delay between consecutive opportunities for drug self-administration,


Effects of chronic ethanol administration in the rat: relative dependency on dietary lipids. II. Paradoxical role of linoleate in the induction of hepatic drug-metabolizing enzymes in vitro.

The effect of chronic ethanol administration on the hepatic microsomal cytochrome P-450 content and activities of NADPH - cytochrome P-450 reductase (EC 1.6.2.4), benzphetamine demethylase, aniline hydroxylase (EC 1.14.14.1), and of the microsomal ethanoloxidizing system were studied in various dietary models. When ethanol was given with linoleate as the only source of dietary lipid, the ethanol induction of these parameters was greater with diets containing 2 or 5% of total calories as linoleate than with diets containing 10% of total calories as linoleate. By contrast, when ethanol was given with high fat (35% of total calories) diets, the ethanol induction of these same parameters was slightly greater when linoleate provided 10% of total calories than when it provided 3% of calories. The apparent effect of dietary linoleate on the induction, by ethanol, of microsomal drug-metabolizing enzymes is markedly different when linoleate is given as the only source of dietary lipid as opposed to when it is given with other dietary lipids. Thus, conclusions on the effect of ethanol on hepatic microsomal drug-biotransformation enzymes, drawn from studies with dietary models in which linoleate provides the only source of dietary lipid, connot be extended to dietary modles of more complex lipid composition. When given as the only source of lipid, 2% of total calories in linoleate appears optimal for basal activity and inductibility, by ethanol, of mixed-function oxidases.


Comparative in vitro metabolism of aflatoxicol by liver preparations from animals and humans.

The metabolism of [14C]aflatoxicol by liver postmitochondrial and microsomal fractions from humans and eight other species was compared. A major metabolic pathway involves the dehydrogenation of aflatoxicol yielding aflatoxin B1. Human liver preparations were more active in this regard than preparations from any of the other species tested. The aflatoxicol dehydrogenase activity was mainly associated with the microsomal fraction and required a hydrogen acceptor (e.g., nicotinamide adenine dinucleotide phosphate), but was not inhibited by carbon monoxide, which implies that it was not dependent on the heme-containing microsomal drug-metabolizing system. It had a pH optimum of 8.0. Postmitochondrial liver fractions also oxidized aflatoxicol (and/or the aflatoxin B1 made from it) to at least five other metabolites that comigrated on thin-layer chromatography plates with authentic standards of aflatoxins Q1,P1,H1,M1, and B2a. None of these oxidative metabolites were formed in the presence of carbon monoxide. We also report on the in vitro reduction of aflatoxin B1 to aflatoxicol by the cytosol fractions from eight species. Most active in this regard were rabbit and trout preparations, while this activity was almost absent in the guinea pig. Preparations from humans and four other species were intermediate between these extremes.


Characterization of rat lung epoxide (styrene oxide) hydrase with a modified radioactive assay of improved sensitivity.

The epoxide hydrase assay developed by Oesch et al. (Biochim. Biophys. Acta, 227: 685-691, 1971) using [3H]styrene oxide as substrate was modified in three ways for use with rat lung microsomes: the substrate was purified before use, the volume of the incubation mixture was scaled down 4-fold, and the incubation time was extended to 45 min (activity was found to be linear for at least 60 min). These modifications increased the sensitivity of the assay procedure 75- to 150-fold. The procedure was found to be linear with lung microsomal protein up to at least 1.8 mg protein per incubation mixture. This modified assay for epoxide hydrase was used to characterize the enzyme in rat lung. Its apparent vmax is 0.5 nmole of styrene glycol formed per min per mg microsomal protein, and its apparent Km was 0.11 to 0.25 mM. The pH optimum is around 9.7. Upon subcellular fractionation of lung tissue, expoxide hydrase distributes in the same manner as a marker for the endoplasmic reticulum (reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase) and in a different way from markers for the nuclei, mitochondria, concentric lamellar organelles, lysosomes, Golgi membranes, plasma membrane and soluble cytoplasm. The specific activity of epoxide hydrase in rough and smooth lung microsomes is aobut the same. Treatment i.p. of rats with methylcholanthrene (3 injections of 20 mg/kg), phenobarbital (5 daily injections of 80 mg/kg) or styrene oxide (5 daily injections of 40 mg/kg), did not induce lung microsomal epoxide hydrase activity. 1,1,1-Trichloropropene 2,3-oxide was shown to be an uncompetitive inhibitor, and cyclohexene oxide was a noncompetitive inhibitor of this enzyme. Ethanol and butanol activate the epoxide hydrase of lung microsomes at low concentrations and inhibit it at higher concentrations.


gamma-L-Glutaminyl-4-hydroxybenzene, an inducer of cryptobiosis in Agaricus bisporus and a source of specific metabolic inhibitors for melanogenic cells.

A stable phenol, gamma-L-glutaminyl-4-hydroxybenzene (GHB), is oxidized by tyrosinase in the gill tissues of the mushroom Agaricus bisporus to a quinone and a second oxidation product which together suppress mitochondrial energy production and the synthesis of proteins and nucleic acids in the zygote, thus establishing dormancy in the spores. Brief incubation of cultured murine L1210 leukemia and B-16 melanoma cells with muM concentrations of the purified quinone notably prolonged survival times or blocked tumor growth in histocompatible mice inoculated i.p. with high concentrations of the exposed cells. The instability of the quinone precluded in vivo administration. The short incubation of cultured B-16 melanoma cells with mM concentrations of GHB markedly prolonged survival times or abolished tumor growth in histocompatible C57BL/6J mice inoculated i.p. with 5 X 10(6) exposed cells. This response did not occur with L1210 leukemia cells, which lack the enzyme tyrosinase. The survival times of mice bearing B-16 melanoma, but not of those with L1210 leukemia, were slightly prolonged by a single injection and were significantly extended by daily i.p. injections of GHB. Normal C57BL/6J mice, given GHB i.p. as single or multiple 400-mg/kg doses, manifested no systemic toxicity but showed depigmentation of the hair after 2 to 3 weeks. These studies provide evidence that GHB exerts cytotoxicity specifically for cells that by their content of tyrosinase convert the phenol to the quinone. This targeted response minimizes systemic toxocity and underscores the potential therapeutic application of this agent to melanocarcinoma.


Induction of resistance to L1210 leukemia in BALB/c X DBA/2Cr F1 mice, with L1210 cells treated with glutaraldehyde and concanavalin A.

Immunization with L1210 mouse leukemia cells treated with glutaraldehyde and concanavalin A resulted in the induction of leukemia-specific immune resistance in BALB/c X DBA/2Cr F1 mice. This was measured by survival of the mice after challenge either with the intact cells or with modified leukemia cells of less malignancy. Factors involved in the induction of immunoprophylaxis were analyzed. To produce an immune response against L1210 cells, preincubation with both glutaraldehyde and concanavalin A was required. Repeated sensitization with this combination produced higher incidences of survivors upon subsequent challenge with L1210 cells than did a single sensitization by equivalent or larger doses. However, the sensitization was rendered less effective by repetition of the immunization process five times.


Similarity of the mechanism of chemical carcinogen-initiated teratogenesis and carcinogenesis in mice.

A maximum tolerated dose (15 mug/g) of the carcinogen 4-nitroquinoline 1-oxide (4NQO) induced neither fetal deaths nor malformations when given to pregnant ICR/Jcl mice at the sensitive stages (Days 9 to 11) for the induction of malformations, although these embryotoxicities were detected with urethan and X-ray. This may not be due to the lack of teratogenic actions of 4NQO, but to the difficulty this compound has in reaching the embryo, because direct injection of 4NQO into the amniotic cavity of the Day-11 embryo, so that exposure was more direct, induced a high incidence of malformations. Similarity of the mechanism of chemical carcinogen-initiated teratogenesis and carcinogenesis was also suggested by the following findings. Urethan-initiated teratogenesis was almost completely inhibited by posttreatment with caffeine during the period of 0 to 24 and 24 to 48 hr after urethan treatment, whereas it was not inhibited during the 48- to 72-hr post-urethan and the 6- to 30-hr pre-urethan period. The results are similar to those of 4NQO-initiated transformation in cultured mouse embryo cells and 4NQO- and urethan-initiated lung tumorigenesis in mice. Cells carrying preteratogenic or pretumorigenic damage produced by some chemical carcinogens may be extremely sensitive to caffeine treatment during and/or after the postcarcinogen DNA replication period, thus resulting in decrease of malformations and tumors. The process may be related to error-prone DNA repair, because caffeine is known to inhibit the postreplication repair in cultured mouse cells.


Cooperation of immune lymphoid and reticuloendothelial cells during Listeria monocytogenes-mediated tumor immunity.

Listeria monocytogenes (LM) in admixture with cells from a murine, chemically induced tumor retarded local tumor development in the syngeneic host. Intra-footpad growth of 10(4) tumor cells was equally inhibited by 4 x 10(4) admixed LM in normal or LM-immune mice indicating that concomitant or prior immunity to LM was equally effective in suppressing tumor growth. Development of cellular immunity to viable LM was required for tumor rejection. Mice prevented from developing anti-LM immunity by inoculation of dead bacteria were also incapable of inhibiting tumor growth. Further, a functionally active reticuloendothelial system was essential for nonspecific inhibition of tumor development as temporary "paralysis" of the reticuloendothelial system by a prior injection of 10(9) heat-killed LM reduced the effectiveness of LM-mediated tumor suppression. Histological examination of LM or LM tumor-injected sites revealed a stepwise development of LM-mediated inflammatory reaction of delayed type associated with gradual degeneration of the adjacent tumor cells.


Evidence for a functional role of RNA in centrioles.

Basal bodies, purified from Chlamydomonas and Tetrahymena, were exposed to various enzymatic treatments and then assayed for their ability to nucleate aster formation upon injection into eggs of Xenopus laevis. Untreated basal bodies injected into frog eggs act as centrioles and induce the formation of asters. The aster-inducing activity of basal bodies was eliminated by treatment with proteolytic enzymes and ribonucleases. Aster-inducing activity was not affected by DNAse and a number of other enzymes. The effect of proteolytic digestion on aster-inducing activity appeared to be directly correlated with the degree of structural damage to the basal body. Low concentrations of pancreatic ribonuclease A, ribonuclease T1, and S1 nuclease also completely abolished aster-inducing activity, although these enzymes had no effect on basal body structure. Ribonuclease-treated basal bodies remained capable of supporting microtubule elongation in vitro. Preliminary evidence indicates that basal bodies from Chlamydomonas and Tetrahymena contain about 5 x 10(-16) g of RNA which co-band with basal bodies and aster-inducing activity by equilibrium density gradient sedimentation. We conclude first, that centrioles contain RNA which is required for initiation of aster formation, and second, that the centriole activity or ability to assemble a mitotic aster is separable from the basal body activity, or ability to serve directly as a template for microtubule growth.


Synthesis of hybrid bacterial plasmids containing highly repeated satellite DNA.

Hybrid plasmid molecules containing tandemly repeated Drosophila satellite DNA were constructed using a modification of the (dA)-(dT) homopolymer procedure of Lobban and Kaiser (1973). Recombinant plasmids recovered after transformation of recA bacteria contained 10% of the amount of satellite DNA present in the transforming molecules. The cloned plasmids were not homogenous in size. Recombinant plasmids isolated from a single colony contained populations of circular molecules which varied both in the length of the satellite region and in the poly(dA)-(dt) regions linking satellite and vector. While subcloning reduced the heterogeneity of these plasmid populations, continued cell growth caused further variations in the size of the repeated regions. Two different simple sequence satellites of Drosophila melanogaster (1.672 and 1.705 g/cm3) were unstable in both recA and recBC hosts and in both pSC101 and pCR1 vectors. We propose that this recA-independent instability of tandemly repeated sequences is due to unequal intramolecular recombination events in replicating DNA molecules, a mechanism analogous to sister chromatid exchange in eucaryotes.


Protein expression in E. coli minicells by recombinant plasmids.

The polypeptides synthesized in E. coli minicells from recombinant plasmids containing DNA fragments from cauliflower mosaic virus, Drosophila melanogaster, and mouse mitochondria were examined. Molecularly cloned fragments of cauliflower mosaic virus DNA directed the synthesis of high levels of three polypeptides, which were synthesized entirely from within the cloned virus DNA fragments independent of their insertion into the plasmid vehicles. Several fragments of D. melanogaster DNA were capable of initiating polypeptide synthesis; however, termination of these polypeptides was dependent upon the insertion into the plasmid vehicle. The majority of D. melanogaster DNA fragments examined did not direct the detectable synthesis of any polypeptides. Insertion of DNA into the Eco RI site of ColE1 and pSC101 plasmids resulted in the altered expression of plasmid-encoded polypeptides. In the case of ColE1, this site of insertion lies within the colicin E1 structural gene, and insertion of foreign DNA into the site results in the synthesis of an inactive truncated colicin E1 molecule. It is probable that the Eco RI site in pSC101 lies within the structural gene for a polypeptide involved in tetracycline resistance, and insertion of DNA into this site may also result in the synthesis of a truncated or elongated polypeptide.


Formation of lipid peroxides in isolated rat liver microsomes by singlet molecular oxygen.

Rat liver microsomes were incubated in neutral aqueous solution of potassium peroxychromate, a system which generates singlet molecular oxygen. Such incubation resulted both in a rapid decline in NADPH-cytochrome c reductase activity, and in an increase in formation of lipid peroxides. These reactions were not inhibited by either superoxide dismutase (SOD) or mannitol, nor were they entirely duplicated by incubating microsomes with hydrogen peroxide. However, a high concentration of 1,4-diazabicyclo-[2,2,2]octane (DABCO), a known scavenger of singlet oxygen, prevented both decline in reductase activity and formation of lipid peroxides. These results suggest that the observed effects are, in fact, attributable to singlet oxygen, and not to hydrogen peroxide, superoxide radical, or hydroxyl radical.


On the inhibition of microsomal drug metabolism by polychlorinated biphenyls (PCB) and related phenolic compounds.

The in vitro metabolism of p-nitroanisole, aminopyrine, and aniline by rat liver microsomal monoxygenases were studied in the presence of different polychlorinated biphenyl (PCB) mixtures and some related hydroxybiphenyls. The tested PCB mixtures contained preferably dichloro- (di-CB), tetrachloro- (tetra-CB), or hexachlorobiphenyls (hexa-CB). All PCB were competitive inhibitors of only aminopyrine demethylation by normal microsomes (Ki 22-39 micron). In microsomes of PCB-pretreated rats the aminopyrine demethylation was inhibited noncompetitively by di-CB and hexa-CB whereas tetra-CB remained a competitive inhibitor (Ki 12 micron). Moreover, after PCB pretreatment all PCB were competitive inhibitors of p-nitroanisole demethylation. 2-OH-biphenyl and 4-OH-biphenyl caused competitive inhibition of aminopyrine demethylation and aniline hydroxylation but failed to inhibit p-nitroanisole metabolism by normal microsomes. Chlorinated 4-hydroxybiphenyls inhibited competitively the metabolism of both type I and type II substrates. However, after PCB pretreatment all phenolic compounds caused uncompetitive inhibition of aniline hydroxylation.


Altered regulation of macromolecular synthesis in methionine-inhibited cultures of Pseudomonas fluorescens UK1.

Effects of high methionine concentrations on growth of Pseudomonas fluorescens UK1 are reported. The following phenomena were observed: (i) Immediate inhibition of growth for a period corresponding to approximately half a generation. Steady-state conditions of growth were no more attained. (ii) In spite of stringency of the macromolecular synthesis in this organism, simultaneously with the growth inhibition, the rate of labelled leucine incorporation into trichloroacetic acid (TCA)-insoluble material was reduced 60% while the rate of labelled uracil incorporation remained constant. (iii) The organism began to liberate methanethiol half a generation after the methionine supplement. Demethiolating activity increased linearly with the cell mass. It is concluded that the inhibition of growth is not due to the liberation of methanethiol from methionine but the amino acid is able to uncouple the mechanism coordinating protein and RNA synthesis in P. fluorescens UK1.


[Formation of omega-muricholic acid and fecal excretion of biliary acids in rats].

Two groups of Rats which were fed the same diet, excreted fecal bile acids of different patterns: in one group, the feces contained a large amount of omega-muricholic acid and very little hydrodeoxycholic acid; in the other group the reversed composition was observed. The first group excreted more total bile acids than the second. This observation suggests that the formation of a large amount of omega-muricholic acid results in an increase in total bile acid fecal excretion.


[Simultaneous isolation of rabbit glomerular and tubular basement membrane].

A method allowing simultaneous isolation of Rabbit alomerular and tubular basement membranes has been perfected. Rabbit tubular basement membrane has also been obtained by tryptic digestion of the renal cortex. Electron microscopic observation and biochemical analysis show that the contamination of the membrane preparations by cellular components is negligible.


[Fahraeus effect and Fahraeus-Lindqvist effect].

A series of experiments has been carried out on the flowing of suspended human erythrocytes into glass-tubes whose diameters varied between 30 mum and 1 mm. Thus, the Fahraeus and Fahraeus-Lindqvist effects emergence fields have been drawn, and it has been shown that, contrary to a widely admitted hypothesis, the Fahraeus effect does not account for the Fahraeus-Lindqvist effect.


[Effect of ethanol on amino acid penetration in to mouse brain].

The uptake of large neutral aminoacids across the blood brain of mouse is increased by previously intraperitonealy given ethanol. The uptake of small neutral, acid and basic aminoacids is not modified. Observed increase is correlated with octanol-water istribution coefficient.


[Chromatographic fractionation and studies on microheterogenity of cow lactotransferrin prepared by an original procedure].

Authors describe an original procedure to prepare pure lactotransferrin from cow milk. Physicochemical properties of this lactotransferrin have been studied and compared with results from others. New data are presented: presence of fucose and N-acetylgalactosamine; C-terminal amino-acid identified with threonine. On the other hand, 4 fractions have been obtained by "DEAE-Sephadex" chromatography, study of which demonstrates that the microheterogeneity of the lactotransferrin depends on the carbohydrate moiety and especially on the N-acetylneuraminic acid content which varies from 0 to 2 residues.


[Elective induction in mice of solid and ascitic carcinoma of the exocrine pancreas by the repeated doses of antibodies against a lipoprotein lipase fraction].

Small doses, repeated for months of anti-esterase-lipoprotein antibodies, from several antisera raised against a crude lipoprotein-lipase from Rabbit adipose tissue (2), induce exocrine pancreatic carcinomas in mice, without any specific barrier. This seems to be the first case of a quite elective carcinogenic process of the pancreas, by an immunological mechanism, with a very high incidence, up to 100%. Such carcinomas are highly metastatic, produce a pancreatic carcinomatous ascites, and are deadly. This immunological pathogeny is discussed., more particulary on an action of immuno-complexes in an abnormal physiological environment.


Response of the cerebral circulation in baboons to changing perfusion pressure after indomethacin.

An earlier study has demonstrated that indomethacin, a prostaglandin synthesis inhibitor, blocks the cerebrovascular response to hypercapnia. This response is believed to be mediated by a lowering of pH in the cerebral interstitial fluid. Should autoregulation of cerebral blood flow (CBF) to changing perfusion pressure also be mediated by a changing interstitial pH (the "metabolic" theory), then indomethacin should impair autoregulation. This hypothesis was tested in anesthetized baboons. CBF was measured by the intracarotid 133Xe clearance technique; the preparation and the indomethacin protocol were identical to those of our previous investigation. Arterial pressure was increased by the intravenous infusion of angiotensin and decreased by controlled hemorrhage. Indomethacin was given by continuous infusion into the internal carotid artery. Although it reduced resting CBF, the cerebrovascular response to changing perfusion pressure was unchanged. Because indomethacin affects the response to changing CO2 but not that to changing perfusion pressure, the mechanisms for these two reactions presumably are different and it is improbable that changing interstitial pH is responsible for autoregulation in the cerebral circulation.


Influence of inhibitors of prostaglandin synthesis on renal vascular resistance and on renal vascular responses to vasopressor and vasodilator agents in the cat.

We determined the effects of indomethacin and meclofenamate, two inhibitors of prostaglandin synthesis, on renal vascular resistance and on renal responses to nerve stimulation, pressor and depressor hormones in the in situ feline kidney under conditions of controlled blood flow. Both inhibitors produced a gradual rise in renal vascular resistance which became maximal 15-20 minutes after administration. The increase in renal resistance after indomethacin was not attenuated during intrarenal infusion of either phentolamine or SQ 20881. Pretreatment with propranolol, in a dose sufficient to inhibit renin secretion, also did not attenuate the increase in renal resistance produced by indomethacin. However, infusion of [Sar1-, Ala8]angiotensin II, an angiotensin II antagonist, did attenuate the indomethacin-induced increase in renal vascular resistance. After indomethacin, the vasoconstrictor response to norepinephrine was enhanced, whereas responses to nerve stimulation and angiotensin were unaffected. Although meclofenamate enhanced renal vascular resistance, its effects on vasoconstrictor responses were inconsistent. After indomethacin, the renal dilator response to bradykinin was enhanced; however, dilator responses to nitroglycerin were unaltered. The present data indicate that the increase in renal vascular resistance after indomethacin does not depend on the adrenergic system but may be dependent on the renin-angiotensin system. The inconsistent effect of the inhibitors of synthesis on renal constrictor responses to nerve stimulation suggests that endogenous prostaglandins do not serve to modulate the effects of the sympathetic nervous system on the feline renal vascular bed. These results also indicate that renal dilator responses to bradykninin are not mediated by prostaglandins in the cat.


Serial serum creatine phosphokinase MB isoenzyme activity after myocardial infarction. Studies in the baboon and man.

Serum CPK-MB iosenzyme activity was measured serially after mercury embolization of the left circumflex coronary artery in five baboons and after clinical acute myocardial infarction (AMI) in 20 patients. The calculated amount of enzyme released into the baboons' circulation (CPK-MBR) correlated well (r = 0.991) with the amount of MB isoenzyme depleted from the myocardium (CPK-MBD) when a previously determined decay constant (Kd) was used, but not when Kd was calculated from individual curves or when CPK-MM values were used. In clinical AMI, CPK-MMR averaged 97% (0 to 350%) greater than CPK-MBR, probably because of release of MM isoenzyme from nonmyocardial sources. The mean Kd for CPK-MB in patients (0.0012 min-1) was significantly (P less than 0.01) lower than that obtained in the baboon following bolus injections (0.0018 min-1), probably reflecting delayed myocardial release of enzyme. Therefore, in both experimental and clinical AMI, serial samples for CPK-MB activity, but not total or CPK-MM activity, could provide an accurate index of myocardial enzyme depletion.


Experiments on the influence of oriented stress on bone formation replacing bone grafts.

A cleft maxilla is healed when it has the attributes of a normal maxilla. Experimental and clinical observations suggest that bone perimeters can be extended by biophysical stimuli induced through functional forces when the tissue is under tension. An experiment was designed to test whether the establishment of bone across the cleft is similarly influenced. Bone most consistently replaced split-rib grafts when tension was applied to the segments of surgically produced maxillary clefts. Additional observations on this model indicate that a suture can be developed in the bone bridge across the cleft margins. While the parameters are not established, the hypothesis cannot be rejected that tension facilitates the extension of bone inducing stimuli and influences the differentiation of tissue in intra-alveolar transplants.