Studies on the transfer of tocopherol between lipoproteins.

The net transfer of labeled alpha-tocopherol from donor to acceptor lipoproteins at physiological concentrations was investigated. Labeled lipoproteins were isolated i) following in vitro addition of [3,4-3H] all rac-alpha-tocopherol to plasma, or ii) from plasma obtained 12-16 h after ingestion by normal subjects of an oral dose (100 mg each) of 2R,4'R,8'R-alpha-[5,7-(C2H3)2]tocopheryl acetate and 2S,4'R,'R-alpha-[5-C2H3]tocopheryl acetate. A constant amount (on a protein basis) of labeled lipoprotein was incubated with an increasing amount of unlabeled acceptor lipoprotein for 2 h at 37 degrees C. No discrimination between stereoisomers of alpha-tocopherol was detected. Labeled VLDL and labeled LDL (very low and low density lipoproteins, respectively) tended to retain their labeled tocopherol. Labeled high density lipoproteins (HDL) readily transferred the labeled tocopherol to VLDL (> 60% transferred), while the transfer to LDL was dependent upon the ratio of labeled HDL/LDL with a lower net transfer at higher ratios. This dependency of the distribution of tocopherol upon the ratio of HDL/LDL was also observed in vivo. The tocopherol/mg HDL protein was measured in 11 subjects with varying HDL levels. As the % HDL in the plasma increased from 14 to 50%, the tocopherol/HDL protein also increased (r2 = 0.37, P < 0.05).


Myogenic differentiation of the muscle clonal cell line BC3H-1 is accompanied by changes in its lipid composition.

Phospholipid and neutral lipid composition was studied in the course of myogenic differentiation of the clonal cell line BC3H-1. Total phospholipid content increased during differentiation, predominantly in the major classes of choline and ethanolamine glycerophospholipids. The contents of other lipids, such as triacylglycerols, diminished more than 50% during this period. The content and distribution of fatty acids also underwent marked differentiation-dependent changes. The polyunsaturated (tetrapenta- and hexaenoic) fatty acid species of several phospholipid classes diminished during differentiation, especially those in choline, serine and inositol glycerophospholipids. Most noticeable were the changes in phosphatidylserine; long-chain fatty acids having 20 to 22 carbon atoms and 4 to 6 double bonds decreased from about 30 to about 10 mol%. Although increased levels of saturation in other phospholipid fatty acyl chains appear to accompany the myogenic changes of BC3H-1 cells, some unsaturated fatty acids, such as oleic acid (18:1), increased by as much as 80% during the same period, suggesting the activation of a delta 9 desaturase. Sphingomyelin contained only saturated and monoenoic fatty acids and exhibited a four- to five-fold decrease in its content of monoenoic acyl groups. Diacylglycerols became enriched in arachidonate and docosahexaenoate. The amount of cholesterol and its esters increased slightly during differentiation of BC3H-1 cells. The data show that several metabolic pathways change during myogenic differentiation of the BC3H-1 clonal cell line, particularly de novo biosynthetic pathways, elongation/desaturation reactions, and acyl chain turnover.(ABSTRACT TRUNCATED AT 250 WORDS)


Occurrence of long and very long polyenoic fatty acids of the n-9 series in rat spermatozoa.

Dietary deficiency of essential fatty acids of the n-3 and n-6 series is known to promote a compensatory increase in polyenoic fatty acids of the n-9 series in the lipids of mammalian tissues. In the present study long-chain n-9 polyenes were found to be normal components of the epididymis and especially of sperm isolated from that tissue, in healthy, well-fed, fertile rats maintained on essential fatty acid-sufficient diets. The n-9 polyenes occurred in large concentrations in the choline glycerophospholipids (CGP), the major phospholipid class of spermatozoa in epididymal cauda, and were highly concentrated in plasmenylcholine, the major subclass of CGP. The uncommon polyene 22:4n-9 was found in the highest proportion, followed in order of relative abundance by 22:3n-9, 20:3n-9 and 24:4n-9. These polyenes were probably derived from oleate (18:1n-9) in much the same way as long-chain polyenes of the n-6 and n-3 series are derived from linoleate (18:2n-6) and linolenate (18:3n-3), respectively.


Isolation and characterization of novel 2-hydroxy fatty acids from the phospholipids of the sponge Smenospongia aurea.

The Caribbean sponge Smenospongia aurea revealed the presence of six novel branched alpha-hydroxy fatty acids: 2-hydroxy-17-methyloctadecanoic acid, 2-hydroxy-21-methyldocosanoic acid, 2-hydroxy-22-methyltricosanoic acid, and 2-hydroxy-22-methyltetracosanoic acid, 2-hydroxy-24-methylpentacosanoic acid, and 2-hydroxy-23-methylpentacosanoic acid. These novel alpha-hydroxy fatty acids were associated with phosphatidylethanolamine. The sponges Aplysina lacunosa and Aplysina fistularis also contained considerable amounts of alpha-hydroxy fatty acids, the very long-chain 5,9,23-tricontatrienoic acid (30:3), and phytanic acid. The sterol composition of the three sponges was also studied. It indicated that A. lacunosa and A. fistularis contained large amounts of aplysterol and verongulasterol, while S. aurea did not show any of these sterols. The results are discussed in terms of the taxonomy of the species.


Cell membranes and multilamellar vesicles: influence of pH on solvent induced damage.

Pigment leakage from sheep and horse erythrocytes and from red beet tissue induced by non-polar solvents was determined as a function of pH. The results were compared to disruption of multilamellar vesicles (MLV) composed of phospholipids with equimolar cholesterol under identical conditions of solvent exposure and pH. Solvent access to cholesterol was used to measure vesicle disruption. MLV were made from 1,2-dioleoyl phosphatidylethanolamine, sphingomyelin (SP) and various phosphatidylcholines to simulate the major lipid components of membranes. Pigment leakage from erythrocytes caused by petroleum hydrocarbon (b.p. 60-80 degrees C) was maximal at pH 2-4 and at pH 10, but minimal at pH 6.8; alcohols caused less pigment leakage than petroleum hydrocarbon. Beta-cyanin leakage from beet tissue induced by petroleum hydrocarbon was maximal at pH 2, with very little leakage at pH 4, 6.6 and pH 10. Alcohols caused minimal damage to beet tissue above pH 2. Cholesterol removal by petroleum hydrocarbon from MLV of mixed lipid composition was maximal at pH 2-4, reduced at pH 6.8 and minimal at pH 10. Lipid mixtures in which fatty acyl side chains of one phospholipid were of a different length than the other lost more sterol than mixtures in which the acyl side chains were of identical chain length. MLV with more than 25% SP lost more sterol than those with less or no SP. Results show that in mixtures of phospholipids, SP exposes the hydrocarbon phase of a bilayer to solvent extraction, a property that was also observed in native membranes.(ABSTRACT TRUNCATED AT 250 WORDS)


Evaluation of an oleic acid water-in-oil-in-water-type multiple emulsion as potential drug carrier via the enteral route.

A water-in-oil-in-water (W/O/W) emulsion composed of oleic acid was used as a carrier of carboxyfluorescein (CF) via the enteral route, as a model for future drug transport. The absorption of CF in the small intestine of rats given the emulsion (W/O/W group) was compared with the absorption in a group administered CF alone (CF group), and a group administered a mixed micelle of oleic acid and a surface-active agent in CF solution (MM group). Higher amounts of CF were absorbed in the W/O/W and MM groups than in the CF group. At 120 min, the amount of CF remaining in the intestinal tract was smaller in the MM group than in the W/O/W group. In the early period, CF excretion into bile was higher in the MM group than in the W/O/W group, but from 120 to 360 min, CF excretion in the W/O/W group was higher than in the MM group (non-specific). The blood CF level was significantly higher at 240 and 360 min in the W/O/W group than in the other two groups. The highest concentration in lymph was found in the W/O/W group. The W/O/W emulsion was considered superior to the micelles because it maintained a higher blood level of CF over long periods and transferred it to the lymph. This suggests that the W/O/W emulsion is applicable as a drug carrier via the enteral route.


Omega-3 fatty acid and cholesterol content of newly hatched chicks from alpha-linolenic acid enriched eggs.

Egg yolk was enriched with alpha-linolenic acid (18:3n-3) by feeding laying hens diets containing flax, canola or soybean seeds. Fertilized eggs were incubated and the fatty acid composition of whole body, liver, plasma, brain and the cholesterol content of plasma and liver tissue of the hatched chicks were studied. Eggs enriched with 18:2n-6 fatty acids by feeding hens diets containing sunflower seeds were used as the controls. Feeding flax enriched (P < 0.05) egg yolk and the developing progeny with 18:3n-3, 20:5n-3, 22:5n-3 and 22:6n-3. Feeding sunflower seeds resulted in an increase (P < 0.05) of 18:2n-6, 20:4n-6, 22:4n-6 and 22:5n-6. The predominant polyunsaturated fatty acid of the brain was docosahexaenoic acid (22:6n-3) which was higher (P < 0.05) in the flax and canola fed group. The cholesterol content of the liver tissue was lower (P < 0.05) in chicks hatched from hens fed flax seeds. This study indicates that 18:3n-3 and 18:2n-6 in the maternal diet are potent modulators of long-chain polyunsaturated n-3 or n-6 fatty acid and of cholesterol content in the developing progeny.


Effect of dietary linoleic acid content on the distribution of triacylglycerol molecular species in rat adipose tissue.

The present study examined the effect of varying dietary linoleate intake (0.01, 0.24, 2.4, 24, 80 or 160 g/kg diet) for 24 weeks on the distribution of triacylglycerol (TG) molecular species in rat epididymal adipose tissue. Adipose TG fractions were purified by thin-layer chromatography and separated into different molecular species by reverse-phase high-performance liquid chromatography. The identification of TG species was based on fatty acid composition, retention time and the theoretical carbon number. When the dietary 18:2n-6 content was equal to or less than 24 g/kg, no significant amounts of n-6 fatty acids (mainly 18:2n-6) were observed in adipose tissue TG despite the fact that the levels of 20:4n-6 in liver phospholipids increased significantly. There were 12 major molecular species in adipose tissue when the dietary 18:2n-6 content was less than 2.4 g/kg. When the dietary 18:2n-6 content reached 24 g/kg, an additional six TG species containing one, two or three molecules of 18:2n-6 were observed. The levels of TG molecules containing two or three 18:2n-6 residues were further increased when the diet contained very large amounts of linoleic acid (160 g/kg). Conversely, those TG species containing only one 18:2n-6 residue became less abundant. It is suggested that the accumulation of these linoleate-rich TG molecular species in adipose tissue, particularly di- and trilinoleoyl containing TG, is the result of an adequate or an excessive intake of linoleic acid.


Site-specific differences in the fatty acid composition of human adipose tissue.

The fatty acid composition of triacylglycerols from fifteen distinct adipose depots taken from each of seven adult male human subjects was compared. Oleic, palmitic, linoleic, stearic, myristic, palmitoleic and vaccenic acids accounted for more than 90% of the triacylglycerol fatty acids in all sites from all subjects; a number of other fatty acids were also identified and quantified. There were large differences in the average fatty acid composition between individual subjects. There were no site-specific differences in the proportions of myristic (3.8-4.7% of triacylglycerol fatty acids), palmitic (23-29%), linoleic (6.7-9.8%) or vaccenic (4.1-4.7%) acids or in the proportions of any of the less abundant fatty acids. There were some significant site-specific differences in the proportions of palmitoleic, oleic and stearic acids. The calf depot contained more palmitoleic acid (6.41 +/- 1.09%) than the trapezius (3.12 +/- 0.55%), perirenal (3.59 +/- 0.50%) and mesenteric (3.70 +/- 0.43%) depots, more oleic acid (42.13 +/- 1.27%) than the trapezius (36.03 +/- 2.18%), perirenal (36.50 +/- 1.56%) and breast (37.13 +/- 1.55%) depots and less stearic acid (5.18 +/- 0.89%) than the trapezius (8.57 +/- 0.97%), perirenal (8.49 +/- 0.75%), mesenteric (7.87 +/- 0.42%), breast (8.02 +/- 0.75%) and clavicular (8.34 +/- 0.78%) depots. The buttock depot contained less stearic acid (6.06 +/- 0.65%) than the perirenal, mesenteric and clavicular depots, while the anterior thigh depot contained less stearic acid (6.07 +/- 0.70%) than the perirenal depot. These findings indicate that, while most human adipose depots differ little in fatty acid composition, some sites, in particular the calf, perirenal, trapezius and mesenteric depots, have site-specific properties.


Inhibition of phenytoin bioactivation and teratogenicity by dietary n-3 fatty acids in mice.

Evidence suggests that the teratogenicity of the anticonvulsant drug phenytoin (DPH) can result from its bioactivation via embryonic prostaglandin synthase and/or maternal cytochromes P450. This study examined whether DPH bioactivation and teratogenicity could be reduced by dietary n-3 fatty acids. Female CD-1 mice were fed diets containing 2 wt% safflower oil and 10 wt% of either hydrogenated coconut oil, safflower oil, or a cod liver oil/linseed oil mixture (CLO/LO) for three weeks prior to impregnation and throughout gestation. DPH (55 or 65 mg/kg) was administered via intraperitoneal injections to pregnant mice at 0900 on gestational days 12 and 13, and on day 19 fetuses were given teratologic assessments. A similar dietary study evaluated in vivo covalent binding of radiolabeled DPH administered on day 12, and dams were killed 24 h later. A reduction in DPH-induced cleft palates and a decrease in DPH covalent binding to embryonic protein was observed in the CLO/LO group. Feeding CLO/LO enhanced incorporation of n-3 fatty acids into embryos and inhibited embryonic prostaglandin synthase activity. No differences in maternal hepatic cytochromes P450 activities were observed among dietary treatments. These data indicate that dietary n-3 fatty acids could reduce DPH teratogenicity via inhibition of embryonic prostaglandin synthase bioactivation of DPH.


Plasma lipids and fatty acids in urbanized Bushmen, Hereros and Kavangos of southern Africa (Namibia).

Thirty-nine urbanized ethnic Namibian people comprising 21 Bushmen (semi-urbanized), 7 Hereros and 11 Kavangos were assessed for plasma lipids and fatty acid (FA) composition. Total cholesterol and triacylglycerol concentrations were measured by enzymatic methods, and neutral lipid FA composition by gas-liquid chromatography. The results demonstrated that while total cholesterol concentrations were not significantly different, significant differences in triacylglycerol concentrations (P < 0.05) were seen between Bushmen and Kavangos. By comparing Bushmen with Hereros and Kavangos, significant differences between Bushmen and Kavangos were also observed in plasma triacylglycerol FA compositions, particularly 16:0 (32.73% vs. 25.05%), 16:1n-7 (7.00% vs. 5.06%), 18:2n-6 (9.30% vs. 22.25%) and 20:3n-6 (0.12% vs. 0.48%), while Kavangos had higher 20:4n-6 levels than Hereros (1.44% vs. 2.00%). In plasma cholesteryl esters, Bushmen were significantly different from Kavangos in 16:1n-7 (8.85% vs. 4.93%), 18:1n-9 (32.06% vs. 23.07%) and 20:4n-6 (6.91% vs. 10.00%). Significant differences were also observed between Bushmen and Hereros in 18:0 (1.08% vs. 1.29%) and 18:2n-6 (35.68% vs. 45.50%). The FA of Namibian groups were also compared with South African reference groups comprising urbanized whites and Xhosas and rural Vendas. The differences in blood lipid values can be explained primarily by excessive alcohol consumption. These results suggest that semi-urbanized Bushmen have changed their diets under urbanized conditions which may increase their risk of coronary heart disease.


Toxoplasmosis and laboratory workers: a case-control assessment of risk.

The health risks to staff employed in a toxoplasma reference unit were studied. Sixteen laboratory staff exposed to Toxoplasma gondii and age/sex-matched controls from the general population and a routine microbiology laboratory were assessed. Multiple assays were performed to establish the presence of toxoplasma infection, and laboratory workers were questioned regarding environmental and work-related exposure to the parasite. Details of all of environmental exposure to toxoplasma between the two groups of laboratory personnel, and seroprevalence rates were comparable in all three groups. Three laboratory accidents were recorded, a rate of one per 9300 hours exposure. One case of presumed work-related infection was identified, but significant illness was not observed. Given adequate training, strict adherence to conventional laboratory protocols and medical supervision, laboratory accidents are infrequent and work-associated toxoplasma infection is uncommon. The risks associated with occupational exposure to T. gondii have been over-estimated and we conclude that this organism does not represent a significant health hazard to laboratory technicians.


Fundamentals of the polymerase chain reaction: a future in clinical diagnostics?

The polymerase chain reaction (PCR) is an example of a technique that is having a profound impact on both fundamental and applied clinical science research. The availability of PCR-based diagnostic kits for the detection of polymorphisms within the HLA-DQA1 locus portends a technology that will undoubtedly become part of the clinical laboratory's diagnostic arsenal, and will extend and/or refine laboratory-based diagnosis in many areas. With current research effort directed to increase our knowledge of the overall structure of the human genome, and the identification of disease-associated genes and sequences, we can anticipate correspondingly rapid advances in its applications. This paper briefly reviews the basic facets of the PCR, which suggest it will fulfill such a role in future clinical diagnosis.


Gel techniques in blood group serology.

Serological investigation involves the interpretation of haemagglutination reactions, conventionally obtained in liquid phase systems. Gel centrifugation techniques utilise buffered dextran gels which separate red cells from their suspension media during a centrifugation phase, negative reactions resulting in the formation of a pellet of cells whereas agglutinations are trapped on top of, or throughout, the gel. The gels may be neutral or impregnated with specific reagents such as antihuman globulin, or antisera for phenotyping red cells. Gel systems have been found to be more sensitive than conventional serological techniques, particularly for the detection and identification of clinically significant antibodies. The elimination of the wash phase in the antiglobulin test, and the stability of the completed tests, provide greater reliability of results and enhance standardisation and control of laboratory procedures.


Measurement of faecal ammonia.

A modified end-point enzymatic method for the measurement of ammonia in stool water is presented. A protein precipitation step was included in order to inactivate urease and faecal enzymes, which oxidise NADH. The modified method is reliable, with acceptable precision and accuracy, and is linear up to a concentration of 1.5 mmol/l.


Factor VIIIc inhibitors: an automated assay.

A semi-automated assay for factor VIIIc inhibitors is described which is easy to perform, suitable for processing infectious risk samples, allows for the measurement of human and porcine inhibitors and may be used for kinetic studies of factor VIIIc inhibitors.


Haemoglobin A2 measurement using high performance liquid chromatography.

Haemoglobin A2 levels were assessed using a modular high performance liquid chromatography (HPLC) system with a protocol designed for the measurement of haemoglobin A1C. There was good correlation (r = 0.96; P < 0.001) between this technique and the International Committee for Standardisation in Haematology (ICSH) recommended method of microchromatography. The range of haemoglobin A2 was found to be 2.3-3.2% in apparently normal individuals, and 4.0-6.7% in those with beta-thalassaemia trait. The HPLC system produced reliable results quickly for haemoglobin A2 with no alteration to the protocol used for measuring haemoglobin A1C.


von Willebrand factor antigen: a radial immunodiffusion method evaluated and compared with an ELISA method.

A new commercial kit method for the quantification of von Willebrand factor antigen (vWFAg) by radial immunodiffusion was compared to an established ELISA technique. Major discrepancies were found between the two methods. The radial immunodiffusion method had poorer intra- and inter-assay coefficients of variation than the ELISA method, although there was adequate inter-method agreement when 100 plasma samples from controls and patients were compared. However, there was a great difference in values obtained for vWFAg in the reference sample supplied with the commercial kit and that obtained directly from the National Institute for Biological Standards and Controls. There were also differences in levels of significance when vWFAg was measured by the two techniques in different clinical groups, and standard deviations were larger when the kit method was used. It is suggested that on scientific and economic grounds, the commercial radial immunodiffusion kit does not offer a competitive advantage over the ELISA method.


Legionella pneumophila species identification using a commercial latex agglutination kit: a potential cross-reaction problem with serogroup 12.

Seventy-nine strains of legionella spp. and 13 non-legionella isolates were tested using a commercial latex agglutination kit. Cross-reactions were observed for all strains of Legionella pneumophila serogroup 12 examined between latex particles coated with rabbit antibodies raised against L. pneumophila serogroup 1 and those coated with rabbit antibodies to serogroups 2-14. Strains of L. pneumophila serogroup 1 did not cross-react with latex particles directed against L. pneumophila serogroups 2-14. This suggests that L. pneumophila serogroup 12 shares common surface antigens with L. pneumophila serogroup 1, and that conversely serogroup 1 isolates do not share major surface antigen determinants with serogroups 2-14. All other isolates tested gave expected results.


Counts of viable tubercle bacilli in sputum related to smear and culture gradings.

Pairs of sputum specimens obtained pre-treatment from 166 smear-positive patients with pulmonary tuberculosis were examined by direct smear, culture on Löwenstein-Jensen medium after decontamination by the Petroff method, and by quantitative colony counting on selective 7H11 medium after digestion with dithiothreitol. The selective medium counts ranged from no growth to 8.3 log10 cfu/ml with the largest numbers in the range 3.5-7.0 log10 cfu/ml. Although there was overlap in counts between specimens with negative and positive direct smears, a specimen with a count of 4.0 log10 cfu/ml or more was likely to have a positive smear while a negative smear was likely if the count was lower. This demarcation value should be increased to 4.5 log10 cfu/ml to account for under-estimation in the selective medium counts. The corresponding demarcation estimate for LJ cultures was 80 colonies. In distinguishing between patients in the bacterial contents of their sputum, the selective medium counts were better than the gradings of either LJ cultures or direct smears.


Use of two-stage incubations to define sequential intermediates in endoplasmic reticulum to Golgi transport.

Identification of the temporal requirement for components through the use of two-stage incubations is valuable in dissecting the overall transport reaction into steps relevant to vesicle fission and those related to vesicle fusion. In the context of semiintact mammalian cells in which a functional vesicle intermediate has not been detected, components playing a role in targeting are presently difficult to identify. However, the two-stage incubations are particularly powerful when either the donor or acceptor compartments can be manipulated independently, as is the case for intra-Golgi transport using enriched Golgi fractions or in the case of ER-to-Golgi transport in perforated yeast, in which a vesicle intermediate can be physically isolated.


Use of sec mutants to define intermediates in protein transport from endoplasmic reticulum.

In this chapter we have discussed the methodology used to identify and characterize three intermediates in protein transport from the ER that represent stages of transport vesicle budding, targeting, and fusion. The intermediates are obtained using a variety of transport inhibitors: low-temperature incubations, addition of chemicals, or inactivation of Sec protein function using temperature-sensitive mutants or specific antibodies. In all cases, the transport block imposed by the inhibitor is reversible, permitting assessment of the requirements for transport from each intermediate stage. Based on the differential requirements for Sec protein function, as well as the distinct kinetics and efficiency of transport from each intermediate, we demonstrate that each transport intermediate is functionally distinct.


Microbial contamination of ambient air by ultrasonic humidifier and preventive measures.

The microbially contaminated ultrasonic humidifier (UH) causes humidifier fever. The number of airborne viable bacteria was determined when the UH was operating, and other methods to humidify the air of hospital wards were also examined. A UH contaminated with 10(5) bacteria ml(-1), a level common in hospitals, increased the bacterial count in the air from 860 m(-3) to 88,000 m(-3) at a distance of 3 m from the humidifier. Thus UH in hospitals may contaminate the air and be a potential hazard to patients. Contamination was slight when a washable and disinfectable ultrasonic nebulizer was used with disinfection at 24 h intervals. In tracheostomy patients requiring a high degree of air humidification, ultrasonic nebulizers which are readily washed and disinfected are recommended.


Involvement of cellular division cycle in the susceptibility of Escherichia coli to cold- and osmotic-shock.

Susceptibility of Escherichia coli to cold-shock, osmotic-shock and cold-osmotic-shock has been evaluated throughout the cellular division cycle for a variety of generation time. Two periods of sensitisation were detected. The first of these was towards cold-shock which occurred 60 min prior to cell division and might correspond to initiation of DNA replication. The second period of sensitisation was towards both cold-, osmotic- and cold-osmotic-shock and occurred immediately prior to and during cell division. Patterns and extents of sensitisation towards each of these three stresses during the second period were suggestive of a common sensitising event associated with constriction of the cell and separation.


Evaluation of the genotoxic spectrum of cisplatin for Candida albicans.

The platinum co-ordination complex cisplatin [cis-diamminedichloroplatinum (II)] is a highly effective anticancer drug whose activity derives from its ability to form adducts crosslinking neighbouring purine bases in DNA. Under in vitro conditions, cisplatin induced cellular inactivation, forward and reverse mutations, reciprocal and nonreciprocal mitotic recombinations and phenotypic switching in the opportunistically pathogenic yeast Candida albicans. Mutant and recombinant yields were higher with post-treatment growth at 25 degrees C rather than 37 degrees C: the reverse was true for cell death or phenotypic switches. These responses comport with prior evidence that, generally, the higher recovery temperature discourages DNA repair processes in C. albicans. Thiosulphate, an agent used therapeutically to reduce nephrotoxic side effects of cisplatin in humans, greatly decreased both the lethality and recombinagenicity of cisplatin for C. albicans. Implications of these observations for possible genetic destabilization of C. albicans populations born naturally by cancer patients undergoing treatments with cisplatin are discussed.


Ecology and distribution of toxigenic Vibrio cholerae in aquatic environments of a temperate region.

The occurrence of a human pathogen, Vibrio cholerae, in aquatic environments and the distribution of toxigenic strains were studied for 2 years. The pathogen was isolated from freshwater, brackish and marine environments. V. cholerae non-01 was frequently recovered but the 01 serogroup was not detected. Of the 57 environmental strains tested, eight (12%) were found to be potentially toxigenic. A number of atypical V. cholerae 01 strains were found which showed higher virulence potentials than the serogroup non-01 strains. A significant correlation of the incidence of the pathogen was noted with water temperature in freshwater environments. In marine environments, a reciprocal correlation of the V. cholerae count and salinity was observed. The present study describes the ecology of V. cholerae in aquatic environments of a temperate region and notes that the occurrence of potentially virulent strains could be of public health significance.


Mycoplasmas in the plaque and saliva of children.

Plaque and saliva samples from 60 children were screened for the presence of mycoplasmas. Each child provided a sample of stimulated saliva and one of plaque from the buccal surface of the most posterior tooth in the upper right quadrant. Mycoplasmas were cultured from 93% of the saliva samples and from 50% of the plaque samples. At the time of sampling, gingivitis was assessed at the site adjacent to that from which the plaque was taken. Mycoplasmas were found significantly more often in plaque from sites in which the adjacent gingivae exhibited inflammation than from sites in which no inflammation was detectable. The proportion of mycoplasmas in plaque from samples adjacent to inflamed gingivae was significantly greater than that in plaque from sites which were clinically healthy. A similar pattern was detected in the corresponding saliva samples. The finding of a greater incidence, together with increased levels, of mycoplasmas in plaque samples from sites with gingivitis may suggest a role for these organisms in this condition.


Identification of formyl Met-Leu-Phe in culture filtrates of Helicobacter pylori.

Helicobacter pylori synthesizes and secretes a substance which co-chromatographs and is antigenically cross-reactive with the bacterial chemotactic peptide fMet-Leu-Phe. Using reverse phase and affinity chromatography this substance has now been purified. Carboxypeptidase Y microsequencing has verified that this material is fMet-Leu-Phe. The infiltration of polymorphonuclear leucocytes to sites of H. pylori infection may be a response to mucosal permeation of soluble, diffusable bioreactive substances such as fMet-Leu-Phe.


An American anesthesia training program in East Africa.

A unique 5-year program designed to improve the quality and availability of anesthesia care in developing nations has been initiated in East Africa. Based upon North American anesthesiologists serving as volunteer teachers, the program emphasizes teaching to the exclusion of taking over anesthesia care. The teaching is practical and relevant to the clinical practice of anesthesia in developing countries. The volunteers teachers serve, one at a time, 12 months of the year for several years in two pre-existing structured East African anesthesia training programs, thereby providing the advantages of consistent, reliable teaching assistance for a period of years to programs of demonstrable interest and experience in anesthesia training.


Premature decline in Morris water maze performance of aging micrencephalic rats.

The rat with methylazoxymethanol-induced micrencephaly is a useful animal model of congenital brain defects and associated cognitive impairment. Born with profound morphological and neurochemical alterations in the forebrain, it shows impaired ability to learn mazes. In order to determine how an animal with such a developmentally damaged brain would function in old age, Long-Evans rats 6, 15, and 24 months of age were tested for their ability to learn to locate a hidden platform in the Morris water maze. The performance of micrencephalic rats of all ages was impaired on acquisition, retention, and transfer trials. Moreover, the magnitude of their acquisition deficit increased with age. It remains to be determined whether the premature decline of the micrencephalic rat in learning the task simply reflects a greater impact on an already compromised brain by neuron loss characteristic of aging brains or whether the prenatal insult alters some basic processes resulting in premature aging.


Deficits in development of central cholinergic pathways caused by fetal nicotine exposure: differential effects on choline acetyltransferase activity and [3H]hemicholinium-3 binding.

Nicotine has been hypothesized to induce neurobehavioral teratology by mimicking prematurely the natural developmental signals ordinarily communicated by the ontogeny of cholinergic synaptic transmission. In the current study, the effects of fetal nicotine exposure (2 mg/kg/day or 6 mg/kg/day) on development of central cholinergic pathways were examined in striatum and hippocampus of animals exposed from gestational days 4 through 20, using maternal infusions with osmotic minipumps. Brain region weights and choline acetyltransferase activity, an enzymatic marker for development of cholinergic nerve terminals, were within normal limits in the nicotine-exposed animals. However, development of [3H]hemicholinium-3 binding which labels the presynaptic high affinity cholinergic transporter, was deficient in both striatum and hippocampus. Abnormalities occurred during two distinct phases; in the early neonatal period, when [3H]hemicholinium-3 binding sites are transiently overexpressed, and during or after the period of rapid synaptogenesis, when binding in controls is rising consequent to the increase in nerve impulse activity. These data thus indicate that fetal nicotine exposure, even at doses that do not cause overt signs of maternal/fetal/neonatal toxicity or growth impairment, influences both specific gene expression of cholinergic nerve terminal markers, as well as indices of neuronal function. Comparison of regional selectivity at the two dose levels indicated greater sensitivity of the striatum, a region with a prenatal peak of neuronal mitosis, as compared to hippocampus, where mitosis peaks postnatally; the regional differences are consistent with vulnerability to nicotine during a critical phase of cell development.


The effect of prenatal alcohol exposure on attention in the rat.

The effect of prenatal exposure to alcohol on the development of basal heart rate and on the elicitation and habituation of the heart rate orienting response was examined in three experiments with rats. In all experiments, Etoh dams consumed large, daily amounts of alcohol and their weight gain during pregnancy was less than that of ad lib or pairfed dams. In addition, Etoh-exposed pups weighed less and grew more slowly than their ad lib or pairfed counterparts. Although prenatal exposure to alcohol had a significant effect on the ontogeny of basal heart rate, there was no effect on the magnitude of the heart-rate response to a novel olfactory stimulus or on habituation of the heart-rate response to that stimulus. Implications of the present findings for models of alcohol-induced attention deficits are discussed.


Maternal psychotropic medication and neonatal behavior.

A sample of 29 psychiatrically ill women and a non-ill comparison group were recruited during pregnancy. Twelve of the ill mothers were receiving antipsychotic and anti-anxiety medication during the final trimester of pregnancy. The behavior of their infants was assessed at 3 and 14 days of age using the Brazelton Neonatal Behavioral Assessment Scale. Compared to infants of non-ill mothers and infants of ill nonmedicated mothers, infants whose mothers received antipsychotic drugs--particularly those in the phenothiazine family--showed a stable pattern of poor neonatal motor functioning that included tremulousness, hypertonicity, and poor motor maturity. It was speculated that this behavior was symptomatic of a neonatal abstinence or withdrawal syndrome.


EEG monitoring of carotid endarterectomy with routine patch-graft angioplasty: an experience in a large series.

Four hundred and thirty-nine carotid endarterectomies (CEAs) with routine use of patchgraft angioplasty were performed in 375 patients; the indwelling shunt was used only in patients showing clamp-related EEG abnormalities. Five patients showed EEG abnormalities just after head positioning, which reversed after removal of head hyperextension; three cases suffered EEG flattening due to severe bradycardia or cardiac arrest before carotid clamping, which promptly reversed after treatment. Clamp-related EEG abnormalities appeared in 106 operations (24.2%) and all reversed after the insertion of the indwelling shunt; patients with occlusion of the contralateral internal carotid artery showed a 68.8% rate of EEG clamp-related changes. The short term follow-up (one month after the operation) showed six minor strokes with complete recovery (1.37%), one intraoperative stroke (0.23%), three delayed major strokes (0.69%) and three neurological deaths (0.69%). The long-term follow-up over an average of 42 months showed a 3.7% rate of relevant neurological complications (ie permanent deficits + death) and a 3.16% rate significant restenosis or occlusion of the operated carotid artery. Our results show that the routine use of EEG monitoring and patch-graft angioplasty allow to perform CEAs with a very high degree of safety, improving the clinical course of the disease.


[Continuous localized EEG discharge during sleep in children without neuropsychological problems].

After having reported continuous localized EEG discharge during slow sleep (CLEDS) in six children with congenital encephalopathy, we observed a similar EEG picture in six children free from both neuropsychological and neuroradiological defects. They suffered from partial idiopathic epilepsy; five presented a familial disposition towards febrile seizures. Continuous paroxysmal activity during sleep was observed from 4.8 yrs to 4.11 yrs (mean age: 4.9 yrs). Spontaneous remission of CLEDS was observed in three cases after 2-25 months, but one or more relapses occurred in two cases, and five children are still suffering from CLEDS. Seizures were controlled by drugs in all cases. Deterioration of intelligence level, although not severe, was observed in one case, after 24 months of CLEDS.


A fostering study of the effects of prenatal cocaine exposure: I. Maternal behaviors.

The effect of rearing condition and prenatal exposure to cocaine on maternal behaviors was examined. Sprague-Dawley dams were given SC injections of 40 mg/kg/3cc cocaine HCl (C40) or saline (LC) daily from gestational days 8-20. Maternal behavior was assessed in treated dams rearing their biological pups (LC/LC; C40/C40), treated dams rearing untreated pups (LC/FOS; C40/FOS), and foster dams rearing treated pups (FOS/LC; FOS/C40). All dams were monitored for home cage behavior (time eating, drinking, and with pups) for 2 h during both the light and dark cycle on postnatal day 4 (P4), pup retrieval on P5-P9, and maternal aggression to a female intruder (latency to the first attack, number of attacks, boxing, pins, intruder time spent submissive and motionless) on P10. No differences were observed in nest behavior or in tests of pup retrieval among the six groups. Dams rearing their biological litter (LC/LC and C40/C40) were significantly quicker to initiate the first attack when compared to all other groups. This increased aggression was maintained throughout the test session in the C40/C40 dams who made significantly more intruder attacks than all other groups, with the intruder spending significantly more time in a submissive posture (lying on back). In contrast, LC/LC dams did not exhibit an increased number of attacks during the test, apparently responding to an increased freezing in their intruders with a reduction in aggressive behavior. Taken together these findings suggest that prior cocaine exposure results in alterations in maternal aggression that is evident when these dams rear their own pups.


[REM-sleep behavior disorder and olivo-ponto-cerebellar atrophy: a case report].

The authors report the observation of REM-sleep behavior disorder in a patient also suffering from an olivo-ponto-cerebellar atrophy. They discuss the place of this sleep disorder among the parasomnias, its pathophysiological basis and its features similar to those of the somnambulism. Only a polysomnography study can help to make the diagnosis.


[Idiopathic peripheral facial paralysis. Prognostic value of the combined study of the blink reflex with stimulation of the facial nerve. 91 cases].

The blink reflex and stimulodetection of the facial nerve was studied during the first ten days, the 30th and 60th day and at the end of the sixth month in 91 patients with Bell's palsy. In patients with present R1 during the first ten days, there was a remarkable tendency towards a satisfactory recovery without complications. On the contrary using this single test, it is impossible to determine with 100% accuracy, which Bell's palsy will have an unsatisfactory recovery with severe dysfunction. The blink reflex seems to be a useful test for predicting the prognosis in an early stage of the paralysis. The association of the blink reflex and stimulodetection of the facial nerve allows to evaluate the risk of sequelae by the means of different electrophysiological patterns.


A fostering study of the effects of prenatal cocaine exposure: II. Offspring behavioral measures.

The impact of rearing condition was assessed in Sprague-Dawley dams given 40 mg/kg cocaine (C40) or saline (LC control) subcutaneously (SC) from gestational days 8-20 and their offspring. Treated pups reared by their biological dams (LC/LC; C40/C40), treated pups reared by surrogate dams (FOS/LC; FOS/C40), and foster pups raised by treated dams (LC/FOS; C40/FOS) were examined. On postnatal day 7 (P7), pups received either 0 (unpaired) 2, 3, or 4 pairings of an odor and footshock and were tested for their aversion to this odor. Foster and LC pups, regardless of rearing condition, exhibited significant odor aversions following either 2, 3, or 4 training trials. In contrast, C40 pups reared by surrogate dams required 4 trials to acquire the aversion, and C40 pups reared by their own dams did not exhibit conditioning even after 4 trials. At P17, no differences were seen among the groups in the aversion formed to an auditory or an olfactory stimulus that was paired with footshock. At P60, shock-elicited aggression among pairs of siblings was examined. Regardless of prenatal exposure condition, offspring reared by dams given cocaine showed a decreased latency to the first aggressive contact, an effect that was evident without any alteration in shock sensitivity. Together these data suggest that being reared by a dam previously exposed to cocaine has an impact on offspring behavioral function apart from the effects of prenatal cocaine exposure per se. The implications of the data regarding the cognitive performance of pups exposed prenatally to cocaine are also discussed.


The effects of prenatal tobacco and marijuana use on offspring growth from birth through 3 years of age.

This is a prospective study of prenatal substance use. Women were interviewed during their fourth and seventh months of pregnancy, at delivery, and at 8, 18, and 36 months postpartum. At birth, there were 763 liveborn, singleton offspring in the sample. At each phase, the offspring were examined and measured for growth. Data are presented on the relationship between tobacco and marijuana use and the size of the offspring at birth, 8, 18, and 36 months of age. At birth, there was a significant inverse relationship between tobacco use and weight, length, and head circumference. At 8 months of age, only length continued to be associated with prenatal tobacco exposure. By 18 months of age, there was no relationship between prenatal tobacco exposure and size of the offspring. Prenatal marijuana exposure was only associated with decreased length at birth. Neither tobacco nor marijuana use predicted gestational age or morphological abnormalities.


Sympathetic skin response in hemispheric lesions.

We recorded the sympathetic skin response (SSR) from electrical nerve stimulation in 16 patients with cerebrovascular accident (CVA). Location and nature of the lesion were documented by computerized tomography (CT). Median (pre-rolandic and parietal) somatosensory evoked potentials (SEP) were also recorded. SSR was absent bilaterally in eight hemiplegics after stimulation of the plegic side and present bilaterally after stimulation of the normal side in the first weeks after CVA. Parietal and pre-rolandic SEPs were absent in the affected hemispheres. SSR was present bilaterally after stimulation of each side in the remaining CVA cases with reduced amplitude SEPs. Absence of the electrically evoked SSR in hemispheric lesions may be due to involvement of central afferent pathways or temporary suppression of suprasegmental excitatory influences.


Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on behavior of monkeys in peer groups.

Adult female rhesus monkeys were fed diets containing 0, 5, or 25 ppt 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for approximately 4 years. They were bred to unexposed males during TCDD exposure (Experiment 1) and again after TCDD exposure ended (Experiment 2). Offspring from both experiments were weaned at 4 months and socialized for 1.5 h/day in groups of four monkeys each beginning at approximately 8 months of age. Each social group contained both control and TCDD-exposed monkeys. In Experiment 2, the offspring were later placed in new social groups containing only monkeys from the same TCDD exposure condition. The TCDD-exposed offspring born concurrent with maternal TCDD exposure (Experiment 1) initiated more rough-tumble play, retreated less during play bouts, and were less often displaced from preferred positions in the playroom. They also engaged in more self-directed behaviors. The behavior of offspring born after maternal TCDD exposure ended (Experiment 2) was not altered when they were socialized with control monkeys. However, some behavioral changes did emerge when they were placed in social groups containing only TCDD-exposed monkeys.


Acylation of 1-palmitoyl-sn-glycerophosphocholine by chick brain microsomes is unaffected by fatty acid binding protein.

Rates of incorporation of exogenously supplied fatty acids into 1-palmitoyl-sn-glycerophosphocholine were measured using the microsomal fraction from brains of 14-15 day old chick embryos. The substrate preferences for reacylation were: 18: 2(n-6) = 20: 4(n-6) > or = 20: 5(n-3) = 18: 3(n-3) > or = 18: 1(n-9) > or  = 22: 6(n-3) > or = 18: 0. The normalized rate with 18: 0 was significantly lower than all other rates except for 22: 6(n-3), and the acylation rate with 22: 6(n-3) was significantly lower than with 18: 2(n-6) and 20: 5(n-3). With the addition of fatty acid binding protein partially purified from brain cytosol, a decrease (not significant) in the rate of incorporation was observed; the substrate preference was unchanged. In the presence of FABP, normalized rates with 18: 2(n-6) were significantly higher than with 18: 0, 18: 1(n-9), or 22: 6(n-3); rates with 20: 4(n-6) were significantly higher than those with 22: 6(n-3). Preliminary data on the acylation of 1-palmitoyl-sn-glycerophosphoethanolamine showed lower rates of incorporation than for the choline analogue and no clear substrate preference, but a similar lack of effect of fatty acid binding protein. These results do not support the proposed function of fatty acid binding protein in the establishment of a phospholipid composition rich in polyunsaturated fatty acids. The results are consistent, however, with the role of the reacylation reaction in the continual turnover of particular substrates [18: 2(n-6) and 20: 4(n-6)] used to generate second messengers.


Metabolism in rat and affinity chromatographic purification of bone sialoprotein secreted by metabolically labelled rat osteoblastic cells (UMR-106).

An osteoblastic, established cell line UMR-106 was shown to synthesize high levels of the bone-specific, bone sialoprotein (BSP). BSP could be radiolabelled to high specific activity by adding 3H-glucosamine and 35S-sulfate to the UMR-106 cultures and was isolated to high purity using ion-exchange and affinity chromatography on immobilized serotonin. The radiolabelled BSP, partially purified by ion-exchange chromatography, was injected intravenously into a rat in order to study its tissue distribution and urinary clearance. About 43% of the total recovered radioactivity was excreted in the urine within 75 h and the remainder was widely distributed, with the liver, kidney, heart and pelt showing the highest concentrations. The use of established cell lines for the synthesis of radiolabelled glycoconjugates, in conjunction with rapid purification on affinity matrix, provides a useful approach for studying the metabolism of glycoconjugates in whole animals.


Albumen gland of the snail Achatina fulica is the site for synthesis of AchatininH, a sialic acid binding lectin.

A sialic acid binding lectin, AchatininH was purified from the hemolymph of Achatina fulica snail. To identify the site of synthesis of AchatininH, in vitro incubation studies in presence of labelled amino acid precursor were performed. Different organs from the snail were sliced and incubated in methionine-deficient Eagle's minimum essential medium containing [35S]-methionine at 25 degrees C for 5 h. After termination of incubation, tissues were homogenized, centrifuged and the de novo synthesized protein was immunoprecipitated with specific AchatininH antibody, followed by protein-A. The precipitated antigen-antibody complex was analysed by SDS-PAGE. Data obtained from native gel electrophoresis and SDS-PAGE radioautographic analysis indicates that AchatininH is synthesized in the albumen gland.


Revisiting the action of bovine ribonuclease A and pancreatic-type ribonucleases on double-stranded RNA.

Single-strand-preferring ribonucleases of the pancreatic type, structurally and/or catalytically similar to bovine RNase A but endowed with a higher protein basicity, are able to degrade double-stranded RNA (dsRNA) or DNA:RNA hybrids under standard assay conditions (0.15 M NaCl, 0.015 M sodium citrate, pH 7), where RNase A is inactive. This enzyme too, however, becomes quite active if assay conditions are slightly modified or its basicity is increased (polyspermine-RNase). In the attempt to review these facts, we have analyzed and discussed the role that in the process have the secondary structure of dsRNA as well as other variables whose influence has come to light in addition to that of the basicity of the enzyme protein, i.e., the ionic strength, the presence of carbohydrates on the RNase molecule, and the structure (monomeric or dimeric) of the enzyme. A possible mechanism by which dsRNAs are attacked by pancreatic-type RNases has been proposed.


Characterization of bone protein components with polyacrylamide gel electrophoresis: effects of zinc and hormones in tissue culture.

An attempt was made to clarify the molecular characterization of zinc-induced bone protein synthesis in tissue culture. Calvaria were removed from weanling rat (3-week-old male) and cultured for periods up to 48 hr in Dulbecco's Modified Eagle Medium (high Glucose, 4500 mg/dl) supplemented with antibiotics and bovine serum albumin. When calvaria cultured in the presence of 10(-5) to 10(-4) M zinc were pulsed with [3H] leucine, zinc caused a significant increase in the incorporation of [3H] leucine into the acid-insoluble residues of bone tissue. The soluble fraction obtained from cultured bone was analyzed with SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The major components in the fraction obtained from control bone were 68 killo-dalton (kDa) and 45 kDa proteins. These components were clearly increased by the presence of zinc (10(-4) M). The effect of zinc was completely abolished by the coexistence of 10(-6) M cycloheximide. Meanwhile, 10(-9) M estrogen or 10(-8) M insulin, which can stimulate bone formation, did not enhance the effect of zinc to increase bone 68 and 45 kDa proteins. The present findings suggest that zinc increases many bone protein components, especially 68 and 45 kDa proteins.


Effect of outlet obstruction on pyruvate metabolism of the rabbit urinary bladder.

Bladder function is dependent upon cellular metabolism of substrates and the adequate generation of high-energy phosphate compounds. Partial outlet obstruction induces a marked decrease in bladder function which is associated with a significant decrease in the oxidative metabolism of glucose. The current investigation was designed to determine whether the time course of the decrease in mitochondrial oxidation in the hypertrophied urinary bladder is similar to the time course of the contractile dysfunction observed. In these studies we determined: 1) the rate of 14C-pyruvate metabolism to 14CO2 in control and obstructed tissue (1, 3, 5 and 7 days), and 2) the mitochondrial enzymatic activities of malate dehydrogenase and citrate synthase. The results can be summarized as follows: 1) The rate of pyruvate metabolism decreases by over 50% within one day following partial outlet obstruction, and remains at this level for the seven day period of study. 2) Kinetic analysis demonstrates that the change in enzymatic activity is related to a decrease in Vmax; the Kd for pyruvate is similar for control and after all time periods of obstruction. 3) The enzymatic activity of malate dehydrogenase and citrate synthase is reduced by over 50% within one day following partial obstruction, and remains at this level throughout the 7 day study period. These metabolic results correlate in time and duration with the decreased ability of the bladder to empty following partial outlet obstruction.


The antineoplastic agent estramustine and the derivative estramustine-phosphate inhibit secretion of interleukin-3 in leukemic cells. Possible roles of MAPs.

The antineoplastic drug estramustine is an adduct of estradiol and nor-nitrogen mustard. It has been shown that this drug interferes with microtubule assembly, an effect mediated by estramustine interaction with microtubule-associated proteins (MAPs). In the present report we demonstrate that estramustine and the phosphorylated derivative of the drug, estramustine-phosphate, inhibit the secretion of interleukin-3 by WEHI-3B cells. These studies also show that the estramustine derivative specifically interacts with a MAPs component found in these cells, which exhibited characteristics ressembling those of tau protein isoforms. Western blots using a unique monoclonal antibody MTB6.22 that recognizes microtubule-binding domains on MAPs, indicated that this WEHI protein factor contained the antigenic determinant that are functionally significant for microtubule assembly. ELISA assays using this antibody, also showed a decrease in the levels of the immunoreactive protein in WEHI cells after treatment with EMP. Interestingly, it has been recently described that the action of estramustine-phosphate is mediated by a direct interaction with MAP-binding sites on the microtubule surface, which are recognized by the site-specific monoclonal antibody. These findings together with immuno-precipitation experiments using anti-interleukin-3 antibodies and the inhibitory effect of the estramustine derivative on WEHI secretion process suggest that this anti-mitotic agent may block IL-3 secretion by a mechanism involving its interaction with a 'tau-like' MAPs component present in these cells.


Effects of exercise training and diabetes on cardiac myosin heavy chain composition.

This study determined whether the beneficial effects of exercise training on the diabetic heart previously observed are associated with alterations in ventricular myosin heavy chain (MHC) isoform composition. Diabetes was induced in rats by i.v. streptozotocin. Trained rats were run on a treadmill for 60 min/day, 27 m/min, 10% grade. After 10 wks, ventricular MHC isoenzyme protein composition was analyzed for MHC composition using gel electrophoresis. alpha-MHC and beta-MHC mRNA were determined by Northern and slot blot hybridization techniques. Both protein and mRNA analyses indicated that sedentary control rats exhibited a predominance of alpha-MHC. Sedentary diabetics exhibited a shift to beta-MHC. Exercise trained diabetic rats showed a predominance of beta-MHC. The results indicate that treadmill exercise training of diabetic rat does not prevent the diabetes-induced shift in MHC composition towards the beta-MHC isoform, thus it is unlikely that the beneficial effects of exercise training on the diabetic heart, previously shown, are due to a normalization of the myosin isoform composition.


Characterization of a novel microsomal glutathione S-transferase produced by Aspergillus ochraceus TS.

Purification and characterization of microsomal glutathione S-transferase produced by Aspergillus ochraceus TS are reported. The isozymes are located in microsomes and were active against 1-chloro-2,4-dinitrobenzene, ethacrynic acid, 1,2-dichloro-4-nitrobenzene, trans-4-phenyl-3-buten-2-one,p-nitrobenzyl chloride and bromosulphophthalein. They were inhibited by N-ethylmaleimide and bromosulphophthalein. The GST isozymes produced by Aspergillus ochraceus TS are indistinguishable in respect of their molecular mass both in native and denatured state. The subunit of the purified protein had an apparent M(r) of 11 kDa while molecular mass of the native protein is around 56 kDa. The substrate specificity and pI values of the isozymes were different. The GSTs produced by Aspergillus ochraceus TS fairly share functional properties with mammalian cytosolic isozymes.


The nucleotide metabolism in lactate perfused hearts under ischaemic and reperfused conditions.

It was examined whether lactate influences postischaemic hemodynamic recovery as a function of the duration of ischaemia and whether changes in high-energy phosphate metabolism under ischaemic and reperfused conditions could be held responsible for impairment of cardiac function. To this end, isolated working rat hearts were perfused with either glucose (11 mM), glucose (11 mM) plus lactate (5 mM) or glucose (11 mM) plus pyruvate (5 mM). The extent of ischaemic injury was varied by changing the intervals of ischaemia, i.e. 15, 30 and 45 min. Perfusion by lactate evoked marked depression of functional recovery after 30 min of ischaemia. Perfusion by pyruvate resulted in marked decline of cardiac function after 45 min of ischaemia, while in glucose perfused hearts hemodynamic performance was still recovered to some extent after 45 min of ischaemia. Hence, lactate accelerates postischaemic hemodynamic impairment compared to glucose and pyruvate. The marked decline in functional recovery of the lactate perfused hearts cannot be ascribed to the extent of degradation of high-energy phosphates during ischaemia as compared to glucose and pyruvate perfused hearts. Glycolytic ATP formation (evaluated by the rate of lactate production) can neither be responsible for loss of cardiac function in the lactate perfused hearts. Moreover, failure of reenergization during reperfusion, the amount of nucleosides and oxypurines lost or the level of high-energy phosphates at the end of reperfusion cannot explain lactate-induced impairment. Alternatively, the accumulation of endogenous lactate may have contributed to ischaemic damage in the lactate perfused hearts after 30 min of ischaemia as it was higher in the lactate than in the glucose or pyruvate perfused hearts. It cannot be excluded that possible beneficial effects of the elevated glycolytic ATP formation during 15 to 30 min of ischaemia in the lactate perfused hearts are counterbalanced by the detrimental effects of lactate accumulation.


High affinity DNA-microtubule interactions: evidence for a conserved DNA-MAP interaction involving unusual high CsCl density repetitious DNA families.

We have examined high affinity interactions of chick brain microtubule proteins with 35S labelled tracer DNAs from chick, mouse and D. melanogaster under equilibrium conditions by the nitrocellulose filter binding technique. Ternary reaction mixtures of the above two components and a third component, an excess of unlabelled competitor DNA from either E. coli., mouse, D. melanogaster or chick, were used to measure small fractions of DNA in each case (1-4%) bound to microtubule protein under high stringency- large competitor DNA concentration and 0.5 M NaCl. As seen in part previously (Marx, K.A. and Denial, T. (1985) in The Molecular Basis of Cancer, 172B, 65-75 (Rein, ed), A. Liss, N.Y.) the measured order of competitor DNA strengths was identical for all three tracer DNAs. That is: chick > mouse > D. melanogaster > E. coli competitor DNA. Since the homologous interaction, chick competitor DNA with chick brain microtubule protein, is always the strongest interaction measured, we interpret this as evidence for a conserved protein-DNA sequence interaction. 35S chick DNA tracer sequences, isolated from nitrocellulose filters following the stringent binding in the presence of 0.9 mM-1 E. coli. competitor DNA, was used in driven reassociation reactions with total chick driver DNA. This fraction was found to be significantly enriched in repetitive chick DNA sequences. Since we have observed a similar phenomenon in mouse, we then compared the stringent binding mouse sequences and showed that the bulk of these sequences did not cross-hybridize with total chick DNA. Finally, all three 35S tracer DNAs binding to nitrocellulose were isolated and sedimented to equilibrium on CsCl density gradients. The CsCl density distributions from all three DNAs showed significant (100-fold) enrichment in classical satellite DNAs as well as higher enrichment in two very unusual high CsCl density families of DNA (1.720-1.740 g/cm3; 1.750-1.765 g/cm3). These families are never observed as distinct bands in total DNA CsCl gradients, nor could we isolate them in purified tubulin control binding experiments. This apparently general phenomena may be identifying some of the sequence families involved in the high affinity microtubule interaction, which appears to be conserved in evolution.


Purification of a soluble casein kinase II from Dictyostelium discoideum lacking the beta subunit: regulation during proliferation and differentiation.

A type II casein kinase has been purified from the soluble fraction of Dictyostelium discoideum vegetative cells. The enzyme has been purified 370 fold and behaves catalytically as casein kinase type II, in the sense that it utilizes GTP as well as ATP as phosphoryl donors, it is inhibited by low heparin concentrations and phosphorylates a specific peptide for CK II. It is a tetramer of 38 kDa-subunits with catalytic activity and ability to autophosphorylate in vitro. The comparison of this activity with the nuclear enzyme previously purified from the same organism indicates that both have the same molecular structure. Both enzymes have antigenic determinants in common with casein kinase II from bovine thymus, suggesting a high degree of conservation during evolution. Studies on the activity of this enzyme during early differentiation, and in the transition from quiescence to proliferation shows an increase in specific activity suggesting a crucial role for the enzyme in this organism.


The accumulation and metabolism of glycosphingolipids in primary kidney cell cultures from beige mice.

In the normal C57BL/6J male mouse a specific subset of the kidney glycosphingolipids which is associated with multilamellar bodies of lysosomal origin and represents about 10% of the total kidney glycolipids, is excreted into the urine each day. This excretion is blocked and glycosphingolipids accumulate in the kidney of bgJ/bgJ mutants of this strain. To examine this process in vitro, glycosphingolipid metabolism and excretion were studied in beige mouse kidney cell cultures. Primary kidney cell cultures from male C57BL/6J control and bgJ/bgJ beige mutants were grown in D-valine medium and glycosphingolipids labeled with [3H]palmitate. As we have shown previously, the giant lysosomes of altered morphology were maintained in cultures of the beige kidney cells. Beige-J and control cells synthesized the same types of glycosphingolipids, but the mutant cells had quantitatively higher levels of these compounds than control cells, as determined by high performance liquid chromatography. Beige-J cells incorporated more [3H]palmitate into glycosphingholipids than control cells on a cpm/mg protein basis and the specific activity (cpm/pmole glycosphingolipid) was lower in beige cells. Medium from beige-J cells accumulated more glycosphingolipids than that from control cells in a 24 h period. The glycosphingolipids released into the medium as determined by HPLC were primarily non-lysosomal types and both control and mutant cells retained the glycosphingolipids associated with lysosomal multilamellar bodies excreted in vivo. The elevated levels of lysosomal glycosphingolipids and the dysmorphic lysosomes in primary cultures of beige cells, then, are not caused by a mutant block in secretion of lysosomes.


Fatty acid utilization by young Wistar rats fed a cafeteria diet.

The content and accretion of fatty acids in 30, 45 and 60-day old Wistar rats fed either reference chow or a cafeteria diet has been studied, together with their actual fatty acid intake during that period. Diet had a small overall effect on the pattern of deposition of fatty acids, but the deposition of fat was much higher in cafeteria rats. The fat-rich cafeteria diet allowed the direct incorporation of most fatty acids into lipid storage, whilst chow-feeding activated lipogenesis and the deposition of a shorter chain and more saturated type of fatty acids. During the second month of the rat's life, the elongation pathway as well as delta 9-desaturase became functional, thus helping to shape the pattern of fatty acids actually accrued. The 60-day rats showed a relative impairment in the operation of delta 5-desaturase, since their lipids had a higher C20:4/C20:3 ratio than those of the diet ingested. Cafeteria-diet feeding minimized this effect since the large supply of dietary polyunsaturated fatty acids made the operation of the elongation-desaturase pathways practically unnecessary.


Quantitation and structures of oligosaccharide chains in human trachea mucin glycoproteins.

Human respiratory mucin glycoproteins from patients with cystic fibrosis were purified and oligosaccharide chains were released by treatment with alkaline borohydride. A neutral oligosaccharide alditol fraction was isolated from mucin obtained from a patient with A blood group determinant by chromatography on DEAE-cellulose and individual oligosaccharide chains were then isolated by gel filtration on BioGel P-6 columns and high performance liquid chromatography with gradient and isocratic solvent systems. The structures of the purified oligosaccharides were determined by methylation analysis, sequential glycosidase digestion and 'H-NMR spectroscopy. The amount of each chain was determined by compositional analysis. A wide array of discrete branched oligosaccharide structures that contain from 3 to 22 sugar residues were found. Many of the oligosaccharides are related and appear to be precursors of larger chains. The predominant branched oligosaccharides which accumulate contain terminal blood group H (Fuc alpha 2Ga1 beta 4) or blood group A (Fuc alpha 2(Ga1NAc alpha 3) (Ga1 beta 4) determinants which stop further branching and chain elongation. The elongation of oligosaccharide chains in respiratory mucins occurs on the beta 3-linked G1cNAc at branch points, whereas the beta 6-linked G1cNAc residue ultimately forms short side chains with a Fuc alpha 2(Ga1NAc alpha 3) Ga1 beta 4 G1cNAc beta 6 structure in individuals with A blood group determinant. The results obtained in the current studies further suggest that even higher molecular weight oligosaccharide chains with analogous branched structures are present in some human respiratory mucin glycoproteins. Increasing numbers of the repeating sequence shown in the oligosaccharide below is present in the higher molecular weight chains. [formula: see text] This data in conjunction with our earlier observations on the extensive branching of these oligosaccharide chains helps to define and explain the enormous range of oligosaccharide structures found in human and swine respiratory mucin glycoproteins. Comparison of the relative concentrations of each oligosaccharide chain suggest that these oligosaccharides represent variations of a common branched core structure which may be terminated by the addition of alpha 2-linked fucose to the beta 3/4 linked galactose residue at each branch point. These chains accumulate and are found in the highest concentrations in these respiratory mucins.


Effects of regression of cardiac hypertrophy on myocardial contractility and ventricular myosin isoenzymes.

The effects of regression of cardiac hypertrophy on myocardial contractility and ventricular myosin isoenzymes were investigated in rats with renovascular hypertension. Six-week-old male Wistar rats were made hypertensive by constriction of one renal artery with a silver clip. Regression of cardiac hypertrophy was induced following the lowering of blood pressure by nephrectomy on the affected side 5-6 weeks after constriction of the renal artery and was maintained for 5-6 weeks. In contrast, myocardial hypertrophy was induced by 10-11 weeks of the hypertensive state. Isometric developed tension of isolated left ventricular papillary muscles was measured, while they were being perfused with Tyrode solution. Left ventricular myosin isoenzymes were separated by pyrophosphate gel electrophoresis. The ventricular to body weight ratio of the nephrectomized group was significantly lower than that of the hypertensive group, although it was greater than that of age-matched normal control rats. There were no significant differences in the isometric developed tension among three groups, the nephrectomized, hypertensive, and normal control rats. However, dT/dtmax tended to decrease in the hypertensive rats and recovered to normal in the nephrectomized rats. The left ventricular myosin isoenzyme pattern was shifted toward VM-3 in hypertensive rats and was shifted back toward VM-1 again in nephrectomized rats.


AAEM Minimonograph #13: H reflexes and F waves: physiology and clinical indications.

Motoneurons can be activated both reflexly and antidromically following electrical stimulation of peripheral nerves. These H reflexes and F waves are clinically useful responses which interface at the level of the peripheral nerves and the spinal cord. Because these responses are commonly employed in the electrodiagnostic evaluation of patients, an understanding of their physiology and clinical applications is important. These are reviewed. Reasoning from the physiology, both the value and limitations of H-reflex and F-wave studies are considered for disorders of peripheral nerves, roots, and the central nervous system. Theoretical concepts about the physiology and pathophysiology of the nervous system based on H-reflex and F-wave data are also discussed.


Surface EMG in the recording of fasciculations.

The usefulness of multichannel surface recording of fasciculations was evaluated by a retrospective study of 116 patients with various neurological disorders. Eight channels of a conventional electroencephalograph were used with plate electrode recordings from the upper arms and legs. Wide-spread fasciculations (defined as five or more of the eight muscle groups) were recorded in 48 of 54 patients with motor neuron disease, spinal muscular atrophy or postpolio syndrome, but noted on routine clinical examination at presentation in only 6. Eleven of 23 patients with peripheral neuropathy or myelopathy had fasciculations in five or more leads compared to one clinically, and 3 of 39 with other neurological diseases had fasciculations electrically but in only one were they clinically observed. The method is a noninvasive and sensitive adjunct to clinical examination for detecting fasciculations. Its diagnostic value is limited by the relatively high incidence of fasciculations in neuropathies and myelopathies. However, this study suggests that "false negatives" are rare and that the diagnosis of motor neuron disease should be reconsidered when less than five leads shows fasciculations.


Lumbrical and interossei recording in carpal tunnel syndrome.

Median motor studies are commonly "normal" in mild carpal tunnel syndrome (CTS). This reflects either the sparing of motor compared to sensory fibers, or the inability of conventional studies to detect an abnormality. A novel approach to demonstrate early motor fiber involvement in CTS is the placement of the same active electrode lateral to the third metacarpal, allowing recording from the second lumbrical or the deeper interossei, when stimulating the median or ulnar nerves at the wrist, respectively. We compared the difference between these latencies in 51 normal control hands to 107 consecutive patient hands referred with symptoms and signs suggestive of CTS, who were subsequently proven to have electrophysiologic CTS by standard nerve conduction criteria. A prolonged lumbrical-interossei latency difference (> 0.4 ms) was found to be a sensitive indicator of CTS in all patient groups. It was also helpful in patients with coexistent polyneuropathy, where localization at the wrist was otherwise difficult.


An experimental model of mitochondrial myopathy: germanium-induced myopathy and coenzyme Q10 administration.

In skeletal muscles from rats treated with germanium for 23 weeks, there were numerous ragged-red fibers and cytochrome-c oxidase (COX)-deficient fibers. Biochemically, germanium reduced the enzyme activities in the mitochondrial respiratory chain. Rotenone-sensitive NADH-cytochrome-c reductase as well as COX activities were markedly reduced, while succinate-cytochrome-c reductase was less severely, but significantly, affected. The histopathological findings in these muscles were similar to those seen in patients with mitochondrial encephalomyopathy, suggesting that germanium-induced myopathy may be a useful experimental model. Coenzyme Q10 administration appeared to be ineffective in preventing this experimental myopathy.


Lower motor neuron dysfunction in patients with multiple sclerosis.

A patient in whom multiple sclerosis (MS) was ultimately diagnosed presented with a lower motor neuron syndrome involving 1 hand, with EMG evidence of denervation. Twelve other patients were subsequently identified with definite MS and asymmetric hand atrophy. These patients were studied clinically and electrophysiologically. Evidence of chronic and ongoing denervation was noted in the hands of 12 of the 13 patients; in only 3 patients could the EMG abnormalities be accounted for by peripheral nerve lesions. Thus, lesions resulting in lower motor neuron damage may occur in the central nervous system in MS patients. We suggest that demyelination in the region of the ventral root exit zone may account for these findings.


Flavivirus induces MHC antigen on human myoblasts: a model of autoimmune myositis?

Infection of human embryonic myoblasts by West Nile virus (WNV), a flavivirus, caused significant upregulation of class I and II MHC expression as determined by flow cytometry. After 48 hours at a multiplicity of infection of 5 pfu/cell, a sixfold increase in MHC class I expression was induced from initially low levels of expression. In contrast, MHC class II was induced de novo to five times the control fluorescence level. At least 70% of the cells were infected as determined using fluorescence microscopy and anti-WNV antibody labeling. Myoblasts were > 90% pure as shown by anti--Leu-19 labeling. MHC class I (but not class II) was increased threefold after exposure to virus-inactivated supernatant from 48-hour--infected cells, indicating the presence of factor(s) contributing to the MHC class I increase. These findings may be important in establishing a link between viral infection of human cells and induction of inflammatory autoimmune disease. We discuss the possibility of using WNV as an in vivo model.


The pterygoid reflex in man and its clinical application.

A technique for eliciting and recording the stretch reflex (R) of the medial pterygoid muscle (Pter) is described. The latency was 6.9 +/- 0.43 ms in 23 healthy volunteers (mean age 23.7 years) showing a side-to-side difference of 0.29 +/- 0.21 ms. The PterR latencies were little shorter and side-to-side differences little greater than of the masseter reflex. Observations in 5 selected patients with small brainstem lesions suggest that the neurons of the PterR afferents form a cluster within the caudal portion of the trigeminal mesencephalic nucleus. Testing the masseter and pterygoid reflexes provides a more precise localization of small ponto-mesencephalic lesions.


Axonal swellings in human intramuscular nerves.

The presence, morphology, distribution, and abundance of axonal swellings in intramuscular nerves were evaluated. Axonal swellings were present in intramuscular nerves in 42% of 127 muscle biopsies from patients with a variety of conditions. The incidence was highest in muscle from patients with peripheral neuropathy, but swellings were present in muscle from patients with motor neuron disease, primary muscle diseases, and some individuals without clinical or histological evidence of neuromuscular disease. The greatest number of swellings in intramuscular nerves was in muscle from patients with chronic inflammatory demyelinating neuropathy. Swellings were spherical or elliptical, 4-20 microns in diameter, 5-30 microns in length, and composed of neurofilaments. Swellings were present only in myelinated axons of intramuscular nerves, proximal to nodes of Ranvier or in internodal regions. Swellings were not associated with axonal degeneration. They were probably not transported. The formation or accumulation of swellings may reflect altered axonal dynamics common to a number of disease processes.


Loss and renewal of thick myofilaments in glucocorticoid-treated rat soleus after denervation and reinnervation.

Denervation of rat soleus muscle and simultaneous administration of high doses of corticosteroids for 7 days caused marked muscle fiber atrophy and selective loss of thick myofilaments from many muscle fibers by light and electron microscopy. Myosin heavy chain/actin ratios were greatly reduced on polyacrylamide gel electrophoresis. Nerve crush instead of cut permitted reinnervation after 2 weeks and demonstrated the reversibility of the muscle changes within a week after reinnervation. There was formation of new thick filaments and their reintegration into myofibrils without further breakdown, although large areas of Z-disc streaming appeared. The mechanism of A-band breakdown remains obscure, but it presumably starts with limited proteolysis and continues with disaggregation of myosin molecules. This is consistent with our observation that the muscle fibers retain a relatively good reactivity to antibodies against myosin heavy chain 1 week after denervation and corticosteroid administration. A syndrome recalling these experiments is seen in severely asthmatic patients receiving corticosteroids and pharmacologically paralyzed for mechanical respiration.


The right time? Chronopharmacology--a new science.

There is convincing scientific work to indicate that more attention should be given to the timing of drug administration. Most prescribers are currently more concerned with "what" to prescribe rather than "when" to prescribe it. Chronotherapeutics certainly holds promise for the creation of the most favourable conditions for drug effects and safety and may therefore represent an important method of improving the treatment of many diseases. The goal of chronotherapeutics is to optimise the therapeutic effect and control or reduce the adverse effects without altering the functioning of the drug in the body.


[Tourette syndrome].

Tourette Syndrome is not as rare as many health professionals may think. On the contrary it is very common, especially among the Afrikaans population. It is possibly the most common inherited condition. Tourette Syndrome is a neurological variation characterized by "tics" or mannerisms, which can also be combined with one or all of the following: frontal lobe involvement particularly the limbic system; learning problems; anxiety; obsessive-compulsive behaviour; sexual deviations. Most authorities regard TS as a dominant inherited condition although Comings describes it as semi-dominant--semi-recessive.


Immunohistochemical, ultrastructural and immunoelectron microscopic studies of spinal cord neurofibrillary tangles in progressive supranuclear palsy.

Immunohistochemical, ultrastructural and immunoelectron microscopic studies of spinal cord neurofibrillary tangles (NFTs) in progressive supranuclear palsy (PSP) were performed. The spinal cord NFTs reacted with antibodies to tau protein (tau-2), ubiquitin and Alzheimer neurofibrillary tangles (ANTs, Ab 39). Ultrastructurally, the NFTs consisted of bundles of straight fibrils. In longitudinal sections, the individual NFT fibrils appeared as straight fibrils with a diameter of approximately 15 nm. In cross sections, circular structures approximately 15 nm in diameter were seen, and some had a central density. Electron microscopic examination of specimens stained with the antibodies and by the modified Bielschowsky method revealed the products of the tau, ubiquitin and ANTs immunoreactions and silver deposits on the NFT fibrils. This is the first demonstration of the ultrastructure of spinal cord NFTs in PSP.


Allocortical involvement in Huntington's disease.

Seven brains of individuals who had suffered from Huntington's disease and seven control brains were examined. Preparations stained for Nissl material and lipofuscin pigment revealed a characteristic layer-specific loss of nerve cells in two allocortical areas, the entorhinal region and the subiculum. The most severe changes occurred in the entorhinal layer Pri-gamma while layer Pre-alpha, layer Pre-gamma and the subiculum showed less severe alterations.


An immunohistochemical study of mononuclear cells in meningiomas.

A consecutive series of 34 meningiomas were re-examined as to subtype and presence of nuclear atypia, mitotic figures, areas of high cellularity and necrosis. Meningioma cells were epithelial membrane antigen (EMA) positive in 31 out of the 34 tumours. The presence of mononuclear cells and macrophages was assessed by immunohistochemistry using the monoclonal antibodies L26 (CD20, B cell marker), DF-T1 (CD43, T cell marker), KP1 (CD68, macrophage marker) and MAC387 (monocytes). L26 positive B cells were observed infrequently. CD43 positive mononuclear cells were infiltrating the parenchyma as individual cells and as groups of cells in 29 (87% of the tumours). CD68 positive macrophages were seen in 19 (59% of the tumours), as scattered single cells or groups of cells. There was a statistically significant association between the number of CD68 positive cells (necrotic areas excluded) and microscopic features of aggressiveness, i.e. high cellularity as well as the combination of nuclear atypia and frequent mitotic figures. MAC387 stained only a few cells; the immunopositive cells were present mainly within and around vessels. Meningioma cells displayed a diffuse immunopositivity for L26 (CD20) in 29 out of 34 meningiomas, but did not stain with macrophage markers. Mast cells were found in 9 out of 32 tumours; when present they were significantly more prevalent in the syncytial subtype. Thus, mononuclear cell infiltrates in meningiomas are mainly composed of T cells and macrophages, indicating an immune system surveillance and response to the tumour cells. The functional and prognostic significance of the presence of CD68 positive cells, macrophages, deserve further study in the search for more reliable histological criteria to predict recurrence and biological aggressiveness in meningiomas.


Methyl bromide intoxication and acute energy deprivation syndromes.

The case reported in this issue of symmetrical brain stem damage associated with exposure to methylbromide has affinities with a number of analogous syndromes associated with tissue energy deprivation. Attention is drawn to topographical and metabolic similarities and differences in these conditions, and suggestions are made for possible ways of mitigating the damage in future cases that may also be of value in Wernicke and Leigh's diseases.


Case report: neuropathology of methyl bromide intoxication.

The neuropathological findings in a man and his dog both of whom died after acute exposure to methyl bromide are presented. The dog had cerebral oedema, most marked in the depths of cortical sulci. The man survived 30 days after the initial illness and his brain contained well-defined symmetrical lesions in the mammillary bodies and inferior colliculi. He also had a peripheral neuropathy and lymphocytic thyroiditis. The pathology in the central nervous system has features resembling Wernicke's encephalopathology; the mechanism by which methyl bromide may produce such lesions is discussed.


The effects of pre-natal exposure to methylazoxymethanol acetate on microglia.

The effects of exposure to a cytotoxic agent, methylazoxymethanol acetate (MAM Ac), on the distribution, density and quantitative morphology of microglia in the rat forebrain have been examined with the aid of a peroxidase-conjugated lectin derived from Griffonia simplicifolia. Following exposure to MAM Ac (25 mg/kg maternal body weight) on embryonic day 13 (E13), round microglia were concentrated around the areas of induced cell death at the outer margins of the ventricular germinal zone, particularly in the striatopallidal angle and dorsal thalamus. By E19, there were no detectable differences in microglia distribution between experimental and control animals. The increase in number of microglial cells in the neocortex and caudatoputamen on exposure to MAM Ac lasted for only 4 to 6 days. Subsequently, the number of microglia dropped below control values in both regions. The density of microglia in these areas was similar in control and experimental animals from 6 days after exposure. The proportion of microglia relative to all other cells was also similar at post-natal day 17 (P17) in both experimental and control animals. These results suggest that the distribution and final size of the microglial population is determined by the microenvironment and not by the extent of cell death which may have acted as the initial stimulus to microglial invasion.


The use of azathioprine to ameliorate post-treatment encephalopathy associated with African trypanosomiasis.

The treatment of human African sleeping sickness is complicated by a post-treatment meningoencephalitis that may be fatal. Using a mouse model this study assesses the use of the non-steroidal anti-inflammatory drug, azathioprine, in the management of this post-treatment reaction. Female NIH mice treated with the trypanocidal compound diminazene aceturate (40 mg/kg), 28 days after infection, developed a similar post-treatment reaction to that seen in humans. Administration of azathioprine (100 mg/kg) for 5 days before and 5 days after trypanocidal chemotherapy abrogated the pathology in the central nervous system although this returned approximately 15 days after cessation of azathioprine. Activated astrocytes associated with the later stages of the infection did not appear to be affected by the use of azathioprine.


Effect of ether stress on growth hormone during development in the neonatal rat.

Stress in adult rats causes an inhibition of growth hormone (GH) secretion which might be mediated by corticotropin-releasing factor (CRF). The response of neonates to stress differs from that observed in adults, including changes in GH secretion that are independent of CRF. The present study examines the effects of ether exposure, a stress known to elicit CRF release, on serum GH in the neonatal period. Preliminary experiments indicated that ether elicits increases in serum adrenocorticotropic hormone and corticosterone, and that the latter response is blocked by pretreatment with dexamethasone. Corticosterone was measured as an indicator of hypothalamo-pituitary-adrenal (HPA) axis stimulation for subsequent studies. Rat pups of 5, 8, 10, 15, 18 or 30 days were divided into three groups. Baseline animals were taken for decapitation directly from the mother. Ether animals were exposed to ether fumes for 1 min, returned to the mother after a brief recovery period, and killed 30 min later. Handled control animals were removed from the mother briefly, returned, and similarly killed at 30 min. Blood was assayed for GH and corticosterone. Handling itself stimulated both GH and corticosterone on postnatal days 10 and 15 and suppressed GH and corticosterone on days 5 and 30. Ether significantly lowered GH and increased corticosterone when compared to this handling control from day 8 to 18, but values in ether-treated animals were different from baseline animals only at 5 and 30 days of age. These results indicate that ether stress produces a mild decrease in GH by day 5 postnatally and throughout the neonatal period which is only apparent in relation to 'handled' controls.(ABSTRACT TRUNCATED AT 250 WORDS)


Growth hormone pulsatility and the endocrine milieu during sexual maturation in male and female rats.

Although a sexually dimorphic pattern of rat growth hormone (rGH) secretion is well recognized in adult rats, episodic rGH release has been incompletely characterized in younger rats. In the present study, 28 to 113-day-old rats received jugular catheters and were housed in chambers designed for stress-free blood sampling. Three to seven days after surgery, 300 microliters of blood was withdrawn every 10 min between 16.00 and 22.00 h. Pulsatile rGH secretion before 33 days of age was similar in males and females. Low baseline rGH secretion was interspersed with infrequent, low amplitude pulses. In early puberty, between 33 and 40 days of age, rGH pulse amplitudes increased more than tenfold in both sexes. The duration of rGH pulses was significantly greater at this time in males versus females, a pattern that continued though adulthood. By late puberty, between 41 and 54 days of age, rGH pulse amplitudes similarly increased twofold in both sexes. Baseline rGH secretion increased at this time in both sexes, with females having a higher baseline compared to males. Only in adult rats over 54 days of age, was the typical pattern of low basal rGH secretion, combined with high amplitude, low frequency rGH pulses in males versus females evident. A sex difference in body weight, i.e. males weighing more than females, became evident after day 33 of age. This sex difference was preceded by an increase in testosterone in males versus females at all ages examined.(ABSTRACT TRUNCATED AT 250 WORDS)


Effect of the duration of streptozotocin-induced diabetes on turnover of central biogenic amines in mice.

The relationship between central catecholaminergic and serotonergic neurons and the development of streptozotocin-induced diabetic mice was examined over periods of 3, 14, 50 and 100 days. The accumulation of 3,4-dihydroxyphenylalanine (DOPA) and 5-hydroxytryptophan (5-HTP) were measured after central decarboxylase activity was inhibited by m-hydroxybenzylhydrazine (NSD-1015). Results indicated that DOPA accumulation in the hypothalamus was not altered during the entire time course of the experiment. On the other hand, DOPA accumulation in the striatum was decreased in 50- and 100-day diabetic mice. The DOPA levels in the pons medulla were not increased until the mice had been diabetic for 100 days. The accumulation of 5-HTP was decreased in the hypothalamus of 14-day diabetic mice and was also present at 50 and 100 days, that in the striatum and pons medulla was not decreased until the mice had been diabetic for 50 days and persisted to 100 days in the striatum. These data showed that both DOPA and 5-HTP accumulation in the striatum and pons medulla were changed only in long-term diabetic mice and suggested that changes in catecholamine and serotonin turn-over rates are not generalized, but restricted only to some particular brain regions and time courses.


Regulation of corticotropin-releasing factor mRNA in neuroendocrine and autonomic neurons by osmotic stimulation and volume loading.

Corticotropin-releasing factor (CRF) has been reported to be expressed in oxytocin-containing magnocellular neurosecretory neurons of the paraventricular (PVN) and supraoptic (SON) nuclei, and in Barrington's nucleus, a pontine micturition center. Each of these cell groups is known to play a role in fluid and electrolyte homeostasis. To gain a better understanding of the role of CRF in this context, the effects of osmotic stimulation and volume loading on CRF mRNA levels in the PVN, SON and Barrington's nucleus were examined using in situ hybridization histochemistry with an 35S-labeled cRNA probe. Adult male rats received either normal tap water (control), or hypertonic (2%) saline (HS) for up to 3 days. In a second experiment, normal saline was infused through a jugular vein cannula at 6 ml/h for 3 days; control rats were cannulated but received no infusion. Relative levels of CRF mRNA were compared by estimating both the number of positively hybridized cells and the density of silver grains in emulsion-dipped autoradiograms. HS treatment resulted in marked increases in CRF mRNA in the magnocellular neurosecretory system. All recognized oxytocinergic cell clusters, i.e., the anterior, medial and posterior magnocellural subdivisions of the PVN, the dorsal aspect of the SON, and components of the accessory magnocellular system, displayed this effect. By contrast, HS treatment resulted in significant decreases in CRF mRNA levels in the parvocellular (hypophysiotropic) division of the PVN and in Barrington's nucleus. By contrast, volume loading, which failed to affect plasma osmolality, significantly increased CRF mRNA levels in Barrington's nucleus.(ABSTRACT TRUNCATED AT 250 WORDS)


Hypothalamic GnRH pulse generator activity during the estradiol-induced LH surge in ovariectomized goats.

Electrophysiological manifestation of the hypothalamic GnRH pulse generator activity was examined during the LH surge induced by estradiol in ovariectomized goats. The characteristic increases in the frequency of multiple unit activity (MUA volley) associated with the pulsatile secretion of LH were recorded using electrodes implanted bilaterally in the medial basal hypothalamus. Estradiol was infused for 16 h at the rate of 3 micrograms/h, to induce an LH surge 10.0-11.5 h later. Regular recurrence of MUA volley was observed throughout the experimental period including the LH surge, but the interval between the MUA volleys became longer (p < 0.01) after the onset of the LH surge as compared with the pretreatment control period (32.9 +/- 2.1 vs. 60.0 +/- 5.1 min). These results suggest that an increased frequency of LH pulses is not a prerequisite for the LH surge in ovariectomized goats given estradiol, and imply that the positive feedback effects of estradiol on LH secretion appear to be exerted through a neuronal mechanism that is intrinsically different from the GnRH pulse generator.


Effect of [4Cl-D-Phe6,Leu17]VIP on the inhibition of pulsatile LH release by VIP and related peptides in the ovariectomized rat.

Pulsatile LH secretion in the ovariectomized (OVX) rat is inhibited by intracerebroventricular (icv) infusion of vasoactive intestinal peptide (VIP). VIP, rat growth hormone-releasing hormone (rGRH) and secretin with and without an antagonist to VIP, [4Cl-D-Phe6,Leu17]VIP (VIPA), were infused icv into OVX rats. Both VIP and rGRH at an infusion rate of 3.5 nmol/h lowered mean LH concentrations and pulse frequency without affecting pulse amplitude, and these effects were blocked by concurrent infusion of VIPA (10.5 nmol/h). Secretin also inhibited pulsatile LH secretion, but was only fully effective at the higher infusion rate of 7 nmol/h. This effect of secretin was also blocked by concurrent infusion of 10.5 nmol/h of VIPA. These results suggest that all three of these peptide hormones inhibit pulsatile LH secretion by an interaction with VIP receptors.