Cerebral utilization of glucose, ketone bodies and oxygen in starving infant rats and the effect of intrauterine growth retardation

Cerebral arteriovenous differences of acetoacetate, D-beta-hydroxybutyrate, glucose, lactate and oxygen and brain DNA content was measured at 20 days of age in intrauterine growth retarded (IUGR) rats and normal littermates after 48 and 72 h of starvation. Cerebral blood flow (CBF) was measured with labeled microspheres in other comparable groups of IUGR and control rats. CBF was similar in IUGR and normal littermates (0.57+/-0.09 and 0.58+/-0.10 ml/min respectively). After 48 h of starvation, arterial glucose was significantly lower in IUGR than control animals but the arterial concentrations of ketone bodies were similar. After 48 h of starvation, cerebral arteriovenous difference of beta-hydroxybutyrate was significantly higher in control than IUGR rats also when expressed per mg brain DNA as was the fractional uptake of D-beta-hydroxybutyrate. After 72 h of starvation, arterial concentrations of ketone bodies were significantly lower in IUGR rats than controls but the fractional uptake of D-beta-hydroxybutyrate was increased compared to IUGR rats starved for 48 h. The average percentage of calculated total substrate uptake (mumol/min) accounted for by ketone bodies increased in control animals from 31.1% after 48 h of starvation to 41.0% after 72 h of starvation. In IUGR rats these percentage values were 26.5 and 25.7 respectively. After 72 h of starvation the fraction of total cerebral uptake of substrates accounted for by ketone bodies was significantly higher in control that IUGR rats. As total cerebral uptake of substrates was similar between IUGR and control animals it is concluded that IUGR rats are more dependent on glucose as a substrate for the brain during starvation.     

Preferential but not exclusive T cell receptor V beta chain utilization of myelin basic protein and peptide-specific T cell clones in mice

The repertoire of T cell receptor (TCR) V beta chain utilization was investigated in PL/J, CXJ-1, SJL/J and B10.S-->SJL/J chimeric mice in response to either myelin basic protein (MBP) or the strain-specific encephalitogenic peptide. Our analysis showed that there was an overlapping predominance in the TCR V beta gene utilization in the MBP-specific responses, which were independent of the major histocompatibility complex (MHC) class II haplotype present, and the immunodominant peptide region recognized in these different strains. In those mice having the TCR V beta b haplotype (PL/J, CXJ-1, and the B10.S-->SJL chimera) either the TCR V beta 4, 8, and 13 or the TCR V beta 4, 6, and 13 predominated. In contrast, in mice with TCR V beta a haplotype (SJL/J) V beta 4, 6, and 17a were found. However, the quantitative distribution of these preferentially utilized TCR V beta chains in each strain was defined by the MHC class II haplotype and the immunodominant peptide recognized. The expression of the V beta 8 gene product in the peripheral TCR repertoire did not always correlate with predominant V beta 8 utilization in the MBP-specific response.     

Preferential interactions of fluorescent probe Prodan with cholesterol

The fluorescent probe Prodan has been widely used as a probe of model and biological membranes. Its fluorescent maxima in phospholipid bilayers vary as a function of phase state, with maxima at 485 for the liquid crystal Lalpha, 435 nm for the gel L'beta, and 507 nm for the interdigitated gel LbetaI phase, with excitation at 359 nm. These spectral changes have been used for the detection of phase changes among these phases. In the present study, the fluorescent properties and partition coefficients of Prodan in model membranes of phosphatidylcholines and phosphatidylethanols have been studied as a function of lipid phase state and cholesterol content. It is shown that the Prodan spectrum in the presence of cholesterol no longer reflects the known phase state of the lipid; in each phase state, the presence of cholesterol leads to a spectrum with the maximum at 435 nm, characteristic of the noninterdigitated gel phase. The partition coefficient of Prodan into these lipids also varies with the phase state, giving values of 0.35 x 10(4) in the interdigitated gel, 1.8 x 10(4) in the noninterdigitated gel, and 7. 6 x 10(4) in the liquid crystal phase. In the presence of cholesterol these partition coefficients are increased to 13 x 10(4) for the liquid crystal and the gel phase, and 5.1 x 10(4) in the presence of 100 mg/ml ethanol. These results suggest that Prodan has preferential interactions with cholesterol, and is thus not a randomly distributed fluorescent reporter probe in membranes containing cholesterol. These results suggest that Prodan should be used only with great caution in complex lipid mixtures, particularly biological membranes.     

Triethyllead-restrained myelin deposition and protein synthesis in the developing rat forebrain

A study was made of the electrogenesis on (Na+C1)-free muscles in potassiumsulfate media, with the membrane potential not corresponding to Ek. It was found that the potassium content in these muscles may change: at 2.5 mM external potassium, the internal potassium content decreases, whereas at 75 mM it increases. Undoubtedly, some other ions (phosphates?) apart from K+ do penetrate the muscle membrane. The evidence obtained disprove a previous idea that under the condition described it is potassium alone which is involved in potential genesis.     

In vivo acylation of rat brain myelin proteolipid protein

Examination of brain myelin proteins by sodium dodecyl sulfate-gel electrophoresis followed by fluorography clearly showed that both proteolipid protein (PLP) and DM-20 were acylated 24 h after the intracerebral injection of 30-day-old rats with [3H]palmitic acid. The radioactivity associated with PLP remained after purification, re-electrophoresis, and fluorography. Most of the radioactivity associated with PLP was removed when the gels were treated with hydroxylamine and then fluorographed, indicating that fatty acids were bound to PLP by ester linkage. Cleavage of purified PLP with methanolic sodium hydroxide readily released almost all protein-bound radioactivity. Thin layer chromatography of this material on both silver nitrate and reverse-phase plates provided evidence that most of the radioactivity co-migrated with methyl palmitate (77%) and methyl stearate (19%); however, some radioactivity was associated with methyl oleate (4%). Gas-liquid chromatography of the fatty acids associated with PLP distinctly revealed the presence of methyl palmitate and a detectable peak of methyl stearate.     

Preferential inhibition of lipid synthesis by the bacteriocin pyocin S2

The effects of pyocin S2, a bacteriocin produced by Pseudomonas aeruginosa strain M47, on several processes in susceptible bacterium have been examined. Lipid synthesis, measured in terms of [32P]phosphate, [14C]acetate or [2-3H]glycerol incorporation into lipid fractions, was halted almost completely soon after pyocin S2 addition. When cell suspensions were treated with various amounts of pyocin S2, the extent of inhibition of lipid synthesis was proportional to the ratio of killed bacteria. Protein synthesis was not essential for the inhibition. Degradation of lipid due to pyocin S2 was not detected. Pyocin S2 also affected protein and nucleic acid syntheses, but these inhibitions appeared with a delay of about 10 min after the cessation of lipid synthesis. The effect of trypsin [EC 3.4.21.4] on the viability of cells which had adsorbed pyocin S2 was also investigated: the cells went through a period when the destruction of pyocin S2 by trypsin restored the colony-forming ability of the cells (stage I). Then transition to a second state in which the cells lost viability irrespective of trypsin treatment (stage II) took place. The transition from stage I to stage II depended on the energy metabolism of the cells and followed first-order kinetics with a rate proportional to the number of killing units of adsorbed pyocin S2. The residual capacity for lipid synthesis in cells which had adsorbed pyocin S2 after trypsin treatment at various times indicated that lipid synthesis was inhibited only in the cells at stage II of pyocin S2 action.     

Regulation of oleoyl-CoA synthesis in the peripheral nervous system: demonstration of a link with myelin synthesis

We studied the regulation of oleic acid synthesis in the PNS. During mouse postnatal development, the proportion of 18:1 rises in the sciatic nerve from 17% at 5 days of age to 33% at 25 days. However, this rise does not occur in the dysmyelinating mutant mouse trembler. In normal mouse development, the total stearoyl-CoA desaturase (SCD) activity measured in sciatic nerve homogenates is high during the first 3 weeks. Yet in trembler nerves, this SCD activity represents only 15% of normal values. Using the RT-PCR technique, we demonstrate that the SCD2 isoform is predominantly expressed in the PNS. Northern blot analysis showed that the mRNA levels for SCD2 parallel those of other specific myelin proteins in both normal mouse and trembler mutant development. Similar experiments in a rat demyelination-remyelination model confirmed that SCD2 mRNA levels are regulated in the PNS in a similar manner to myelin-specific proteins.     

Preferential in vivo accumulation of sn-2,3-diacylglycerols in postheparin plasma of rats

The stereochemical course of in vivo hydrolysis of triacylglycerols by lipoprotein lipase was investigated by determining the structure of diacylglycerol intermediates in postheparin plasma of rats which had been fed [3H]glycerol-labeled Intralipid 2 h before an injection of heparin or had been given an injection of a mixture of [3H]glycerol-Intralipid and heparin. During the clearance of both the natural chylomicrons and the artificial emulsion, sn-2,3-diacylglycerols (60-80%) were found to be the dominant enantiomers. Similar results were obtained when the contribution of the hepatic lipase was altered, either by tying off the mesentery artery and portal vein before injection of heparin, or by injection of heparin directly into the portal vein. These findings are consistent with a preferential release of the acyl group from the sn-1 position of the triacylglycerol molecule as demonstrated previously in vitro. A preferential orientation of the substrate in the enzyme-substrate complex or at the oil-water interface is discussed as a possible basis for these effects.     

Screening of drugs inhibiting cholesterol synthesis

The biosynthesis of fusidin, an antibiotic of steroid structure, was used as a model of steroid synthesis. Screening of compounds modifying the fusidin synthesis included 80 strains of mycelial fungi. A high frequency of such fungal cultures was observed. 9 cultures forming compounds which inhibited the synthesis of cholesterol in the cell culture of human hepatocytes were screened. A method for biological estimation of the activity of cholesterol synthesis inhibitors was developed on rabbits with high blood levels of cholesterol. It was shown that the cholesterol synthesis inhibitors, isolated as a result of the specific screening were not toxic and markedly lowered the cholesterol blood levels.     

Feed-back control of cholesterol synthesis in partially hepatectomized rats

Partial hepatectomy caused a marked stimulation of cholesterol and fatty acid syntheses without affecting serum total cholesterol, total phospholipid and triacylglycerol concentrations of rats so far examined 48 h after the operation. Serum free cholesterol level, however, was increased by the treatment and the ratio of lysophosphatidylcholine to phosphatidylcholine was concomitantly decreased, suggesting the impairment of serum lecithin: cholesterol acyltransferase activity. The lipid content in the liver, especially triacylglycerol and ester cholesterol, was increased markedly by the operation. Feeding of a high cholesterol diet which elevated serum cholesterol and phospholipid levels to the partially hepatectomized rats, accelerated the accumulation of hepatic triacylglycerol and ester cholesterol by the partial hepatectomy. The weight of the regenerating liver was not influenced by cholesterol feeding, which suggested that cholesterol feeding did not inhibit the regeneration mechanism of the liver. The increase of cholesterol synthesis after partial hepatectomy was inhibited by cholesterol feeding. Therefore, it is conceivable that the negative feed-back control of cholesterol synthesis is induced by cholesterol feeding under the stimulated cell divisions of the liver after partial hepatectomy. It is suggested from the present data that a large amount of the cholesterol which is necessary for cell growth can be taken up from serum, when serum cholesterol concentration is high.     

Preferential regulation of protein synthesis initiation complex formation by purine nucleotides

Adult male volunteers with a prior history of either moderate (N = 12) or heavy (N = 14) marihuana use were systematically observed before, during and after a 21-day period of free access to 1 g 2% delta-9 THC marihuana cigarettes. A matched sample of casual alcohol drinkers (N = 11) served as a control group. Sleep and other molar behaviors were observed hourly to obtain a representative sample of daily activity. Both moderate and heavy users were less active immediately after marihuana use and slept more on days following heavier consumption. Heavy users reduced their waking activity on days following heavier consumption, as well as during the entire period of marihuana availability. These reactions did not persist beyond the period of availability for either group. The findings suggest a dose-related delayed reaction to heavy marihuana consumption which disappears following the cessation of regular use. However, changes in activity following single doses of marihuana may be related more to the social circumstances of its use than to its pharmacological action.     

Plasma lipids, ketone bodies, and glucose concentrations in calves fed high- and low-fat milk replacers

Twenty-four 5-day-old male calves were fed twice daily milk replacers containing either 5% (low-fat) or 25% (high-fat) lard. Plasma lipids, blood glucose, and ketone bodies were determined in jugular blood before feeding and every hour during 8 h after feeding. The high-fat diet caused in the 1st h after feeding a sharp increase of triglycerides and phospholipids followed by a sharp decrease; these two increased slowly during the following 5 h. Within the first 2 h after feeding, there was an increase of cholesterol esters, free cholesterol, and nonesterified fatty acids. With the low-fat diet, triglycerides and cholesterol esters showed a small increase during the 4 h following meal whereas phospholipids, free cholesterol, and nonesterified fatty acids were not affected significantly. With both diets, blood glucose reached a maximum of 110 mg/100ml 1 h after feeding; ketone bodies were not altered significantly. With the high-fat diet, lipid digestion would occur in two phases; firstly, part of the fat would be lipolyzed quickly by pregastric esterase before clot formation in the abomasum; secondly, the rest of the lipids, slowly released by progressive lysis of the coagulum would be digested under the action of gastric and pancreatic lipases. The first phase did not occur with the low-fat diet.     

In vivo clonal expression of T lymphocytes specific for an immunodominant N-terminal myelin basic protein epitope in healthy individuals

The role of myelin basic protein (MBP) T cell recognition in the induction of experimental allergic encephalomyelitis (EAE) has been well established in mice and rats. A remarkable restriction has been observed in T cell receptor (TCR) genes utilized by encephalitogenic T cell lines (TCLs) specific for immunodominant epitopes in these species. Pathological similarities between this animal model and multiple sclerosis (MS) has led to consider MBP as a major candidate autoantigen in this human disorder. Unlike in inbred strains of animals, the T cell response to MBP in humans is quite heterogenous with regard to fine epitope specificity. The existence of V alpha and/or V beta restriction in MBP-specific T cells, from MS patients and healthy controls, is still a matter of debate. In this study we generated 77 MBP-specific TCLs from nine healthy donors and showed that peptide 7-27 is one of the most frequently recognized epitopes. 37% of all epitope-specific TCLs recognized this peptide and p7-27 specific TCLs were generated from seven out of the nine subjects studied. A high level of in vivo clonal expansion was observed in p7-27-specific TCLs in several subjects, which however is not specific of this epitope since this phenomenon was also observed in p85-104- and 149-162-specific TCLs.     

Microbial amino acid synthesis and utilization in rats: the role of coprophagy

The Substance Abuse Monitor is a comprehensive community-based addictions information system that forms the basis for an innovative approach to community needs assessment. This paper describes: (1) the conditions that created the need and support for the monitoring system; (2) the community development strategy that was employed to secure the commitment of agencies to the project; (3) the specifics of the needs assessment procedures; (4) some of the theoretical, clinical and methodological issues on which the procedures are based; (5) the practical applications of the system; and (6) the limitations of the system. The establishment of the database was based upon the idea that there would be benefits at several levels: the community, the participating agencies, the individual counsellors and the clients. The realization of these benefits requires that the information collected and the results generated must be capable of addressing specific service delivery problems with practical and tangible solutions. It is argued that the project has been successful in generating such solutions, and has considerable potential as an ongoing needs assessment tool.     

Aqueous primary standard for use in measuring cholesterol by the cholesterol oxidase method

An aqueous primary standard is needed for measuring cholesterol by enzymatic procedures. Sodium deoxycholate solubilizes cholesterol in water. Crystalline cholesterol (1.00, 3.00, 4.00, and 5.00 g/L), added to a solution containing 150 g of this compound per liter of a 9 g/L saline solution, was measured by a cholesterol oxidase procedure, with a centrifugal analyzer. The solubilizer did not interfere. When compared to an isopropanol-based commercial cholesterol standard, the cholesterol standard in solubilizer showed excellent correlation (r = 0.986; m = 0.989). Day-to-day variation for the mixture during nine days was small (CV, 2.9% at 100 g/L, 3.7% at 3.00 g/L, and 1.8% at 4.00 g/L). Linearity was maintained up to 5.00 g/L. The cholesterol concentration in four reference sera so analyzed maintained CV's of less than 4%. The viscosity of the mixture was similar to that of serum. The standard mixtures, stored at room temperature for 360 days, remained stable. The solubilized cholesterol standard is shown to be suitable for use in the enzymatic procedure.     

The regional distribution of myelin oligodendrocyte glycoprotein (MOG) in the developing rat CNS: an in vivo immunohistochemical study

Using an immunohistochemical approach we have characterized the in vivo developmental distribution of myelin oligodendrocyte glycoprotein within the rat CNS. Myelin oligodendrocyte glycoprotein expression emerged in a non-uniform manner during the first 3 postnatal weeks. Although it was absent throughout the CNS of the newborn rat at postnatal day 0(P0), it had appeared in the spinal cord and brainstem by P7. The forebrain and cerebellum remained devoid of immunoreactivity until after P14. Myelin oligodendrocyte glycoprotein emerged at different times within the closely associated fasciculi of the dorsal funiculus. It appeared in the fasciculus cuneatus during the first postnatal week and in the fasciculus gracilis and corticospinal tracts during weeks 2 and 3 respectively. Myelin oligodendrocyte glycoprotein expression developed along a caudo-rostral gradient from spinal cord to forebrain and along an antero-posterior gradient within the CNS in general. The relationship between the onset of myelin oligodendrocyte glycoprotein expression and myelinogenesis was also investigated. In most regions, myelin oligodendrocyte glycoprotein expression lagged behind the initial appearance of myelin basic protein and Luxol Fast Blue-stained myelin by at least 1 week. These observations support the idea that myelin oligodendrocyte glycoprotein is the latest myelin protein to appear in development, only being expressed during the final stages of oligodendrocyte differentiation. Furthermore, the pattern of staggered expression within the dorsal columns indicates that localized, region-specific interactions may comprise a key element in the control of the terminal phases of oligodendrocyte differentiation.