Measurement of 2-deoxyglucose and 2-deoxyglucose 6-phosphate in tissues

The enzymatic methods previously described for 2-deoxyglucose (DG) and 2-deoxyglucose 6-phosphate have been refined and adapted to measurements of brain samples ranging from 50 mg wet weight to less than a microgram dry weight. Procedures for preparing such samples for assay are described. Analytical properties of the enzymes employed are given together with means for overcoming their possible short comings. Emphasis is placed on information useful for employing DG to assess rapid changes in glucose metabolism.     

How can storage time and temperature affect enzymic activities in urines?

We measured a tubular brush-border enzyme (alanine aminopeptidase, AAP) and a lysosomal hydrolase (N-acetyl-beta-D-glucosaminidase, NAG) in morning urines from 15 healthy normal subjects to check if different storage times and temperatures could modify enzyme concentrations. Short-term (24 h) storage time at room temperature or 4 degrees C does not affect AAP and NAG activities. Both enzymes are well preserved at -70 degrees C. AAP dramatically falls after 1 month at -20 degrees C, lowering to about 8% of the initial value after only 4 days of storage. On the contrary, NAG is well preserved at these storage conditions. Centrifugation has revealed not critical for measurement of these two enzymes.     

The T cell receptor V alpha 11 gene family. Analysis of allelic sequence polymorphism and demonstration of J alpha region-dependent recognition by allele-specific antibodies.

Allelic polymorphism in TCR loci may play an important role in shaping the T cell repertoire and in disease susceptibility. We have used a combination of antibody and sequence analysis to investigate polymorphism in the murine V alpha 11 family. Two different antibodies have been analyzed that recognize particular V alpha 11 family members of the V alpha b and V alpha d haplotypes. One antibody shows J alpha dependency, suggesting a conformational element to the epitope. Investigation of the anti-V alpha 11 staining pattern on different mouse strains indicates that there is a marked influence of MHC haplotype on V alpha 11 selection and that V alpha 11 is preferentially expressed on CD4+ cells. Sequence analysis of V alpha 11 genes from the V alpha a, V alpha b, and V alpha d haplotypes shows two potential regions for the haplotype-specific epitopes. The relatedness of the different V alpha 11 family members from different haplotypes suggests that the V alpha 11.1/11.2 gene duplication is relatively recent, but that V alpha 11.3 separated much earlier. Differences between V alpha 11.3 and V alpha 11.1/11.2 are concentrated in the putative complementarity determining regions (CDR), whereas differences between alleles are not clearly clustered. However, the V alpha 11.1a and V alpha 11.1d alleles differ from V alpha 11.1b and V alpha 11.2b in CDR1. A V alpha 11.2-expressing anti-cytochrome c T cell has the same V-J junction as a V alpha 11.1-bearing cell with a similar fine specificity, indicating that V alpha 11.1b and V alpha 11.2b do not contribute different Ag specificities.     

Myelotoxicity of epirubicin]

Epirubicin (pharmorubicin, India), an antitumor antibiotic of the anthracycline group, was studied in regard to its effect on peripheral blood, bone marrow and lymphoid organs (the thymus and spleen) of CBA mice after its intraperitoneal administration in a single dose equal to the MIC (7.8 mg/kg) and in a course dose (1/5 of the MIC 5 times a day). The cytogenetic impairments induced by the cytostatic were estimated on metaphase plates with the bone marrow specimens and by counting the peripheral blood erythrocytes with micronuclei (the micronuclear test). It was shown that epirubicin induced cytogenetic disturbances in the hemopoietic cells within the first 72 hours. The antibiotic had a marked reversible effect on the erythroid population and lymphoid tissues and a moderate toxic action on the granulocyte population. The antibiotic did not affect thrombocytopoiesis. The single administrations had a more pronounced and prolonged myelotoxic and lymphotropic effect.     

Effect of inserted oxysterols on phospholipid packing in normal and sickle red blood cell membranes

Fourier transform infrared (FTIR) spectroscopy was used to examine the effect of oxysterol insertion into normal and sickle RBC membranes and the total lipid extracts of the membranes. Examination of the FTIR C-H stretch and fingerprint regions reveal that the insertion of 7 alpha- and 7 beta-hydroxycholesterol has the greatest effect on the fluidity of RBC membranes and lipid extracts. The results confirm the observation that sterol molecules are oriented in the membrane so that the 7 position is located in the phospholipid head group region at the lipid/water interface. The substitution of a keto for a hydroxy group at the number seven position decreases the effect of the sterol on membrane packing.     

Organotin compounds induce aneuploidy in human peripheral lymphocytes in vitro

In vitro exposure of PHA-stimulated human lymphocytes to organotin compounds resulted in statistically significant increases in the frequencies of hyperdiploid cells. When taken together with our previous study demonstrating spindle inhibiting effects of the same organotin compounds by an indirect method (Jensen et al., 1989), the present study strongly indicates that organotin compounds are able to induce aneuploidy, probably by affecting spindle function.     

Differentiation of dairy strains of the Bacillus cereus group by phage typing, minimum growth temperature, and fatty acid analysis

A total of 130 Bacillus strains were isolated from dairy products, the dairy environment and from packaging boards and board-producing machines. Ninety-eight of these were members of the B. cereus group ( B. cereus, B. mycoides and B. thuringiensis ) as determined by whole cell fatty acid composition. Fatty acid composition did not differentiate between the three species. Of the 98 strains, which were indistinguishable by biochemical tests, 87 could be assigned into 21 different phage types (11 strains remained untypable) when tested with 12 B. cereus, B. mycoides and B. thuringiensis phages. The distribution of phage types between strains from different sources showed that the source of contamination of the dairy products was of milk origin and not from the packaging materials. Most strains isolated from the dairy products were able to grow below 10°C, whereas strains from the dairy environment and from board mills had higher minimum growth temperatures.     

Conversion of human low density lipoprotein into a very low density lipoprotein-like particle in vitro.

The lipid substrate specificity of Manduca sexta lipid transfer particle (LTP) was examined in in vitro lipid transfer assays employing high density lipophorin and human low density lipoprotein (LDL) as donor/acceptor substrates. Unesterified cholesterol was found to exchange spontaneously between these substrate lipoproteins, and the extent of transfer/exchange was not affected by LTP. By contrast, transfer of labeled phosphatidylcholine and cholesteryl ester was dependent on LTP in a concentration-dependent manner. Facilitated phosphatidylcholine transfer occurred at a faster rate than facilitated cholesteryl ester transfer; this observation suggests that either LTP may have an inherent preference for polar lipids or the accessibility of specific lipids in the donor substrate particle influences their rate of transfer. The capacity of LDL to accept exogenous lipid from lipophorin was investigated by increasing the high density lipophorin:LDL ratio in transfer assays. At a 3:1 (protein) ratio in the presence of LTP, LDL became turbid (and aggregated LDL were observed by electron microscopy) indicating LDL has a finite capacity to accept exogenous lipid while maintaining an overall stable structure. When either isolated human non B very low density lipoprotein (VLDL) apoproteins or insect apolipophorin III (apoLp-III) were included in transfer experiments, the sample did not become turbid although lipid transfer proceeded to the same extent as in the absence of added apolipoprotein. The reduction in sample turbidity caused by exogenous apolipoprotein occurred in a concentration-dependent manner, suggesting that these proteins associate with the surface of LDL and stabilize the increment of lipid/water interface created by LTP-mediated net lipid transfer. The association of apolipoprotein with the surface of modified LDL was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, and scanning densitometry revealed that apoLp-III bound to the surface of LDL in a 1:14 apoB:apoLp-III molar ratio. Electron microscopy showed that apoLp-III-stabilized modified LDL particles have a larger diameter (29.2 +/- 2.6 nm) than that of control LDL (22.7 +/- 1.9 nm), consistent with the observed changes in particle density, lipid, and apolipoprotein content. Thus LTP-catalyzed vectorial lipid transfer can be used to introduce significant modifications into isolated LDL particles and provides a novel mechanism whereby VLDL-LDL interrelationships can be studied.     

Genetic control of hexokinase variation in insects

Hexokinase variation in insects appears to be under the control of a single locus in some species and under multiple-locus control in others. It is often difficult to distinguish the number of loci controlling hexokinase expression. Analysis of hexokinase electrophoretic patterns in six species of mosquitoes and five species of crickets, as well as a review of hexokinase variation in other insect species, is used to emphasize the importance of interspecific comparisons when making genetic inferences. Evidence is provided which adds support for multiple hexokinase loci in dipterans. Hexokinase control by multiple loci may be difficult to determine in some species because of tight linkage, disequilibrium, and/or posttranslational modification.This work was supported by NIH Grant AI 11132-09, NSF Grant DEB-7811188, and Yale Sesil Anonymous Funds.