Genetic analysis of metabolic defects in the spontaneously hypertensive rat

Abnormalities in carbohydrate and lipid metabolism are common in patients with essential hypertension and in the spontaneously hypertensive rat (SHR). To identify chromosome regions contributing to this clustering of cardiovascular risk factors in the SHR, we searched for quantitative trait loci (QTL) associated with insulin resistance, glucose intolerance, and dyslipidemia by using the HXB/BXH recombinant inbred (RI) strains. Analysis of variance in RI strains suggested significant effects of genetic factors. A genome screening of the RI strains with more than 700 markers revealed QTL significantly associated with insulin resistance on Chromosomes (Chrs) 3 and 19. The Chr 19 QTL was confirmed by testing a previously derived SHR-19 congenic strain: transfer of a Chr 19 segment delineated by markers D19Rat57 and D19Mit7 from the Brown Norway (BN/Cr) strain onto the genetic background of the SHR/Ola was associated with decreased insulin and glucose concentrations and ameliorated insulin resistance at the tissue level. These findings suggest that closely linked genes on Chr 19, or perhaps even a single gene with pleiotropic effects, influence the clustering of metabolic disturbances in the SHR-BN model.     

Topological and Functional Characterization of the ssSPTs,Small Activating Subunits of Serine Palmitoyltransferase

The topological and functional organization of the two isoforms of the small subunits of human serine palmitoyltransferase (hssSPTs) that activate the catalytic hLCB1/hLCB2 heterodimer was investigated. A variety of experimental approaches placed the N termini of the ssSPTs in the cytosol, their C termini in the lumen, and showed that they contain a single transmembrane domain. Deletion analysis revealed that the ability to activate the heterodimer is contained in a conserved 33-amino acid core domain that has the same membrane topology as the full-length protein. In combination with analysis of isoform chimera and site-directed mutagenesis, a single amino acid residue in this core (Met²⁵ in ssSPTa and Val²⁵ in ssSPTb) was identified which confers specificity for palmitoyl- or stearoyl-CoA, respectively, in both yeast and mammalian cells. This same residue also determines which isoform is a better activator of a mutant heterodimer, hLCB1^S331F/hLCB2a, which has increased basal SPT activity and decreased amino acid substrate selectivity. This suggests that the role of the ssSPTs is to increase SPT activity without compromising substrate specificity. In addition, the observation that the C-terminal domains of ssSPTa and ssSPTb, which are highly conserved within each subfamily but are the most divergent regions between isoform subfamilies, are not required for activation of the heterodimer or for acyl-CoA selectivity suggests that the ssSPTs have additional roles that remain to be discovered.     

Effects of microwaves on membranes of hematopoietic cells in their structural and functional organization

The role of cell membranes in stimulating and inhibiting the effects of microwaves was investigated in experiments carried out with a suspension of murine bone marrow cells irradiated with microwaves in vitro [f = 2.45 GHz, CW, specific absorption rate (SAR) = 12 W/kg]. Results obtained by means of a structural probe, 2.4-TNS, indicate that no structural changes occur in the region of the protein-lipid interphase under conditions of short-term irradiation with microwaves that induced temperatures in the range 36–45°C (exposure time 315 and 525 s, respectively). Investigation of one functional parameter—the ability to produce hematopoietic colonies in the spleen after transplantation of the bone marrow irradiated in vitro by microwaves—indicated the possibility of affecting stimulatory and inhibitory effects of microwaves by using a blocker of cell receptors, Trimepranol. The role of microwaves as a physical factor interfering in the process of cell proliferation at the level of receptor regulation is discussed. © 1993 Wiley-Liss, Inc.     

Separation of multiple genes controlling the T-cell proliferative response to IL-2 and anti-CD3 using recombinant congenic strains

T lymphocytes of the strain BALB/cHeA exhibit a low proliferative response to IL-2 and a high response to the anti-CD3 monoclonal antibodies, while the strain STS/A lymphocyte response to these stimuli is the opposite. We analyzed the genetic basis of this strain difference, using a novel genetic tool: the recombinant congenic strains (RCS). Twenty BALB/c-c-STS/Dem (CcS/Dem) RCS were used, each containing a different random set of approximately 12.5% of the genes from STS and the remainder from BALB/c. Consequently, the genes participating in the multigenic control of a phenotypic difference between BALB/c and STS become separated into different CcS strains where they can be studied individually. The strain distribution patterns of the proliferative responses to IL-2 and anti-CD3 in the CcS strains are different, showing that different genes are involved. The large differences between individual CcS strains in response to IL-2 or anti-CD3 indicate that both reactions are controlled by a limited number of genes with a relatively large effect. The high proliferative response to IL-2 is a dominant characteristic. It is not caused by a larger major cell subset size, nor by a higher level of IL-2R expression. The response to anti-CD3 is known to be controlled by polymorphism in Fc receptor 2 (Fcgr2) and the CcS strains carrying the low responder Fcgr2 allele indeed responded weakly. However, as these strains do respond to immobilized anti-CD3, while the STS strain does not, and as some CcS strains with the BALB/c allele of Fcgr2 are also low responders, additional gene(s) of the STS strain strongly depress the anti-CD3 response. In a backcross between the high responder and the low responder strains CcS-9 and CcS-11, one of these unknown genes was mapped to the chromosome 10 near D10Mit14. The CcS mouse strains which carry the STS alleles of genes controlling the proliferative response to IL-2 and anti-CD3 allow the future mapping, cloning, and functional analysis of these genes and the study of their biological effects in vivo.     

The Coxsackievirus-Adenovirus Receptor Protein Can Function as a Cellular Attachment Protein for Adenovirus Serotypes from Subgroups A, C, D, E, and F

Attachment of an adenovirus (Ad) to a cell is mediated by the capsid fiber protein. To date, only the cellular fiber receptor for subgroup C serotypes 2 and 5, the so-called coxsackievirus-adenovirus receptor (CAR) protein, has been identified and cloned. Previous data suggested that the fiber of the subgroup D serotype Ad9 also recognizes CAR, since Ad9 and Ad2 fiber knobs cross-blocked each other’s cellular binding. Recombinant fiber knobs and ³H-labeled Ad virions from serotypes representing all six subgroups (A to F) were used to determine whether the knobs cross-blocked the binding of virions from different subgroups. With the exception of subgroup B, all subgroup representatives cross-competed, suggesting that they use CAR as a cellular fiber receptor as well. This result was confirmed by showing that CAR, produced in a soluble recombinant form (sCAR), bound to nitrocellulose-immobilized virions from the different subgroups except subgroup B. Similar results were found for blotted fiber knob proteins. The subgroup F virus Ad41 has both short and long fibers, but only the long fiber bound sCAR. The sCAR protein blocked the attachment of all virus serotypes that bound CAR. Moreover, CHO cells expressing human CAR, in contrast to untransformed CHO cells, all specifically bound the sCAR-binding serotypes. We conclude therefore that Ad serotypes from subgroups A, C, D, E, and F all use CAR as a cellular fiber receptor.     

Regulation of glutamate dehydrogenase level in excised pea roots by some exogenously supplied compounds with auxin activity

Besides IAA, other compounds with auxin activity (of both indole and non-indole nature) can also cause an increase in glutamate dehydrogenase level in excised pea roots. The effect of these substances is probably not mediated by ethylene, depends on proteosynthesis, is independent of the regulatory effect of sugars and need not be influenced by the antagonism between auxins and cytokinins.     

The effect of kinetin and auxin on the chloroplast structure and chlorophyll content in wheat coleoptiles

Kinetin and auxin when applied to excised segments of wheat coleoptiles bring about changes in chloroplast structure and chlorophyll content of parenchymatous cells. Auxin (IAA) alone at a concentration of 10^-5M stimulated the elongation (growth), but the chloroplast membrane system was less developed and the chlorophyll content was lowest in comparison with control and other variants. Kinetin (KIN) exhibited various effects depending on the concentration used. 10^-6M KIN somehow stimulated the elongation and enhanced the amount of grana coming to one chloroplast section, but the individual grana were relatively small and the chlorophyll content a little higher than in the control. On the other hand 10^-5M KIN which did not promote the elongation of wheat coleoptiles, had the maximum stimulatory effect on the chloroplast membrane system, especially on the occurrence of large grana, and the chlorophyll content was highest in comparison with the other variants. The occurrence of starch grains in chloroplasts was lower than in the other variants. The effect of a joint treatment of KIN and IAA did not exceed that of KIN (10^-5M) alone. Thus the development of chloroplasts and the accumulation of chlorophyll in wheat coleoptiles are stimulated by the concentration of KIN which does not promote the elongation of coleoptiles.