Isolation and characterization of rat cDNA clones for two distinct thyroid hormone receptors

Two distinct v-erbA-related cDNA clones representing the products of different genes were isolated from a rat liver cDNA library. The first, rc-erbA-alpha, was 82% identical to v-erbA and encoded a polypeptide with a calculated molecular mass of 45,000 daltons. This cDNA clone arises from the same gene product as a v-erbA-related cDNA isolated from rat brain by Thompson et al. (Thompson, C. C., Weinberger, C., Lebo, R., and Evans, R. (1987) Science 237, 1610-1614). The second cDNA clone, rc-erbA-beta, was 76% identical to v-erbA and encoded a polypeptide with a calculated molecular mass of 52,000 daltons. Both rc-erbA-alpha and rc-erbA-beta translational products bound 3,5,3'-triiodo-L-thyronine with affinities equal to each other (Kd approximately equal to 0.4 nM) and comparable to the nuclear thyroid hormone receptor extracted from rat liver. The relative affinities of a series of thyroid hormone analogs for both translational products were also identical. In various tissues and cell lines, the relative levels of rc-erbA-beta RNA, but not rc-erbA-alpha RNA, correlated with measurements of nuclear 3,5,3'-triiodo-L-thyronine binding sites. Based on this correlation, we suggest that rc-erbA-beta may encode the "classical" nuclear thyroid hormone receptor, whereas rc-erbA-alpha may encode an isoreceptor species with differing functional properties.     

Normal mouse strains differ in the site of initiation of closure of the cranial neural tube

The scanning electron microscopic study of day 9 embryos reported here documents differences among normal mouse strains in morphology of cranial neural tube closure. The site of initiation of contact and fusion of the cranial neural folds, previously defined as Closure 2 (Macdonald et al., '89), is located in the region of the junction between the forebrain (prosencephalon) and midbrain (mesencephalon) in three normal strains: LM/Bc, AEJ/RkBc, and ICR/Bc. However in a fourth normal strain, SWV/Bc, Closure 2 is initiated much further rostral, in the prosencephalic region. In addition, the anterior neuropore, rostral to Closure 2, closes late in ICR/Bc embryos, relative to the posterior progress of development of the Closure 2 seam. Initiation of closure from the most rostral end of the neural tube (Closure 3) appears to be relatively delayed in ICR/Bc embryos. We hypothesize that the observed genetic polymorphism in location of the first site of fusion between the cranial neural folds in normal mouse embryos may be one basis for differences among normal strains in liability to exencephaly induced by teratogens.     

Stimulation of insulin release from pancreatic islets by quinones

Coenzyme Q (CoQ₀) and other quinones were shown to be potent insulin secretagogues in the isolated pancreatic islet. The order of potency was CoQ₀benzoquinonehydroquinonemenadione. CoQ₆ and CoQ₁₀ (ubiquinone), duroquinone and durohydroquinone did not stimulate insulin release. CoQ₀'s insulinotropism was enhanced in calcium-free medium and CoQ₀ appeared to stimulate only the second phase of insulin release. CoQ₀ inhibited inositol mono-, bis- and trisphosphate formation. Inhibitors of mitochondrial respiration (rotenone, antimycin A, FCCP and cyanide) and the calcium channel blocker verapamil, did not inhibit CoQ₀-induced insulin release. Dicumarol, an inhibitor of quinone reductase, did not inhibit CoQ₀-induced insulin release, but it did inhibit glucose-induced insulin release suggesting that the enzyme and quinones play a role in glucose-induced insulin release. Quinones may stimulate insulin release by mimicking physiologically-occuring quinones, such as CoQ₁₀, by acting on the plasma membrane or in the cytosol. Exogenous quinones may bypass the quinone reductase reaction, as well as many reactions important for exocytosis.     

Randomised controlled trial of effects of coordinating care for terminally ill cancer patients.

OBJECTIVES--To measure effects on terminally ill cancer patients and their families of coordinating the services available within the NHS and from local authorities and the voluntary sector. DESIGN--Randomised controlled trial. SETTING--Inner London health district. PATIENTS--Cancer patients were routinely notified from 1987 to 1990. 554 patients expected to survive less than one year entered the trial and were randomly allocated to a coordination or a control group. INTERVENTION--All patients received routinely available services. Coordination group patients received the assistance of two nurse coordinators, whose role was to ensure that patients received appropriate and well coordinated services, tailored to their individual needs and circumstances. MAIN OUTCOME MEASURES--Patients and carers were interviewed at home on entry to the trial and at intervals until death. Interviews after bereavement were also conducted. Outcome measures included the presence and severity of physical symptoms, psychiatric morbidity, use of and satisfaction with services, and carers'' problems. Results from the baseline interview, the interview closest to death, and the interview after bereavement were analysed. RESULTS--Few differences between groups were significant. Coordination group patients were less likely to suffer from vomiting, were more likely to report effective treatment for it, and less likely to be concerned about having an itchy skin. Their carers were more likely to report that in the last week of life the patient had had a cough and had had effective treatment for constipation, and they were less likely to rate the patient''s difficulty swallowing as severe or to report effective treatment for anxiety. Coordination group patients were more likely to have seen a chiropodist and their carers were more likely to contact a specialist nurse in a night time emergency. These carers were less likely to feel angry about the death of the patient. CONCLUSIONS--This coordinating service made little difference to patient or family outcomes, perhaps because the service did not have a budget with which it could obtain services or because the professional skills of the nurse-coordinators may have conflicted with the requirements of the coordinating role.     

Microcirculatory pathways in normal human spleen, demonstrated by scanning electron microscopy of corrosion casts

Confusion regarding microcirculatory pathways in normal human spleen has arisen due to extrapolation from pathological material and from other mammalian spleens, not to mention difficulties in tracing intricate three-dimensional routes from the study of thin sections or cut surfaces of tissue. We examined microcirculatory pathways in normal human spleens freshly obtained from organ transplant donors. A modified corrosion casting procedure was used to obtain an open view of vessels and their connections. Our results demonstrate: 1) "arteriolar-capillary bundles" within lymphatic nodules and extensive branching of arterioles in the marginal zone (MZ); 2) the marginal sinus around lymphatic nodules; 3) the peri-marginal cavernous sinus (PMCS) outside the MZ or immediately adjacent to the nodule itself; the PMCS receives flow via ellipsoid sheaths and MZ, or directly from arterial capillaries, and drains into venous sinuses; 4) fast pathways for flow into venous sinuses via ellipsoid sheaths; 5) arterial capillary terminations in the reticular meshwork of the red pulp or MZ ("open" circulation); direct connections to venous sinuses also occur ("closed" circulation), although rarely; and 6) numerous open-ended venous sinuses in the MZ, allowing a large proportion of the splenic inflow to bypass the red cell filtration sites in the reticular meshwork and at venous sinus walls.     

A membrane-bound diacylglycerol kinase that selectively phosphorylates arachidonoyl-diacylglycerol. Distinction from cytosolic diacylglycerol kinase and comparison with the membrane-bound enzyme from Escherichia coli

The membrane-bound diacylglycerol kinase from Swiss 3T3 cells (M-DG kinase) was characterized with a mixed micellar assay system, and compared with the cytosolic diacylglycerol kinase from 3T3 cells and with the membrane-bound diacylglycerol kinase from Escherichia coli. M-DG kinase selectively phosphorylated arachidonoyl-diacylglycerols, at a rate 2- to 8-fold higher than that for other naturally occurring long-chain diacylglycerols. In contrast, the cytosolic 3T3 enzyme exhibited little or no selectivity among long-chain diacylglycerols but had higher activity with more soluble substrates such as 1,2-didecanoylglycerol. Comparison of the properties of M-DG kinase with those of the bacterial membrane-bound enzyme revealed that selectivity for arachidonoyl-diacylglycerol was unique to the mammalian enzyme. All three kinases were activated by phosphatidylserine, but activation did not alter the arachidonoyl selectivity of M-DG kinase. Phosphatidylserine activated M-DG kinase by increasing Vm and decreasing the apparent Km for diacylglycerol. High concentrations of diacylglycerol reduced the Ka for phosphatidylserine, but did not abolish the phosphatidylserine requirement for maximum activity. Examination of the thermal lability of M-DG kinase revealed that this enzyme was rapidly and selectively inactivated by preincubation with its preferred substrate. This novel effect may have obscured previous attempts to discern substrate selectivity. Taken together, the results provide evidence that M-DG kinase is an arachidonoyl-diacylglycerol kinase that may participate in the formation of arachidonoyl-enriched species of phosphatidylinositol.     

Structural characterization of the rat carboxypeptidase A1 and B genes. Comparative analysis of the rat carboxypeptidase gene family

Nucleotide sequencing of a rat carboxypeptidase B (CPB) cDNA and direct sequencing of the CPB mRNA via primer extension on pancreatic polyadenylated RNA has yielded the complete amino acid sequence of rat CPB. The rat enzyme is synthesized as a precursor species containing a large amino-terminal fragment (108 amino acids) that contributes a putative signal sequence and an activation peptide. The mature form of rat CPB is homologous to bovine CPB (77% identity); the amino acids in bovine CPB which have been previously implicated in catalysis or ligand binding are invariant in the rat orthologue. The rat CPB cDNA was used as a probe for the isolation of the rat CPB gene. Detailed characterization of three overlapping rat genomic clones demonstrated that the coding region for the rat CPB precursor is sequestered in 11 exons which are dispersed throughout 34 kilobase pairs of genomic DNA. The nucleotide sequence of a large part of the gene has been determined including that of the exons, the exon/intron boundaries, and the 5' flanking region. We also report the partial nucleotide sequence of the rat CPA1 gene. Comparative analysis of the structural organization of the rat CPB, rat CPA1, and rat CPA2 genes (Gardell, S. J., Craik, C. S., Clauser, E., Goldsmith, E. J., Stewart, C.-B., Graf, M., and Rutter, W. J. (1988) J. Biol. Chem. 263, 17828-17836) reveals that, with one exception, the number, position, and sequence composition of the exons in these three carboxypeptidase genes are conserved in spite of considerable divergence with respect to the lengths of their corresponding intervening sequences. Conserved sequences in the 5' flanking regions of the rat CPA1, CPA2, CPB, and other pancreas-specific genes have been identified.     

Regulation of malate dehydrogenase activity by glutamate, citrate, alpha-ketoglutarate, and multienzyme interaction

Binding experiments indicate that mitochondrial aspartate aminotransferase can associate with the alpha-ketoglutarate dehydrogenase complex and that mitochondrial malate dehydrogenase can associate with this binary complex to form a ternary complex. Formation of this ternary complex enables low levels of the alpha-ketoglutarate dehydrogenase complex, in the presence of the aminotransferase, to reverse inhibition of malate oxidation by glutamate. Thus, glutamate can react with the aminotransferase in this complex without glutamate inhibiting production of oxalacetate by the malate dehydrogenase in the complex. The conversion of glutamate to alpha-ketoglutarate could also be facilitated because in the trienzyme complex, oxalacetate might be directly transferred from malate dehydrogenase to the aminotransferase. In addition, association of malate dehydrogenase with these other two enzymes enhances malate dehydrogenase activity due to a marked decrease in the Km of malate. The potential ability of the aminotransferase to transfer directly alpha-ketoglutarate to the alpha-ketoglutarate dehydrogenase complex in this multienzyme system plus the ability of succinyl-CoA, a product of this transfer, to inhibit citrate synthase could play a role in preventing alpha-ketoglutarate and citrate from accumulating in high levels. This would maintain the catalytic activity of the multienzyme system because alpha-ketoglutarate and citrate allosterically inhibit malate dehydrogenase and dissociate this enzyme from the multienzyme system. In addition, citrate also competitively inhibits fumarase. Consequently, when the levels of alpha-ketoglutarate and citrate are high and the multienzyme system is not required to convert glutamate to alpha-ketoglutarate, it is inactive. However, control by citrate would be expected to be absent in rapidly dividing tumors which characteristically have low mitochondrial levels of citrate.     

The effect of three hypolipidemic drugs on catalase activity and peroxisomal and mitochondrial palmitate oxidation in rat cardiac and skeletal muscle

Catalase activity and peroxisomal and mitochondrial palmitate oxidation have been investigated in cardiac and skeletal muscle from rats fed clofibrate, ciprofibrate or nafenopin in an unrefined diet for different periods of time. Nafenopin was also added to either a high carbohydrate (70% of kilocalories from glucose) or high fat (70% of kilocalories from lard) diet and fed to rats for either 1 or 3 weeks. Catalase activity was elevated in all muscles from rats fed the hypolipidemic drugs. The response of catalase activity in muscle to clofibrate was dose-dependent. The response time of catalase activity was different in individual muscles. Peroxisomal palmitate oxidation was elevated in the heart and soleus muscle from rats fed nafenopin in either the high-carbohydrate or the high-fat diet. There was no change in peroxisomal palmitate oxidation in psoas or extensor digitorum longus muscle from rats fed the drugs. Mitochondrial palmitate oxidation was only slightly increased by nafenopin in the heart and soleus muscles after 3 weeks of nafenopin feeding. The results suggest that the cardiac muscle, like the liver, responds to hypolipidemic drug treatment with an increase in peroxisomal fat oxidation. The skeletal muscle response is less specific and that tissue may not contribute to the hypolipidemic effect of the drugs. The findings also suggest that these drugs do not induce peroxisome proliferation in skeletal muscle.