Correlation of enzymatic,metabolic, and behavioral deficits in thiamin deficiency and its reversal

To clarify the enzymatic mechanisms of brain damage inthiamin deficiency, glucose oxidation, acetylcholine synthesis, and the activities of the three major thiamin pyrophosphate (TPP) dependent brain enzymes were compared in untreated controls, in symptomatic pyrithiamin-induced thiamin-deficient rats, and in animals in which the symptoms had been reversed by treatment with thiamin. Although brain slices from symptomatic animals produced¹⁴CO₂ and¹⁴C-acetylcholine from [U-¹⁴C]glucose at rates similar to controls under resting conditions, their K⁺-induced-increase declined by 50 and 75%, respectively. In brain homogenates from these same animals, the activities of two TPP-dependent enzymes transketolase (EC 2.2.1.1) and 2-oxoglutarate dehydrogenase complex (EC 1.2.4.2, EC 2.3.1.61, EC 1.6.4.3) decreased 60–65% and 36%, respectively. The activity of the third TPP-dependent enzyme, pyruvate dehydrogenase complex (EC 1.2.4.1, EC 2.3.1.12, EC 1.6.4.3.) did not change nor did the activity of its activator pyruvate dehydrogenase phosphate phosphatase (EC 3.1.3.43). Although treatment with thiamin for seven days reversed the neurological symptoms and restored glucose oxidation, acetylcholine synthesis and 2-oxoglutarate dehydrogenase activity to normal, transketolase activity remained 30–32% lower than controls. The activities of other TPP-independent enzymes (hexokinase, phosphofructokinase, and glutamate dehydrogenase) were normal in both deficient and reversed animals.Thus, changes in the neurological signs during pyrithiamin-induced thiamin deficiency and in recovery paralleled the reversible damage to a mitochondrial enzyme and impairment of glucose oxidation and acetylcholine synthesis. A more sustained deficit in the pentose pathway enzyme, transketolase, may relate to the anatomical abnormalities that accompany thiamin deficiency.Dedicated to Henry McIlwain.     

Endogenous proteinases modulate the function of the β-adrenergic receptor-adenylate cyclase system

Photoaffinity labeling techniques have recently demonstrated that mammalian β₁- and β₂-adrenergic receptors reside on peptides of M_r 62 000–64 000. These receptor peptides are susceptible to endogenous metalloproteinases which produce peptides of M_r 30 000–55 000. Several proteinase inhibitors markedly attenuate this process, specifically EDTA and EGTA. In this study we investigated the functional significance of this proteolysis (and its inhibition) in the β₂-adrenergic receptor-adenylate cyclase system derived from rat lung membranes. Membrane preparations containing proteolytically derived fragments of the receptor of M_r 40000–55 000 are fully functional with respect to their ability to bind β-adrenergic antagonist radioligands such as [³H]dihydroalprenolol and β-adrenergic antagonist photoaffinity reagents such as p-azido-m-[^{125I]iodobenzylcarazolol}. They retain the ability to form a high-affinity, agonist-promoted, guanine nucleotide-sensitive complex thought to represent a ternary complex of agonist, receptor and guanine nucleotide regulatory protein. Nonetheless, after proteolysis, GTP is less able to revert this high-affinity receptor complex to one of lower affinity, and all aspects of adenylate cyclase stimulation are reduced. In addition, the functional integrity of the N protein in membranes prepared without proteinase inhibitors is reduced as assessed by reconstitution studies with the cyc[su− variant of S49 lymphoma cell membranes. These results suggest that endogenous proteolysis does not directly impair the ability of β-adrenergic receptors to either bind ligands or interact with the guanine nucleotide regulatory protein. However, they imply that endogenous proteolysis likely impairs the functionality of other components of the adenylate cyclase system, such as the nucleotide regulatory protein.     

Studies on sex-organ development. Oestrogenic effect on ornithine decarboxylase activity in the differentiating Müllerian ducts and other organs of the chick embryo.

The activity of ornithine decarboxylase in the differentiating left and right Müllerian ducts was assayed and compared with that in other embryonic organs, i.e. the liver and the brain throughout the stages of development. In general the enzyme activity was high in the early stages and decreased extensively in the late stages of development. Specifically, in the left and righ Müllerian ducts, the enzyme activity was high from day 8 to day 9 of incubation. In the right duct the enzyme activity started to decline on day 9 and then continuously decreased to an almost undetectable value on day 18 of incubation. In the left duct the enzyme activity also decreased slightly from day 9 to day 12; however, it increased from day 13 to day 15 and finally decreased to a constant value from day 18 until hatching. The alteration in enzyme activity in the Müllerian duct as assayed in vitro during development is not due to the effect of the size of the endogenous ornithine pool. When the enzyme activity was subjected to oestrogen stimulation, an increase of 5--10-fold for the left duct and of 5--3-fold for the right duct was observed during the course of development. No such stimulation was observed with the treatment of progesterone. Testosterone consistently caused a 25--30% inhibition of the enzyme activity in the Müllerian duct. Oestrogen slightly stimulated the enzyme activity in the developing liver but inhibited that of the brain. The concentration of the three polyamines measured in the Müllerian duct corresponds to the activity of the enzyme determined.     

Effect of carbon source on enzymes involved in glycerol metabolism inNeurospora crassa

Specific activities of eight enzymes involved in glycerol metabolism were determined in crude extracts of three strains ofNeurospora crassa after growth on six different carbon sources. One of the strains was wild type, which grew poorly on glycerol as sole carbon source; the other two were mutant strains which were efficient glycerol utilizers. A possible basis for this greater effeciency of glycerol utilization was catabolite repression of glyceraldehyde kinase by glycerol in wild type, and two-fold higher glycerate kinase activity in the mutant strains after growth on glycerol, thus apparently allowing two routes for glyceraldehyde to enter the glycolytic pathway in the mutant strains but only one in wild type. The preferential entry of glyceraldehyde to the glycolytic pathway through glycerate was suggested by the lack of glyceraldehyde kinase in all three strains after growth on one or more of the carbon sources and the generally higher levels of aldehyde dehydrogenase and of glycerate kinase than of glyceraldehyde kinase.     

Influence of 0.5 ppm nitrogen dioxide exposure of mice on macrophage congregation in the lungs

Summary The lungs of 12 mice, half of which were exposed to continuous 0.5 ppm nitrogen dioxide for 3 weeks, were explanted in culture, and the instances of macrophage congregation were quantitated according to numbers of target cells involved, categories of congregation from three to 11 or more, numbers of macrophages participating in each category for the total cultures, and the influence of delaying explantation for 24 and 96 hr. A total of 9042 macrophages and 2140 epithelial and spindle target cells were counted in the outgrowths from 306 explants. The incidence of macrophage congregation (or numbers of target cells) was greater for the cultures from the NO₂-exposed animals, both with respect to total incidences between groups (p→0), and the 0-hr (p<0.001) and 24-hr (p<0.01) culture subgroups. In addition, the values for T₃ to T₆ macrophage congregation were individually and consistently greater for the exposed animal group. Postmortem interval stress at 96 hr appeared to result in large colonies, but they were reduced greatly in number. Also the incidence of macrophage congregation fell by 28% as compared to 0-hr and 24-hr subgroups. Supported by Grants NHLI No. HL 17412 and EPA No. R. 800881.     

Rat hepatocyte primary cultures

Summary Primary cultures of rat hepatocytes survived well for up to 4 days in defined medium in the presence of dexamethasone but not in its absence. The loss of viability was accompanied by a loss of ultrastructural features characteristic of hepatocytes. The cultures began producing plasminogen activator and a neutral protease after 24 hr in culture. Dexamethasone inhibited the production of both of these substances. The deterioration of the cultures appeared not to be related to plasminogen activator, but prolongation of survival by a variety of protease inhibitors suggested that the neutral protease might contribute to deterioration. Dr. Goldblatt was supported by Grant No. SG-87 from the American Cancer Society as an American Cancer Society Scholar while on sabbatical leave from the Department of Pathology, University of Connecticut Health Center, Farmington, Connecticut. This study was supported by Contracts NO1-CP-55705 from the National Cancer Institute and 68-02-2483 from the Environmental Protection Agency.     

Studies of cyclic AMP action using mutant tissue culture cells

Summary S49 mouse lymphoma cell mutants, each with a specific defect in its ability to generate or respond to cyclic AMP, have been isolated. Analysis of the properties of these cells has begun to provide information on complex and significant biologic problems related to the cyclic AMP system. Presented in the Opening Symposium on Nutritional Factors and Differentiation at the 28th Annual Meeting of the Tissue Culture Association, New Orleans, Louisiana, June 6–9, 1977. The work was supported in part by National Science Foundation Grant BMS 75-06764 and National Institutes of Health Grants GM 16496 and GM 00001. P.C. is the recipient of National Institutes of Health Research Career Development Award GM 00308. P. A. I. is an Established Investigator of the American Heart Association.     

Prostaglandin-mediated hyperemia and renin-mediated hypertension during acute ureteral obstruction

Acute elevation of ureteral pressure to 100 mm Hg in anesthetized dogs (n=7) resulted in an increase (P<0.005) in systemic blood pressure from 151±7 to 163 ± 7 mm Hg, a transient (15 min) increase (P<0.05) in renal blood flow from 413 ± 27 to 465 ± 27 ml/min C and a rise (P<0.05) in plasma renin activity from 6.0 ± 1.6 to 10.3 ± 2.1 ng/ml/hr. Pretreatment with a competitive inhibitor of angiotensin II, i.e. sar¹gly⁸AII, abolished the hypertensive response to acute ureteral obstruction, and pretreatment with 2 mg/kg of either indomethacin (n=6) or meclofenamate (n=3), 15 min before obstruction, prevented the hyperemic response. These results suggest that acute ureteral obstruction leads to hypertension via activation of the renin-angiotensin system and hyperemia via a prostaglandin-initiated mechanism.     

Significant changes in 16 S RNA conformation accompanying assembly of the 30 S ribosome in vitro

Our previous studies have shown that 16 S RNA can assume two different conformational forms as detected by agarose gel electrophoresis, and that these two forms vary in their ability to bind individual 30 S ribosomal proteins specifically. In this paper we show that the faster electrophoretic form can be converted to the slower electrophoretic form by the binding of either protein S4, S8, S7 or S15. The slower form can then be transformed into a fast form by heat-activating the reconstitution intermediate (RI) particle, which has been constructed under reconstitution conditions at 0 °C, to RI¹. We demonstrate that the transformation of the 16 S RNA conformation by binding of protein S7 permits the subsequent binding of protein S9 following deproteination. We propose that many of the classical assembly-dependent relationships are due to induced changes in the 16 S RNA conformation.     

ISOLATION AND IDENTIFICATION OF γ-AMINOBUTYRYL-CYSTATHIONINE FROM HUMAN BRAIN AND CSF

Abstract— A new dipeptide, γ-aminobutyryl-cystathionine, has been identified in human brain and CSF. The compound was isolated from peptide concentrates which were prepared by removing free a-amino acids from deproteinized brain extracts on a copper Sephadex column. The isolated peptide was shown to be GABA-Cysta by standard chemical methods, and its identification was confirmed by mass spectrometry. GABA-Cysta is present in both biopsy and autopsy specimens of adult human brain, its content in some brain areas being as high as 0.090 μrnol/g wet weight. Its concentration in CSF is much lower. What physiologic role this unusual peptide plays in brain remains to be determined.