On the location of the tetrapyrrole macrocycle of chlorophyll a in phospholipid vesicles and in hexadecane

Summary The state of chlorophyll a in phosphatidylcholine vesicles was examined. The results indicate that the chlorophylls are present in monomeric form. A kinetic study of chlorophyll reactions with K₂S₂O₈ and piperidine showed that these substances react with the porphyrin rings of pigments located on both vesicle faces, most probably within the polar headgroup region.Acknowledgments. This work was supported by the Research Committee and the Biophysics Research Group, University of Québec at Trois-Rivières.     

Specificity in the hydrolysis of N-acyl-L-phenylalanine 4-nitroanilides by chymotrypsin

Summary The affinity of N-acyl-L-phenylalanine 4-nitroanilides for chymotrypsin is enhanced as the hydrophobicity of non-amino acid residues in the P₂-position of the substrates increases, whereas k_cat remains nearly constant. On the other hand, if alanine or leucine is in the P₂-position k_cat increases with decreasing K_M.     

Adaptive heart and breathing frequencies in 4 ecologically differentiating chromosomal species of mole rats in Israel

Summary Breathing (f_R) and heart (f_H) frequencies decreases as aridity increases in 4 chromosomal species of theSpalax ehrenbergi that inhabits humid to arid habitats in Israel in the order 2n=52, 58, 54, 60. Breathing frequencies were 50.0, 46.9, 45.9, and 43.4% of the expected values, and f_H were 37.6, 32.7, 27.8, and 25.8% for 2n=52, 58, 54, and 60, respectively. The decrease of f_R and f_H has a genetic basis and correlates with the metabolism of the mole rat.     

Developmental changes of aldehyde dehydrogenase isozymes in human livers: Mitochondrial ALDH2 isozyme is expressed in fetal livers

Summary Previous reports suggested that the major cytosolic aldehyde dehydrogenase (ALDH₁) was present in fetal and infant livers, but the major mitochondrial isozyme (ALDH₂) was absent or severely diminished. Re-examination by means of starch gel electrophoresis followed by enzyme activity staining, and by means of dot blot immuno-hybridization of liver samples with known genotypes of theALDH ₂ locus, indicated that bothALDH ₁ andALDH ₂ genes are expressed in fetal and infant livers. In addition, ALDH₄ isozyme was also observed. The results imply that a fetus with the usual homozygousALDH ₂ ¹ /ALDH ₂ ¹ genotype, but not one with the atypicalALDH ₂ ¹ /ALDH ₂ ² orALDH ₂ ² /ALDH ₂ ² , is capable of detoxifying acetaldehyde transferred from the mother.     

Agglutination of leukemic cells and daunomycin entrapped erythrocytes with lectin in vitro and in vivo

Summary Wheat germ agglutinin (WGA) agglutinated the L₁₂₁₀ leukemic cells and daunomycin entrapped erythrocytes in vitro. Comparing with control preparations, the greatest increase in survival was obtained in vivo when the daunomycin entrapped erythrocytes and WGA were given to BDF₁ mice bearing L₁₂₁₀ cells.     

Influence of ethanol on thyroxine accumulation in the hypothalamus,pituitary gland and cerebrospinal fluid in the newborn rabbit

Summary The effect of ethanol on thyroxine (T₄) accumulation in the hypothalamus (H), pituitary gland (P) and cerebrospinal fluid (CSF) has been investigated in 1–15-day-old rabbits. It has been found that H or CSF serum ratios decreased with age by about 2 in the course of 13 postnatal days. Stable T₄ resulted in an increase of¹²⁵I-T₄ in H, P and CSF. Ethanol per se caused an increase in transfer and accumulation of radiothyroxine or made the changes after loading animals with carrier T₄ more pronounced.     

A126 in the active site and TI167/168 in the TI loop are essential determinants of the substrate specificity of PTEN

PTEN prevents tumor genesis by antagonizing the PI3 kinase/Akt pathway through D3 site phosphatase activity toward PI(3,4)P₂ and PI(3,4,5)P₃. The structural determinants of this important specificity remain unknown. Interestingly, PTEN shares remarkable homology to voltage-sensitive phosphatases (VSPs) that dephosphorylate D5 and D3 sites of PI(4,5)P₂, PI(3,4)P₂, and PI(3,4,5)P₃. Since the catalytic center of PTEN and VSPs differ markedly only in TI/gating loop and active site motif, we wondered whether these differences explained the variation of their substrate specificity. Therefore, we introduced mutations into PTEN to mimic corresponding sequences of VSPs and studied phosphatase activity in living cells utilizing engineered, voltage switchable PTEN_CiV, a Ci-VSP/PTEN chimera that retains D3 site activity of the native enzyme. Substrate specificity of this enzyme was analyzed with whole-cell patch clamp in combination with total internal reflection fluorescence microscopy and genetically encoded phosphoinositide sensors. In PTEN_CiV, mutating TI167/168 in the TI loop into the corresponding ET pair of VSPs induced VSP-like D5 phosphatase activity toward PI(3,4,5)P₃, but not toward PI(4,5)P₂. Combining TI/ET mutations with an A126G exchange in the active site removed major sequence variations between PTEN and VSPs and resulted in D5 activity toward PI(4,5)P₂ and PI(3,4,5)P₃ of PTEN_CiV. This PTEN mutant thus fully reproduced the substrate specificity of native VSPs. Importantly, the same combination of mutations also induced D5 activity toward PI(3,4,5)P₃ in native PTEN demonstrating that the same residues determine the substrate specificity of the tumor suppressor in living cells. Reciprocal mutations in VSPs did not alter their substrate specificity, but reduced phosphatase activity. In summary, A126 in the active site and TI167/168 in the TI loop are essential determinants of PTEN’s substrate specificity, whereas additional features might contribute to the enzymatic activity of VSPs.     

Induction of neovascularization in vivo by glycerol

Glycerol, injected into a site between the femoral vessels of the rat, induced neovascularization, both from the preexisting microcirculation and from the side of the femoral vein facing the artery-vein interstitium where the glycerol was administered. The use of glycerol together with a known angiogenic substance (PGE₂) did not modify the neocapillary density (NCD) obtained with glycerol alone. In contrast, the lower level of NCD achieved with an acylglycerol (triacetylglycerol) was increased when the latter was associated with PGE₂. Values reached were similar to, but never higher than, those for glycerol alone, or combined with PGE₂. The results suggest that glycerol and some substances containing glycerol, amongst which 1-butyrylglycerol has been previously considered¹, may stimulate angiogenesis by a direct or indirect mechanism of action.     

Sperm enzyme activity analysis in individual sperm for detection of heritable mutations in mammals

Summary Male mice were injected i.p. with 2.5 mg/kg mitomycin C, 100 mg/kg ethyl nitrosourea or saline and mated with untreated virgin females five weeks later. Sperm from 64 of the F₁ male progeny were analyzed histochemically for acrosin, succinic dehydrogenase and alpha-glycerophosphate dehydrogenase activity. The frequency of F₁ males with sub-normal sperm enzyme activity was significantly higher among progeny from treated males than in controls. These results show that analysis of sperm enzyme activity in F₁ males is a practical method for detection of transmitted mutations induced in a treated parent.We gratefully acknowledge USPHS, NIEHS grant 1 RO1 ES02607-02 and technical assistance by G. M. Oldford.     

Efficacy of tumor cell vaccine after incorporating monophosphoryl lipid A (MPL) in tumor cell membranes containing tumor-associated ganglioside

Murine B16 melanoma expresses the ganglioside. GM₃. GM₃ shed from tumor cells is immunosuppressive and promotes tumor growth¹. Reduction or elimination of the shed GM₃ could be therapeutic, and the anti-GM₃ antibodies may reduce and clear the shed ganglioside. To test this hypothesis, mice were challenged with tumor cells, with or without inducing anti-GM₃ antibody response. Since gangliosides are poor immunogens and T-cell independent antigens, an adjuvant (monophosphoryl lipid A (MPL), a non-toxic lipid A ofSalmonella), directed against B-cells, was employed. MPL was incorporated onto liposomes and into the surface membrane of B16 mouse melanoma cells; both are rich in GM₃. C57BL/6J mice immunized with MPL-liposomes or MPL-B16 cells responded with elevated levels of anti-GM₃ IgM. Non-immunized mice or mice immunized with B16 cells alone or ganglioside GM₃ alone (without MPL) elicited poor anti-GM₃ IgM response, confirming the GM₃'s immunologic crypticity and MPL's immunopotentiating effect. MPL's immunopotentiating effect was improved by coupling it to melanoma cell membranes C57BL/6J mice were immunized with irradiated B16 alone or MPL alone or MPL-conjugated irradiated B16. After three weekly immunizations, each mouse received a challenge dose of viable syngeneic B16. Neither MPL alone nor B16 alone had a significant effect on tumor growth or host survival; however, administration of MPL-conjugated B16 cells significantly prevented tumor growth and prolonged survival. Our results indicate that MPL-incorporated B16 cells augment the anti-GM₃ IgM response, which may reverse GM₃-induced immunosuppression by eliminating tumor-derived GM₃, and restore immunocompetence.     

Augmentation of natural antiganglioside IgM antibodies in lower motor neuron disease (LMND) and role of CD5+ B cells

IgM antibodies directed against neuronal gangliosides GM₁ , GM₂ , GD_1a , GD_1b and GT_1b occur in normal individuals and their level significantly decreases with age. Patients with lower motor neuron disease (LMND) produce high levels of these autoantibodies. AntiGM₁ IgM is selectively augmented. In these patients, the CD5+ (B1) and CD5− (B2) subsets of B cells are not distinct entities but range from those without detectable CD5 marker to those with high CD5+ expression. B1 B cells were sorted to homogeneity, but B2 B cell cannot be isolated to homogeneity because of the presence of B1 cells with low CD5 expression. In short term cultures both the subsets produced IgM antibodies, but the antibodies reacted better with desialylated GM₁ than with GM₁ . Cycloheximide (Cx) (0.35 mM) largely blocked IgM synthesis of the B1 B cells but inhibition of the B2 B cells was incomplete, possibly due to shedding of cytophilic antibodies as well as to the presence of B1 phenotype with loss of CD5 expression. CD5+ B cells may be involved in the production of antiglycolipid IgM. Received 9 June 1997; received after revision 21 July 1997; accepted 28 July 1997     

Role of nitric oxide in the functional response to ischemia-reperfusion of heart mitochondria from hyperthyroid rats

We investigated the role of nitric oxide (NO) in the mitochondrial derangement associated with the functional response to ischemia-reperfusion of hyperthyroid rat hearts. Mitochondria were isolated at 3000 g from hearts subjected to ischemia-reperfusion, with or without N-nitro-L-arginine (L-NNA, an NO synthase inhibitor). During reperfusion, hyperthyroid hearts displayed tachycardia and low functional recovery. Their mitochondria exhibited O₂ consumption similar to euthyroid controls, while H₂O₂ production, hydroperoxide, protein-bound carbonyl and nitrotyrosine levels, and susceptibility to swelling were higher. L-NNA blocked the reperfusion tachycardic response and increased inotropic recovery in hyperthyroid hearts. L-NNA decreased mitochondrial H₂O₂ production and oxidative damage, and increased respiration and tolerance to swelling. Such effects were higher in hyperthyroid preparations. These results confirm the role of mitochondria in ischemia-reperfusion damage, and strongly suggest that NO overproduction is involved in the high mitochondrial dysfunction and the low recovery of hyperthyroid hearts from ischemia-reperfusion. L-NNA also decreased protein content and cytochrome oxidase activity of a mitochondrial fraction isolated at 8000 g. This and previous results suggest that the above fraction contains, together with light mitochondria, damaged mitochondria coming from the heaviest fraction, which has the highest cytochrome oxidase activity and capacity to produce H₂O₂. Therefore, we propose that the high mitochondrial susceptibility to swelling, favoring mitochondrial population purification from H₂O₂-overproducing mitochondria, limits hyperthyroid heart oxidative stress.Received 24 March 2004; received after revision 9 June 2004; accepted 5 July 2004     

Induction of nuclear styrene monooxygenase and epoxide hydrolase in rat liver

Summary The apparent K_m and V_max of styrene monooxygenase and styrene epoxide hydrolase were determined in intact nuclear preparations from male rat liver after in vivo treatment with phenobarbital and -naphthoflavone, which are known to induce microsomal cytochrome P-450 and cytochrome P-448 respectively. Treatment with phenobarbital does not alter the apparent K_m, but greatly increases the V_max of both nuclear styrene monooxygenase and styrene epoxide hydrolase. Almost the same pattern is observed for styrene monooxygenase after treatment with -naphthoflavone, whereas the same treatment slightly increases both the V_max and K_m value of styrene epoxide hydrolase.This work was supported by the CNR (National Research Council) Contract No. 78.02864.96 within the special program Control of Cancer Growth.     

Specific immunosuppression of IgE response to hapten DNP by DNP linked to monoclonal IgG1 in rats

Summary The induction of the anti-DNP IgE in rat was suppressed by pretreatment of rats with the tolerogen synthesized by coupling DNP to rat IgG, i.e.; DNP_7–10-IgG. It was found that DNP₁₀-IgG₁ was an effective tolerogen, whereas other DNP conjugates, i.e. DNP₉-IgM, DNP₉-IgA, DNP₁₀-IgE, DNP₁₀-IgG_2c and DNP₁₀-IgG_2a were ineffective.This work was partly supported by a research grant from the Medical Council of Canada to W.Y.L.     

Functional and biochemical characteristics of mitochondrial fractions from rat liver in cold-induced oxidative stress

We determined characteristics of rat liver mitochondrial fractions, resolved at 1000 (M₁), 3000 (M₃), and 10,000 g (M₁₀) after 2 and 10 days cold exposure. In all groups, the M₁ fraction exhibited the highest oxidative capacity, oxidative damage, H₂O₂ production rate, and susceptibility to stress conditions, and the lowest antioxidant levels. Cold exposure increased cytochrome oxidase activity in all fractions and succinate-supported O₂ consumption in the M₁ and M₁₀ fractions during state 3 and state 4 respiration, respectively. With succinate, the H₂O₂ release rate increased in all fractions during state 4 and state 3 respiration, whereas with pyruvate/malate, it increased only during state 4 respiration. Increases in tissue mitochondrial proteins caused a faster H₂O₂ flow from the mitochondrial to cytosolic compartment, which was limited by the reduction in the M₁ fraction. Despite increased liposoluble antioxidant levels, cold also caused enhanced oxidative damage and susceptibility to oxidative challenge and Ca²⁺-induced swelling in all fractions. These changes leading to elimination of H₂O₂-overproducing mitochondria avoided excessive tissue damage. We propose that triiodothyronine, whose levels increase in the cold environment, brings about the biochemical changes producing oxidative damage and those limiting its extent.Received 16 July 2004; received after revision 27 September 2004; accepted 18 October 2004     

Etamsylate as inhibitor of prostaglandin biosynthesis in pregnant human myometrium in vitro

Summary The effects of etamsylate on prostaglandin (PG) biosynthesis in microsomes of pregnant human myometrium in vitro have been determined, and compared with those of indomethacin. Both drugs inhibited PG biosynthesis, indomethacin being the more potent inhibitor of the two. Etamsylate inhibited synthesis of 6-oxo-PGF₁, PGF₂, PGE₂, and thromboxane B₂; increasing the concentration of etamsylate increased the inhibition of synthesis. It is suggested that etamsylate has no anti-cyclo-oxygenase activity, but acts by inhibiting the activity of prostacyclin synthetase, endoperoxide reductase, endoperoxide isomerase, and thromboxane synthetase.     

Release of 3,5,3′-triiodothyronine,thyroxine and thyroglobulin from TSH-stimulated mouse thyroids in the perifusion system

Summary We established a perifusion system using mouse thyroid glands. In this system, TSH increased the release of T₃ and T₄ significantly, and the response of thyroglobulin to TSH was delayed in comparison with that of T₃ and T₄.     

Changes in the T4/T3 molar ratio following thyrotrophin releasing hormone injection in cattle

Summary The injection of thyrotropin releasing hormone into cattle resulted in a rapid decrease in the T₄/T₃ molar ratio. 2 breeds of cattle, Shorthorn and Africander Cross were studied. The decrease in the T₄/T₃ molar ratio was significantly greater in the Shorthorn breed. It is concluded that acute stimulation of the thyroid gland with TRH results in enhanced release of both T₃ and T₄ and that T₃ is discharged more rapidly than T₄.     

Alterations in the fraction of oxygen in inspired gas affect rates of renal prostaglandin E2 synthesis in anesthetized dogs

Summary Reductions of oxygen in inspired gas from 20% to 15%, in anesthetized dogs, reduced arterial PO₂ and increased the renal efflux of PGE₂ but not PGF_2a . Renal blood flow, blood pressure, plasma renin activity as well as arterial pH and PCO₂ were unaffected. PGs may mediate the renal hemodynamic or excretory consequences of alterations in PO₂. In addition, minor variations in PO₂ might account, in part, for the variable renal venous PGE₂ concentrations reported under basal conditions.Authentic prostaglandins were supplied by Dr John E. Pike of the Upjohn Company. This work was supported by grants from the Veterans Administration, the Southern Medical Association and U.S. Public Health Service. Dr. Brash is an American Heart Association, Missouri Affiliate Fellow.     

Down-regulation of sodium current in chronic heart failure: effect of long-term therapy with carvedilol

Evidence has accumulated recently about the importance of alterations in Na⁺ channel function and slow myocardial conduction for arrhythmias in the infarcted and failing heart. The present study tested a hypothesis that Na⁺ current (I_Na/C) density decreases in chronic heart failure (HF) and that Na⁺ channel (NaCh) functional density can be restored by long-term therapy with carvedilol, a mixed α- and β-adrenergic blocker. Studies were performed using a canine model of chronic HF produced in dogs by sequential intracoronary embolizations with microspheres. HF developed approximately 3 months after the last embolization (left ventricle, LV, ejection fraction = 28 ± 1 %). Ventricular cardiomyocytes (VCs) were isolated enzymatically from LV mid-myocardium, and I_Na was measured by whole-cell patch-clamp. The maximum I_NA/C was decreased in failing (n = 19) compared to normal (n = 12) hearts (33.1 ± 1.6 vs 48.5 ± 5.1 pA/pF, mean ± SE, p < 0.001). The steady-state inactivation and activation of I_Na remained unchanged in failing compared to normal hearts. Long-term treatment with carvedilol (1 mg/kg, twice daily for 3 months) normalized I_Na/C in dogs with HF. I_Na/C in HF dogs (n = 6) treated with carvedilol was higher compared to that of non-treated HF dogs (n = 6) (49.4 ± 0.9 vs 29 ± 4.8 pA/pF, p < 0.007). In vitro culture of VCs of failing hearts for 24 h did not restore I_Na/C. However, I_Na/C was partially restored when VCs were incubated for 24 h with BAPTA-AM, an intracellular Ca²⁺ buffer. Thus, we conclude that experimental chronic HF in dogs results in down-regulation of the functional density of NaCh that can be restored by long-term therapy with carvedilol. The mechanism of NaCh down-regulation in HF may be linked to poor Ca²⁺ handling in this stage of disease. Received 4 June 2002; received after revision 1 July 2002; accepted 17 July 2002 RID="*" ID="*"Corresponding author.