Association of lactate, intracellular pH, and intracellular calcium during capacitation and acrosome reaction: contribution of hamster sperm dihydrolipoamide dehydrogenase, the E3 subunit of pyruvate dehydrogenase complex.

The role of dihydrolipoamide dehydrogenase (DLD), the E3 subunit of the pyruvate dehydrogenase complex (PDHc) in hamster sperm capacitation and acrosome reaction has been implicated previously. In this study, attempt has been made to understand DLD/PDHc involvement from the perspective of pyruvate/lactate metabolism. Inhibition of DLD was achieved by the use of a specific inhibitor, 5-methoxyindole-2-carboxylic acid. It was seen that 5-methoxyindole-2-carboxylic acid-treated spermatozoa with inhibited DLD (and PDHc) activity had lactate accumulation, which caused an initial lowering of the intracellular pH and calcium and an eventual block in capacitation and acrosome reaction. Collectively, the data reveal a significant contribution of the metabolic enzymes DLD and PDHc to lactate regulation in hamster spermatozoa during capacitation and acrosome reaction. Additionally, the importance of lactate regulation in the maintenance of sperm intracellular pH and calcium, two important physiologic factors essential for sperm capacitation and acrosome reaction, has also been established.

Distinct changes in intranuclear lamin A/C organization during myoblast differentiation.

Intranuclear lamin foci or speckles have been observed in various cell types. In order to explore the possibility of changes in internal lamin organization during muscle differentiation, we have examined the appearance of A-type lamin speckles that associate with RNA splicing factor speckles in C2C12 myoblasts and myotubes. Lamin speckles were observed in dividing myoblasts but disappeared early during the course of differentiation in postmitotic myocytes, and were absent in myotubes and muscle fibers. However, no changes were seen in the typical peripheral organization of lamins A/C or B1 or in RNA splicing factor speckles. Lamin speckles were also absent in quiescent myoblasts but reappeared as cells were reactivated to enter the cell cycle. These changes were not observed in other quiescent cell types. Immunoblot analysis indicated that the abundance and migration of lamins A and C was not altered in differentiated myoblasts. When myotube or quiescent myoblast nuclei were extracted with nucleases and detergent, a uniformly stained internal lamina was revealed, indicating that lamins A/C were antigenically masked in these cells, probably owing to structural reorganization of the lamina during differentiation or quiescence. Our results suggest that muscle cell differentiation is accompanied by regulated rearrangements in the organization of the A-type lamins.

Early localization of NPA58, a rat nuclear pore-associated protein, to the reforming nuclear envelope during mitosis.

We have studied the mitotic reassembly of the nuclear envelope, using antibodies to nuclear marker proteins and NPA58 in F-111 rat fibroblast cells. In earlier studies we have proposed that NPA58, a 58 kDa rat nuclear protein, is involved in nuclear protein import. In this report, NPA58 is shown to be localized on the cytoplasmic face of the envelope in interphase cells, in close association with nuclear pores. In mitotic cells NPA58 is dispersed in the cytoplasm till anaphase. The targeting of NPA58 to the reforming nuclear envelope in early telophase coincides with the recruitment of a well-characterized class of nuclear pore proteins recognized by the antibody mAb 414, and occurs prior to the incorporation of lamin B1 into the envelope. Significant protein import activity is detectable only after localization of NPA58 in the newly-formed envelope. The early targeting of NPA58 is consistent with its proposed role in nuclear transport.

Colocalization of intranuclear lamin foci with RNA splicing factors.

The lamins form a fibrous network underlying the inner nuclear membrane termed the nuclear lamina. In order to gain insights into the role of lamins in nuclear organization, we have characterized a monoclonal antibody (LA-2H10) raised against recombinant rat lamin A that labels nuclei in a speckled pattern in all cells of unsynchronized populations of HeLa and rat F-111 fibroblast cells, unlike the typical nuclear periphery staining by another monoclonal antibody to lamin A, LA-2B3. In immunolocalization studies the lamin A speckles or foci were found to colocalize with the RNA splicing factors SC-35 and U5-116 kD, but not with p80 coilin found in coiled bodies. Lamin B1 was also associated with these foci. These foci dispersed when cells entered mitosis and reformed during anaphase. The differential reactivity of LA-2H10 and LA-2B3 was retained after nuclei were extracted with detergents, nucleases and salt to disrupt interactions of lamins with chromatin and other nuclear proteins. Using deletion fragments of recombinant lamin A, the epitope recognized by LA-2H10 was located between amino acids 171 and 246. Our findings are consistent with a structural role for lamins in supporting nuclear compartments containing proteins involved in RNA splicing.

Curcumin mediated apoptosis in AK-5 tumor cells involves the production of reactive oxygen intermediates.

Curcumin, the active ingredient of the rhizome of Curcuma longa has anti-inflammatory, antioxidant and antiproliferative activities. Although its precise mode of action remains elusive, studies have shown that chemopreventive action of curcumin might be due to its ability to induce apoptosis in cancer cells. Curcumin was shown to be responsible for the inhibition of AK-5 tumor (a rat histiocytoma) growth by inducing apoptosis in AK-5 tumor cells via caspase activation. This study was designed to investigate the mechanism leading to the induction of apoptosis in AK-5 tumor cells. Curcumin treatment resulted in the hyperproduction of reactive oxygen species (ROS), loss of mitochondrial membrane potential (delta psi(m)) and cytochrome c release to the cytosol, with the concomitant exposure of phosphatidylserine (PS) residues on the cell surface. This study suggests redox signalling and caspase activation as the mechanisms responsible for the induction of curcumin mediated apoptosis in AK-5 tumor cells.

"Resultant bond moment" as a newly developed electronic parameter in the design of antibacterial, antiprotozoal nitroimidazole derivatives.

Nineteen nitroimidazole derivatives have been reported to be effective antibacterial against one strain each of Bacteroides fragilis and Clostridium perfringens. The negative logarithms of minimum inhibitory concentrations (MICs) of these nitroimidazoles were fitted by a biostatistical relation involving a newly designed structural descriptor, the Resultant Bond Moment (RBM). RBM was developed on the basis of the chemical attribute of bond moments of heteroatoms. RBM reflects the electronic characteristic of the molecule. Apart from this, steric, electronic, and hydrophobic parameters were also utilised in the search of a best fit regression equation. On the basis of these equations new investigative nitroimidazole derivatives have been predicted concerning their antibacterial and antiprotozoal efficacy.

Responsiveness of vaginal cells to estradiol during postnatal development of rat.

Ontogeny of responsiveness to hormones is mainly regulated by the presence of receptors; their type, number, and location in the hormone target cells. Some of these parameters have been used to study the responsiveness of rat vagina to estradiol. The estrogen binding sites (EBS) in the cytosol of rat vagina are present immediately at birth, however the animal becomes responsive to the hormone only after 25 +/- 5 d of age. The authors demonstrate how the serum and tissue levels of estradiol affect the EBS in this tissue during the postnatal development of the rat. The various responses observed after a single i.p. injection of estradiol in the immature rats are explained based on the status of the binding sites for estrogen in this tissue.

Alpha 1-adrenergic receptor involvement in norepinephrine-ethanol inhibition of rat brain Na+ -K+ ATPase and in ethanol tolerance.

Norepinephrine (NE) sensitization of rat brain Na+ -K+ ATPase to ethanol (EtOH) inhibition appears to be mediated by alpha 1-adrenoreceptors, since it was reversed by prazosin and WB-4101 (alpha 1-receptor antagonists) in a concentration-dependent manner, but not by yohimbine and piperoxan (alpha 2-receptor antagonists). In addition, clonidine (alpha 2-agonist) and methoxamine (central receptor type uncertain) produced very little sensitization. Chronic EtOH administration to rats for 3 weeks produced tolerance to the hypothermic effect of test doses of EtOH (3 g/kg, i.p.) and a decreased inhibitory effect of NE + EtOH on the enzyme in vitro. This inhibition was still prevented by prazosin and WB-4101. However, the binding of tritiated WB-4101 and prazosin to brain membrane preparations from control and EtOH-tolerant rats revealed that the maximum number of binding sites (Bmax) and the dissociation constant (KD) of alpha 1-adrenoreceptors were decreased after tolerance development. These changes in numbers and binding properties of alpha 1-adrenoreceptors probably account for the decreased NE sensitization of the ATPase to EtOH inhibition in preparations from EtOH-tolerant rats.

Effect of ethanol tolerance on norepinephrine-ethanol inhibition of (Na+ + K+)-adenosine triphosphatase in various regions of rat brain.

Brain (Na+ + K+)-adenosine triphosphatase activity from untreated rats was inhibited by a combination of 1 microM norepinephrine + 50 mM ethanol (NE + EtOH), in preparations from cerebral cortex (CX), cerebellum (CB), hippocampus (HC), hypothalamus (HT), thalamus-midbrain and pons-medulla, but not from striatum. The rank order of inhibition in these regions was more similar to that of alpha-1 receptor density than of regional NE content. EtOH administration for 3 weeks produced tolerance to the hypothermic effect of EtOH; increased basal adenosine triphosphatase activity in CX, CB and HC, as measured 24 hr after withdrawal; and decreased the inhibitory effect of NE + EtOH in CX, HT, HC and CB preparations. Tolerance to the inhibitory effects of high concentrations (0.22 or 0.44 M) of EtOH alone was found only in CX, HT and HC preparations. Tolerance to NE + EtOH or to EtOH alone was greatest in HC and CX, intermediate in HT and CB and least or absent in other regions. Temperature-dependence of (Na+ + K+)-adenosine triphosphatase activity was studied in preparations from CX (high initial sensitivity to NE + EtOH, high tolerance development), CB (intermediate initial sensitivity, intermediate tolerance) and striatum (no initial sensitivity, no tolerance). Arrhenius plots showed differences between these regions, with respect to changes in transition temperature and activation energy after chronic EtOH treatment in vivo. These changes did not explain the regional differences in tolerance development. Therefore it seems unlikely that a single mechanism, such as "stiffening" of the cell membrane, can explain the varied pattern of tolerance development in different brain regions.

Temporal correlation of changes in rat brain sialic acid and in inhibition of Na+-K+ ATPase with ethanol tolerance.

Administration of ethanol to rats by gavage, in daily doses increasing gradually from 3 to 6 g/kg, led to progressive development of tolerance to the hypothermic effect of test doses of ethanol (3 g/kg, i.p.), between days 4 and 21 of treatment. Tolerance was decreased by day 3 of withdrawal, and had disappeared by day 7. Basal levels of brain microsomal Na+-K+ ATPase activity increased progressively during tolerance development, and returned to normal by 4 days after ethanol withdrawal. Conversely, inhibition of the ATPase by ethanol alone (0.22-0.66 M), or by 1 microM norepinephrine (NE) combined with much lower concentrations of ethanol (0.0125-0.1 M), decreased progressively during tolerance development, and returned to normal after withdrawal. The best temporal correlation with the degree of tolerance was shown by the magnitude of NE sensitization of the enzyme to ethanol inhibition. The chronic sucrose control treatment resulted in an unexplained increase in the NE sensitization. Since synaptosomal membrane concentration of sialic acid showed a similar time course during ethanol treatment, it is possible that changes in membrane sialic acid contribute to the alteration in NE sensitization by altering NE receptor properties.

Effect of chronic ethanol treatment on temperature dependence and on norepinephrine sensitization of rat brain (Na+ + K+)-adenosine triphosphatase.

Rat brain (Na+ + K+)-adenosine triphosphatase is inhibited by ethanol (EtOH) in vitro, the inhibition being greater in the presence of norepinephrine (NE). Enzyme preparations from EtOH-tolerant rats show less inhibition by EtOH in vitro and less sensitization by NE. To investigate the mechanism of these changes, the enzyme activity of brain microsomes from tolerant and sucrose-control rats was measured at temperatures from 10-40 degrees C. Preparations from nonwithdrawn and 24-hr withdrawn rats were studied in the absence of in vitro additions, and in the presence of 1 microM NE, 50 mM EtOH or 440 mM EtOH separately, and of 1 microM NE + 50 mM EtOH. From Arrhenius plots of the results, the transition temperature (Td) was calculated by a method of successive approximations, and the activation energies were calculated from the segments above and below Td. Chronic EtOH treatment significantly decreased Td, but increased activation energies below Td. These findings suggest different effects on membrane matrix lipids than on boundary lipids adjacent to the enzyme. However, EtOH-tolerant preparations showed less effect of EtOH in vitro than did control preparations, on both Td and activation energies. Preparations from EtOH-tolerant and withdrawn rats behaved almost identically, indicating that the changes accompany tolerance and are not withdrawal effects. NE + 50 mM EtOH produced the same effects as 440 mM EtOH alone, in all preparations. EtOH tolerance reduced the sensitizing effect of NE. EtOH is interpreted as affecting both the boundary lipids and the apoenzyme itself.

Interaction of catecholamines and ethanol on the kinetics of rat brain (Na+ + K+)-ATPase.

The effects of catecholamines (CA) and ethanol (EtOH), singly and in combination, on the kinetics of rat brain (Na+ + K+)-ATPase were studied. Addition of 0.05 M EtOH alone did not change Vmax or Km for K+, Na+, Mg2+ and ATP. Addition of 0.1 mM dopamine (DA) or noradrenaline (NA) alone stimulated the enzyme activity in presence of vanadium-containing ATP as substrate, but not with vanadium-free ATP except in the presence of high Mg2+ : ATP ratios. CA alone decreased the Km slightly for K+ and by about 50% for ATP, increased it for Mg2+ and did not change it for Na+. However, the combination of DA or NA + EtOH produced a marked inhibition which was competitive for K+, and uncompetitive or mixed for Mg2+, Na+ and ATP. The inhibitory effect of NA + EtOH was abolished in 20 mM K+. These findings suggest that NA sensitizes the enzyme to EtOH inhibition at physiological K+ concentrations, by conformational change away from the outwardly facing K+-binding E2P for to the inwardly facing Na+-binding E1P form.

Alpha adrenoreceptor mediated alteration of ethanol effects on (Na+ + K+)-ATPase of rat neuronal membranes.

Noradrenaline (NA) sensitizes rat brain (Na+ + K+)-ATPase to inhibition by ethanol (EtOH). This effect of NA increases with the degree of enrichment of the enzyme: 0.1 mM NA + 0.05 M EtOH produced 27% inhibition in whole brain homogenates, 40% in 2.5-fold purified P2 fractions, and 45% in 5-fold purified microsomal fractions. The sensitization by NA was prevented by 0.1 microM phentolamine but not by 100 microM propranolol. Adrenaline and phenylephrine also sensitized the enzyme to EtOH inhibition in all of the fractions but isoproterenol did not. For all three alpha agonists the degree of sensitization was concentration dependent and the degree of reversal of this effect varied with the concentration of phentolamine added. These findings suggest that the NA + EtOH interaction is a direct effect on the membrane, probably mediated by an alpha receptor modified perturbation of the membrane microenvironment of the enzyme.

Acute and chronic catecholamine-ethanol interactions on rat brain (Na+ + K+)-ATPase.

Noradrenaline (N) sensitizes rat brain (Na+ + K+)-ATPase to inhibition by low concentrations of ethanol (E). Only 1-N and not d-N was effective. The sensitization is also produced by other alpha-adrenergic agonists (adrenaline, phenylephrine), but not by isoproterenol, and is prevented by phentolamine but not by propranolol. The sensitization is greater with partially purified enzyme than with crude homogenates. N + E, like much higher concentrations of E alone, produced competitive inhibition with respect to K+ but uncompetitive or mixed inhibition with respect to Na+, Mg++ and ATP, and a reduced "physiological efficiency" of ATP utilization. All these changes were abolished by increasing K+ to 20 mM. After 3-week E treatment, with or without withdrawal, the N + E interaction was markedly reduced, though basal ATPase activity was increased only after withdrawal. Temperature-dependence studies (Arrhenius plots) indicated that sensitization occurs by alteration of activation energy only above the transition temperature. These findings suggest that alpha-agonists fluidize membrane lipids and thus facilitate conformational change of the enzyme by E, resulting in inhibition.