Malate dehydrogenase isoforms from ribbed mussel (Modiolus demissus) gill tissue

• 1.1. The malate dehydrogenase (MHD) activity from the ribbed mussel gill is polymorphic with two distinct mitochondrial forms (M1 and M2) and five forms that could be resolved from cytosolic extracts (C1 to C5) by DEAE-cellulose chromatography and starch gel electrophoresis.
• 2.2. Two of the cytosolic forms (C3 and C4) may represent interchangeable conformational states.
• 3.3. With kinetic analysis there appear to be three distinct cytosolic forms (C1, C2 and C3–C4), with C2 possibly behaving as a heterodimer.
• 4.4. The identity of C5 is uncertain.
• 5.5. The forms isolated from the mitochondria (M1 and M2) exhibited lower apparent K_ms for oxaloacetate (OAA) than the cytosolic forms.
• 6.6. For all isozymic forms, the apparent K_ms for OAA increased as the pH increased between pH 6 and 9
• 7.7. Increasing the salt concentration raised the K_m for OAA for all forms.
• 8.8. The mMDHs were more sensitive to inhibition by NaCl than the cMDHs.
• 9.9. Representative cMDH (C1) and mMDH (M2) isozymes exhibited substrate inhibition by high concentrations of OAA with the mMDH possessing lower K_is for substrate inhibition than the cMDH at each pH tested.
• 10.10. Differences and similarities in K_m app. for OAA at the different pHs and salt concentrations indicated that C1, C2 and C3–C4 and C5 were distinct forms, that M1 and M2 were distinct but very similar to each other, and that C1, C2, C3–C4 and C5 were distinct from M1 and M2.

     

Antibacterial activities in various tissues of the horse mussel, Modiolus modiolus

A search for antibacterial activity in different organs/tissues of the horse mussel, Modiolus modiolus, was conducted. Dried samples were extracted with 60% (v/v) acetonitrile, containing 0.1% (v/v) trifluoroacetic acid. Due to high salt content, two liquid phases were obtained; an acetonitrile-rich phase (ACN extract) and an aqueous phase. The aqueous phase was further subjected to solid phase extraction (SPE). Eluates from SPE and ACN extracts were tested for antibacterial, lysozyme, and toxic activity. Antibacterial activity was demonstrated in extracts from several tissues, including plasma, haemocytes, labial palps, byssus, mantle, and gills. Some of the extracts were sensitive to proteinase K treatment, indicating antibacterial peptides and/or proteins. Lysozyme-like activity and toxic activity against Artemia salina nauplii was detected in fractions from the gills, mantle, muscle, and haemocytes. Results from this study indicate that M. modiolus is a promising source for identifying novel drug lead compounds.     

Tumor-like lesions in the mantle of the mussel Modiolus difficilis from the Sea of Japan

Two inner growths in the mantle beneath the epithelium were found in 1 of 1000 mussels Modiolus difficilis from Amursky Bay, Sea of Japan, within the city precincts of Vladivostok. Both growths were about 2000 microns in maximal diameter in section and elevated slightly above the mantle surface. The mantle epithelium near the growths formed deep invaginations, and clusters of mucous cells were numerous beneath the epithelium. Histological and histochemical methods were employed. Two different kinds of growth were revealed. The off-white growth consisted of cells with thin granular or vesicular cytoplasm containing glucosaminoglycans, proteins and a small amount of neutral polysaccharides. Growth cells were pure white in color after treatment of preparations with 1% H2SO4 and differed markedly from the mantle cells. The yellow growth consisted of large granular cells with neutral polysaccharides and proteins. Although growths were composed of different kinds of cells, they seemed to be derived from subepithelial mucous cells. This was supported by histological and histochemical staining reactions of some tumor and mantle epithelial cells. Mitotic indices (MI) of growths and subepithelial mucous cells were zero, MI of ciliated mantle epithelium reached 0.07%. The lesions were areas of strongly altered mucous cells of mantle epithelium and were non-neoplastic.     

Electrical excitability of taste cells. Mechanisms and possible physiological significance

Neuronal, muscle and some endocrine cells are electrically excitable. While in muscle and endocrine cells AP stimulates and synchronizes intracellular processes, neurons employ action potentials (APs) to govern discontinuous synapses located distantly. Meanwhile, such axonless sensory cells as photoreceptors and hair cells exemplify afferent output, which is not driven by APs; instead, gradual receptor potentials elicited by sensory stimuli control the release of afferent neurotransmitter glutamate. Mammalian taste cells of the type II and type III are electrically excitable and respond to stimulation by firing APs. Since taste cells also have no axons, physiological significance of the electrical excitability for taste transduction and encoding sensory information is unclear. Perhaps, AP facilitates transmitter release, ATP in type II cells and 5-HT in type III cells, although via different mechanisms. The ATP release is mediated by connexin hemichannels, does not require a Ca²⁺ trigger, and largely gated by membrane voltage. 5-HT secretion is driven by intracellular Ca²⁺ and involves VG Ca²⁺ channels. Here, we discuss ionic mechanisms of excitability of taste cells and speculate on a likely role of APs in mediating their afferent output.     

The possible role of ADP ribosylation in physiological regulation of sporulation in Streptomyces griseus.

The role of ADP ribosylation of proteins in the physiological regulation of sporulation in Streptomyces griseus was studied. We report here that both the activity of NAD+: arginine ADP-ribosyltransferase (ADPRT) and the pattern of ADP-ribosylated proteins showed characteristic changes during the life cycle in S. griseus 2682. Analysis off ADP-ribosylated proteins revealed that in a nonsporulating mutant of the parental wild-type (wt) strain (Bld7 mutant), both the activity of ADPRT and the pattern of ADP-ribosylated proteins were different from those of the parental strain. Addition of 3-aminobenzamide (3AB), the most potent inhibitor of ADPRT, inhibited sporulation of S. griseus 2682 and the A-factor (AF)-induced sporulation of S. griseus Bld7, but in both cases the inhibitory effect of 3AB was strictly age-dependent. Using [alpha-32P]GTP, we have demonstrated the presence of GTP-binding proteins in purified cell membranes of S. griseus 2682 and S. griseus Bld7. The same GTP-binding proteins were observed in Bld7 and the wt. AF stimulated the basal GTPase activity of cell membranes of S. griseus 2682 in a concentration-dependent manner, suggesting that GTP-binding proteins might be involved in the AF-induced sporulation process.     

Localization, physiological significance and possible clinical implication of gastrointestinal melatonin.

The gastrointestinal tract (GIT) is a major source of extrapineal melatonin. In some animals, tissue concentrations of melatonin in the GIT surpass blood levels by 10-100 times and the digestive tract contributes significantly to melatonin concentrations in the peripheral blood, particularly during the day. Some melatonin found in the GIT may originate from the pineal gland, as the organs of the digestive system contain binding sites, which in some species exhibit circadian variation. Unlike the production of pineal melatonin, which is under the photoperiodic control, release of GI melatonin seems to be related to periodicity of food intake. Melatonin and melatonin binding sites were localized in all GI tissues of mammalian and avian embryos. Postnatally, melatonin was localized in the GIT of newborn mice and rats. Phylogenetically, melatonin and melatonin binding sites were detected in GIT of numerous mammals, birds and lower vertebrates. Melatonin is probably produced in the serotonin-rich enterochromaffin cells (EC) of the GI mucosa and can be released into the portal vein postprandially. In addition, melatonin can act as an autocrine or a paracrine hormone affecting the function of GI epithelium, lymphatic tissues of the immune system and the smooth muscles of the digestive tube. Finally, melatonin may act as a luminal hormone, synchronizing the sequential digestive processes. Higher peripheral and tissue levels of melatonin were observed not only after food intake but also after a long-term food deprivation. Such melatonin release may have a direct effect on the various GI tissues but may also act indirectly via the CNS; such action might be mediated by sympathetic or parasympathetic nerves. Melatonin can protect GI mucosa from ulceration by its antioxidant action, stimulation of the immune system and by fostering microcirculation and epithelial regeneration. Melatonin may reduce the secretion of pepsin and the hydrochloric acid and influence the activity of the myoelectric complexes of the gut via its action in the CNS. Tissue or blood levels of melatonin may serve as a marker of GI lesions or tumors. Clinically, melatonin has a potential for a prevention or treatment of colorectal cancer, ulcerative colitis, irritable bowel syndrome, children colic and diarrhea.     

Glutamate dehydrogenase from the hyperthermophile Pyrococcus furiosus. Thermal denaturation and activation.

Pyrococcus furiosus is a marine hyperthermophile that grows optimally at 100 degrees C. Glutamate dehydrogenase (GDH) from P. furiosus is a hexamer of identical subunits and has an M(r) = 270,000 +/- 5500 at 25 degrees C. Electron micrographs showed that the subunit arrangement is similar to that of GDH from bovine liver (i.e. 3/2 symmetry in the form of a triangular antiprism). However, GDH from P. furiosus is inactive at temperatures below 40 degrees C and undergoes heat activation above 40 degrees C. Both NAD+ and NADP+ are utilized as cofactors. Apparently the inactive enzyme also binds cofactors, since the enzyme maintains the ability to bind to an affinity column (Cibacron blue F3GA) and is specifically eluted with NADP+. Conformational changes that accompany activation and thermal denaturation were detected by precision differential scanning microcalorimetry. Thermal denaturation starts at 110 degrees C and is completed at 118 degrees C. delta(cal) = 414 Kcal [mol GDH]-1. Tm = 113 degrees C. This increase in heat capacity indicates an extensive irreversible unfolding of the secondary structure as evidenced also by a sharp increase in absorbance at 280 nm and inactivation of the enzyme. The process of heat activation of GDH from 40 to 80 degrees C is accompanied by a much smaller increase in absorbance at 280 nm and a reversible increase in heat capacity with delta(cal) = 187 Kcal [mol GDH]-1 and Tm = 57 degrees C. This absorbance change as well as the moderate increase in heat capacity suggest that thermal activation leads to some exposure of hydrophobic groups to solvent water as the GDH structure is opened slightly. The increase in absorbance at 280 nm during activation is only 12% of that for denaturation. Overall, GDH appears to be well adapted to correspond with the growth response of P. furiosus to temperature.     

ADP and glucose as possible synergistic partners in the stimulation of liver glycogen synthase activation

Glucose administered either intravenously or orally causes liver glycogen synthase activation independent of a rise in circulating insulin. In vitro, physiological concentrations of glucose stimulate synthase phosphatase activity but only in the presence of a second effector which reduced the A0.5 for glucose. Caffeine and certain methylxanthines have been in vitro models for a putative natural effector. The present study demonstrates that, in vitro, ADP also reduced the A0.5 for glucose comparable to the effect of caffeine. The maximum stimulation by glucose in the presence of caffeine or ADP was comparable. The effect of ADP was specific among the major nucleoside diphosphates. However, the A0.5 for ADP was greater than the normal liver concentration which does not change in response to either glucose or insulin administration. The effect of ADP appeared distinct from that of the methylxanthines since it was observed that at near saturating concentrations of ADP and of glucose, stimulation was increased by addition of theophylline. Similarly, addition of adenosine, a natural cell constituent, caused increased stimulation. Subsequently, it was shown that adenosine reduced the A0.5 for ADP to a nearly physiological concentration. Thus, while ADP is not the inducible putative effector which has been predicted it may be part of an intracellular amplification system for glycogen synthase activation which increases the sensitivity to an induced effector. The present work suggests that the effective concentration of the natural ligand may be less than originally anticipated. This work also suggests that the putative effector could be structurally related to adenosine. Phosphorylase phosphatase activity known to be stimulated by ADP and glucose is further stimulated by the combination which may be acting in synergy.     

Several dehydrogenases and kinases compete for endocytosis from plasma by rat tissues.

Plasma contains many enzymes that are probably derived from damaged cells. These enzymes are cleared at characteristic rates. We showed previously that in rats the rapid clearance of alcohol dehydrogenase, lactate dehydrogenase M4 and the mitochondrial and cytosolic isoenzymes of malate dehydrogenase is largely due to endocytosis by macrophages in liver, spleen and bone marrow. We now demonstrate that uptake of each of the enzymes by these tissues is in general decreased by simultaneous injection of a high dose of one of the other dehydrogenases or a high dose of adenylate kinase or creatine kinase. A similar dose of colloidal albumin did not significantly decrease uptake of the four dehydrogenases. Nor was uptake of colloidal albumin, apo-peroxidase from horseradish or multilamellar liposomes influenced by a high dose of mitochondrial malate dehydrogenase. These results indicate that the four dehydrogenases and the two kinases are specifically endocytosed via the same receptor. We suggest that this receptor contains a group, possibly a nucleotide, with affinity for the nucleotide-binding sites of the enzymes.     

The release of ATP triggered by transmitter action and its possible physiological significance: retrograde transmission.

1. When a slice of electric organ of Torpedo is stimulated and superfused with a solution containing a firefly lantern extract, it is possible to measure the release of ATP after each nerve impulse as a light emission. 2. The postsynaptic action of released ACh induces the release of ATP by the postsynaptic cell. Most of the released ATP is of postsynaptic origin. 3. Ion fluxes associated with depolarization, or depolarization itself, trigger the release of ATP from postsynaptic and presynaptic membranes (synaptosomes). 4. ATP is able to block ACh release; a postsynaptic "retrograde transmission" able to control presynaptic transmitter release is possible.     

Isoelectric focusing studies of aldehyde dehydrogenases, alcohol dehydrogenases and oxidases from mammalian anterior eye tissues

1. Isoelectric focusing (IEF) and zymogram methods were used to examine the tissue distribution, multiplicity and substrate specificities of alcohol dehydrogenases (ADHs), aldehyde dehydrogenases (ALDHs) and ocular oxidases (EOXs) from mammalian anterior eye tissues. 2. Baboon, cattle, pig and sheep corneal extracts exhibited high ALDH activities; the corneal ALDHs were distinct from the major liver ALDHs and distinguished by their preference for medium-chain aldehydes. 3. Baboon and pig corneal extracts also showed high ADH activities, by comparison with ovine and bovine samples. Moreover, the ADHs were distinct from the major liver isozymes in pI value and substrate specificity. 4. Mammalian lens extracts exhibited significant ALDH activity of a form corresponding to the major liver cytosolic isozyme. Minor activity of the corneal enzyme was also observed in some species. 5. Lens ADH phenotypes were species-specific, and consisted of either Class II activity (baboon and sheep), Class III ADH activity (pig), or activities of both ADH classes (cattle). 6. Lens extracts also exhibited a complex pattern of ocular oxidase (EOX) activities following IEF. 7. A role in peroxidatic aldehyde detoxification is proposed for these enzymes in anterior eye tissues.