Glucagon-related peptides in the rat hypothalamus

Summary Immunohistochemically, nerve fibers and terminals reacting with anti-N-terminal-specific but not with anti-C-terminal-specific glucagon antiserum were observed in the following rat hypothalamic regions: paraventricular nucleus, supraoptic nucleus, anterior hypothalamus, arcuate nucleus, ventromedial hypothalamic nucleus and median eminence. Few fibers and terminals were demonstrated in the lateral hypothalamic area and dorsomedial hypothalamic nucleus. Radioimmunoassay data indicated that the concentration of gut glucagon-like immunoreactivity was higher in the ventromedial nucleus than in the lateral hypothalamic area. In food-deprived conditions, this concentration increased in both these parts. This was also verified in immunostained preparations in which a marked enhancement of gut glucagon-like immunoreactivity-containing fibers and terminals was observed in many hypothalamic regions. Several immunoreactive cell bodies were found in the ventromedial and arcuate nuclei of starved rats. Both biochemical and morphological data suggest that glucagon-related peptides may act as neurotransmitters or neuromodulators in the hypothalamus and may be involved in the central regulatory mechanism related to feeding behavior and energy metabolism.     

Siblings with renal tubular acidosis and nerve deafness. The first family in Japan

Summary Two siblings with renal tubular acidosis (RTA) and nerve deafness were examined. It was found by ammonium chloride and bicarbonate loading tests that the 6-year-old brother had a hybrid type of RTA and his 4-year-old sister, a distal type of RTA. Enzyme activity and amount of enzyme protein of carbonic anhydrase isoenzyme I and II in red blood cells, measured using an immunoadsorbent method, were normal in both cases. Although this indicated that the RTAs of these patients are not generated by the carbonic anhydrase deficiency, an investigation with renal tissue is necessary to arrive at a final conclusion.     

(-)-Epigallocatechin gallate amplifies interleukin-1-stimulated interleukin-6 synthesis in osteoblast-like MC3T3-E1 cells

We previously reported that interleukin-1 (IL-1), a potent bone resorptive cytokine, stimulates the synthesis of interleukin-6 (IL-6) via activation of p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase in osteoblast-like MC3T3-E1 cells, and that AMP-activated protein kinase (AMPK) negatively regulates the IL-1-induced IL-6 synthesis through the inhibitor of κB (IκB)/nuclear factor-κB (NF-κB) pathway. On the other hand, it is recognized that catechin possesses a beneficial property for bone metabolism. Among them, (-)-epigallocatechin gallate (EGCG) is an abundant and major bioactive component. In the present study, we investigated the effect of EGCG on the IL-1 stimulated IL-6 synthesis in osteoblast-like MC3T3-E1 cells. EGCG significantly enhanced the IL-1-stimulated IL-6 synthesis in a dose-dependent manner in the range between 50 and 100 μM. EGCG increased the mRNA levels of IL-6 stimulated by IL-1. IL-1-induced phosphorylation of IκB and NF-κB were suppressed by EGCG. On the other hand, EGCG failed to affect the IL-1-induced phosphorylation of p44/p42 MAP kinase, p38 MAP kinase and AMPK. These results strongly suggest that EGCG enhances IL-1-stimulated IL-6 synthesis through inhibiting the AMPK-IκB/NF-κB pathway at the point between AMPK and IκB/NF-κB in osteoblasts.     

Protein binding to amphoteric polymer brushes grafted onto a porous hollow-fiber membrane

Three kinds of ampholites, i.e., 3-aminopropionic acid (NH2C2H4COOH), (2-aminoethyl)phosphonic acid (NH2C2H4PO3H2), and 2-aminoethane-1-sulfonic acid (NH2C2H4SO3H), were introduced into an epoxy group-containing polymer brush grafted onto a porous hollow-fiber membrane with a porosity of 70% and pore size of 0.36 microm. The amphoteric group density of the hollow-fiber ranged from 0.50 to 0.72 mmol/g. Three kinds of proteins, i.e., lactoferrin (Lf), cytochrome c (Cyt c), and lysozyme (Ly), were captured by the amphoteric polymer brush during the permeation of the protein solution across the ampholite-immobilized porous hollow-fiber membrane. Multilayer binding of the protein to the amphoteric polymer brush, with a degree of multilayer binding of 3.3, 8.6, and 15 for Lf, Cyt c, and Ly, respectively, with the (2-aminoethyl)phosphonic acid-immobilized porous hollow-fiber membrane, was demonstrated with a negligible diffusional mass-transfer resistance of the protein to the ampholite immobilized. The 2-aminoethane-1-sulfonic acid-immobilized porous hollow-fiber membrane exhibited the lowest initial flux of the protein solution, 0.41 m/h at a transmembrane pressure of 0.1 MPa and 298 K, and the highest equilibrium binding capacity of the protein, e.g., 130 mg/g for lysozyme. Extension and shrinkage of the amphoteric polymer brushes were observed during the binding and elution of the proteins.     

Fourteen morphotypes of Entodinium ovumrajae (Ophryoscolecidae, Entodiniomorphida) found in the Dromedary camel of Egypt

During a survey of the ciliate protozoal composition of the stomach contents of nine dromedary camels of Egypt, fourteen morphotypes of Entodinium ovumrajae, which has been considered as a species peculiar to camels, were found in six camels. Except for five morphotypes including one originally described as an independent species and its forms, these were newly detected. These morphotypes, divided into three groups, can be identified mainly by the morphology of their ectoplasmic processes. Each camel had on average, about five morphotypes of this species.     

Tyrosine dephosphorylation and ethanol inhibition of N-Methyl-D-aspartate receptor function

The inhibitory effect of ethanol on N-methyl-d-aspartate receptors (NMDARs) is well documented in several brain regions. However, the molecular mechanisms by which ethanol affects NMDARs are not well understood. In contrast to the inhibitory effect of ethanol, phosphorylation of the NMDAR potentiates channel currents (Lu, W. Y., Xiong, Z. G., Lei, S., Orser, B. A., Dudek, E., Browning, M. D., and MacDonald, J. F. (1999) Nat. Neurosci. 2, 331-338). We have previously shown that protein kinase C activators induce tyrosine phosphorylation and potentiation of the NMDAR (Grosshans, D. R., Clayton, D. R., Coultrap, S. J., and Browning, M. D. (2002) Nat. Neurosci. 5, 27-33). We therefore hypothesized that the ethanol inhibition of NMDARs might be due to changes in tyrosine phosphorylation of NMDAR subunits. In support of this hypothesis, we found that tyrosine phosphorylation of both NR2A and NR2B subunits was significantly reduced following in situ exposure of hippocampal slices to 100 mm ethanol. Specifically, phosphorylation of tyrosine 1472 on NR2B was reduced 23.5%. These data suggest a possible mechanism by which ethanol may inhibit the NMDAR via activation of a tyrosine phosphatase. Electrophysiological studies demonstrated that ethanol inhibited NMDAR field excitatory postsynaptic potential slope and amplitude to a similar degree as previously reported by our laboratory and others (Schummers, J., Bentz, S., and Browning, M. D. (1997) Alcohol Clin. Exp. Res. 21, 404-408). Inclusion of bpV(phen), a potent phosphotyrosine phosphatase inhibitor, in the recording chamber prior to and during ethanol exposure significantly reduced the inhibitory effect of ethanol on NMDAR field excitatory postsynaptic potentials. Taken together, these data suggest that phosphatase-mediated dephosphorylation of NMDAR subunits may play an important role in mediating the inhibitory effects of ethanol on the N-methyl-D-aspartate receptor.     

Changes in mutagenicity of herbicide chlornitrofen during biodegradation

We used the Ames assay to investigate changes in the mutagenicity of chlornitrofen during its aerobic biodegradation. Although a mixed culture of bacteria obtained from river water degraded chlornitrofen and reduced its concentration from 39 to 6 microg/l in 21 days, the indirect mutagenicity of the solution to Salmonella strains TA98, YG1021, and YG1026 increased gradually. This finding suggests that mutagenic metabolites were produced during the aerobic biodegradation. The increase in the mutagenicity was, however, much smaller under aerobic than under anaerobic conditions. The differing sensitivities of our test strains to the functional groups on the mutagens showed that the mutagenic metabolites were indirect frameshift-type mutagens that might have neither nitro nor amino groups.     

Molecular characterization of mammalian dicarbonyl/L-xylulose reductase and its localization in kidney

In this report, we first cloned a cDNA for a protein that is highly expressed in mouse kidney and then isolated its counterparts in human, rat hamster, and guinea pig by polymerase chain reaction-based cloning. The cDNAs of the five species encoded polypeptides of 244 amino acids, which shared more than 85% identity with each other and showed high identity with a human sperm 34-kDa protein, P34H, as well as a murine lung-specific carbonyl reductase of the short-chain dehydrogenase/reductase superfamily. In particular, the human protein is identical to P34H, except for one amino acid substitution. The purified recombinant proteins of the five species were about 100-kDa homotetramers with NADPH-linked reductase activity for alpha-dicarbonyl compounds, catalyzed the oxidoreduction between xylitol and l-xylulose, and were inhibited competitively by n-butyric acid. Therefore, the proteins are designated as dicarbonyl/l-xylulose reductases (DCXRs). The substrate specificity and kinetic constants of DCXRs for dicarbonyl compounds and sugars are similar to those of mammalian diacetyl reductase and l-xylulose reductase, respectively, and the identity of the DCXRs with these two enzymes was demonstrated by their co-purification from hamster and guinea pig livers and by protein sequencing of the hepatic enzymes. Both DCXR and its mRNA are highly expressed in kidney and liver of human and rodent tissues, and the protein was localized primarily to the inner membranes of the proximal renal tubules in murine kidneys. The results imply that P34H and diacetyl reductase (EC ) are identical to l-xylulose reductase (EC ), which is involved in the uronate cycle of glucose metabolism, and the unique localization of the enzyme in kidney suggests that it has a role other than in general carbohydrate metabolism.     

A growth factor-dependent nuclear kinase phosphorylates p27(Kip1) and regulates cell cycle progression

The cyclin-dependent kinase inhibitor, p27(Kip1), which regulates cell cycle progression, is controlled by its subcellular localization and subsequent degradation. p27(Kip1) is phosphorylated on serine 10 (S10) and threonine 187 (T187). Although the role of T187 and its phosphorylation by Cdks is well-known, the kinase that phosphorylates S10 and its effect on cell proliferation has not been defined. Here, we identify the kinase responsible for S10 phosphorylation as human kinase interacting stathmin (hKIS) and show that it regulates cell cycle progression. hKIS is a nuclear protein that binds the C-terminal domain of p27(Kip1) and phosphorylates it on S10 in vitro and in vivo, promoting its nuclear export to the cytoplasm. hKIS is activated by mitogens during G(0)/G(1), and expression of hKIS overcomes growth arrest induced by p27(Kip1). Depletion of KIS using small interfering RNA (siRNA) inhibits S10 phosphorylation and enhances growth arrest. p27(-/-) cells treated with KIS siRNA grow and progress to S/G(2 )similar to control treated cells, implicating p27(Kip1) as the critical target for KIS. Through phosphorylation of p27(Kip1) on S10, hKIS regulates cell cycle progression in response to mitogens.