Absence of mechanical allodynia and Abeta-fiber sprouting after sciatic nerve injury in mice lacking membrane-type 5 matrix metalloproteinase

Matrix metalloproteinases (MMPs) are a family of endopeptidases that degrade extracellular matrix components. Membrane-type 5 MMP (MT5-MMP/MMP-24) was identified as neuron-specific, and is believed to contribute to neuronal circuit formation and plasticity. To elucidate its function in vivo, we have generated mice lacking MT5-MMP by gene targeting. MT5-MMP-deficient mice were born without obvious morphological abnormalities. No apparent histological defects were observed in the nervous system either. However, MT5-MMP-deficient mice did not develop neuropathic pain with mechanical allodynia after sciatic nerve injury, though responses to acute noxious stimuli were normal. Neuropathic pain induced by peripheral nerve lesions is known to accompany structural reorganization of the nervous system. Intraneural injection of cholera toxin B subunit, a transganglionic tracer, into the injured sciatic nerve of wild-type mice revealed that the myelinated Abeta-fiber primary afferents sprouted from laminae III-VI of the dorsal horn of the spinal cord and invaded lamina II. However, no such sprouting and invasion of Abeta-fibers were observed in MT5-MMP-deficient mice. These findings suggest that MT5-MMP is essential for the development of mechanical allodynia and plays an important role in neuronal plasticity in this mouse model.     

Hydrogen peroxide production in Streptococcus pyogenes: involvement of lactate oxidase and coupling with aerobic utilization of lactate

Streptococcus pyogenes strains can be divided into two classes, one capable and the other incapable of producing H2O2 (M. Saito, S. Ohga, M. Endoh, H. Nakayama, Y. Mizunoe, T. Hara, and S. Yoshida, Microbiology 147:2469-2477, 2001). In the present study, this dichotomy was shown to parallel the presence or absence of H2O2-producing lactate oxidase activity in permeabilized cells. Both lactate oxidase activity and H2O2 production under aerobic conditions were detectable only after glucose in the medium was exhausted. Thus, the glucose-repressible lactate oxidase is likely responsible for H2O2 production in S. pyogenes. Of the other two potential H2O2-producing enzymes of this bacterium, NADH and alpha-glycerophosphate oxidase, only the former exhibited low but significant activity in either class of strains. This activity was independent of the growth phase, suggesting that the protein may serve in vivo as a subunit of the H2O2-scavenging enzyme NAD(P)H-linked alkylhydroperoxide reductase. The activity of lactate oxidase was associated with the membrane while that of NADH oxidase was in the soluble fraction, findings consistent with their respective physiological roles, i.e., the production and scavenging of H2O2. Analyses of fermentation end products revealed that the concentration of lactate initially increased with time and decreased on glucose exhaustion, while that of acetate increased during the culture. These results suggest that the lactate oxidase activity of H2O2-producing cells oxidizes lactate to pyruvate, which is in turn converted to acetate. This latter process proceeds presumably via acetyl coenzyme A and acetyl phosphate with formation of extra ATP.     

Munc13-4 is a GTP-Rab27-binding protein regulating dense core granule secretion in platelets

Platelets store self-agonists such as ADP and serotonin in dense core granules. Although exocytosis of these granules is crucial for hemostasis and thrombosis, the underlying mechanism is not fully understood. Here, we show that incubation of permeabilized platelets with unprenylated active mutant Rab27A-Q78L, wild type Rab27A, and Rab27B inhibited the secretion, whereas inactive mutant Rab27A-T23N and other GTPases had no effects. Furthermore, we affinity-purified a GTP-Rab27A-binding protein in platelets and identified it as Munc13-4, a homologue of Munc13-1 known as a priming factor for neurotransmitter release. Recombinant Munc13-4 directly bound to GTP-Rab27A and -Rab27B in vitro, but not other GTPases, and enhanced secretion in an in vitro assay. The inhibition of secretion by unprenylated Rab27A was rescued by the addition of Munc13-4, suggesting that Munc13-4 mediates the function of GTP-Rab27. Thus, Rab27 regulates the dense core granule secretion in platelets by employing its binding protein, Munc13-4.     

An engineered hyaluronan synthase: characterization for recombinant human hyaluronan synthase 2 Escherichia coli

The Class I hyaluronan synthase (HAS) is a unique glycosyltransferase synthesizing hyaluronan (HA), a polysaccharide composed of GlcUA and GlcNAc, by using one catalytic domain that elongates two different monosaccharides. As for the synthetic mechanism, there are two alternative manners for the sugar elongation process. Some bacterial HASs add new sugars to the non-reducing end of the acceptor to grow polymers. On the other hand, some vertebrate enzymes seem to transfer sugars to the reducing end. Expression of vertebrate HASs as active and soluble proteins will accelerate further precise insight into mechanisms of sugar elongation reactions by natural HASs. Since large scale production of HA polymers and oligomers would become powerful tools both for basic studies and new biotechnology to create functional carbohydrates in medicinal purposes, advent of an efficient method for the expression of HASs in Escherichia coli is strongly expected. Here we communicate the first success of the production of recombinant human HAS2 proteins composed of only the catalytic region in E. coli as the active form. It was demonstrated that an engineered HAS2 expressed in E. coli exhibited significant activity to synthesize a mixture of HAS oligomers from 8-mer (HA8) to 16-mer (HA16). Engineered HAS2 prepared herein elongated sugars from exogenous tetrasaccharide to form polymers with a direction to the non-reducing end. According to the present results, large scale production of engineered recombinant HASs is to be performed using E. coli that will provide practical and economic advantages in manufacturing enzymes for use in the synthesis of various oligomeric HA molecules and their industrial applications.     

Cdk4 is indispensable for postnatal proliferation of the anterior pituitary

For proper development and tissue homeostasis, cell cycle progression is controlled by multilayered mechanisms. Recent studies using knock-out mice have shown that animals can develop relatively normally with deficiency for each of the G1/S-regulatory proteins, D-type and E-type cyclins, cyclin-dependent kinase 4 (Cdk4), and Cdk2. Although Cdk4-null mice show no embryonic lethality, they exhibit specific endocrine phenotypes, i.e. dwarfism, infertility, and diabetes. Here we have demonstrated that Cdk4 plays an essential non-redundant role in postnatal proliferation of the anterior pituitary. Pituitaries from wild-type and Cdk4-null embryos at embryonic day 17.5 are morphologically indistinguishable with similar numbers of cells expressing a proliferating marker, Ki67, and cells expressing a differentiation marker, growth hormone. In contrast, anterior pituitaries of Cdk4-null mice at postnatal 8 weeks are extremely hypoplastic with markedly decreased numbers of Ki67+ cells, suggesting impaired cell proliferation. Pituitary hyperplasia induced by transgenic expression of human growth hormone-releasing hormone (GHRH) is significantly diminished in the Cdk4+/- genetic background and completely abrogated in the Cdk4-/- background. Small interfering RNA (siRNA)-mediated knockdown of Cdk4 inhibits GHRH-induced proliferation of GH3 somato/lactotroph cells with restored expression of GHRH receptors. Cdk4 siRNA also inhibits estrogen-dependent cell proliferation in GH3 cells and closely related GH4 cells. In contrast, Cdk6 siRNA does not diminish proliferation of these cells. Furthermore, Cdk4 siRNA does not affect GHRH-induced proliferation of mouse embryonic fibroblasts or estrogen-dependent proliferation of mammary carcinoma MCF-7 cells. Taken together, Cdk4 is dispensable for prenatal development of the pituitary or proliferation of other non-endocrine tissues but indispensable specifically for postnatal proliferation of somato/lactotrophs.     

Identification and characterization of a novel component of the cornified envelope, cornifelin

Psoriasis is recognized as a chronic inflammatory disease characterized by epidermal hyperproliferation. To identify psoriasis-related genes, we compared the mRNA populations of normal and psoriatic skin. We identified one gene, designated as cornifelin, which showed increased expression in psoriatic skin. Human cornifelin contains 112 amino acids and is expressed in the uterus, cervix, and skin. In situ hybridization analysis demonstrated the presence of human cornifelin in the granular cell layer of the epidermis. To investigate the function of cornifelin, we established a transgenic mouse line overexpressing human cornifelin. Using these mice, we have shown that cornifelin is directly or indirectly cross-linked to at least two other cornified envelope proteins, loricrin and involucrin, in vivo. Overexpression of human cornifelin correlated with decreased loricrin expression and increased involucrin expression in the transgenic mouse. However, abnormality of epidermal differentiation was not observed in the transgenic mouse.     

Flavonoid-induced reduction of ENaC expression in the kidney of Dahl salt-sensitive hypertensive rat

Flavonoid, a plant extract, exhibits various biological actions. Dietary flavonoid intake is reported to reduce an elevated blood pressure, however the mechanism is unknown. The epithelial Na+ channel (ENaC) in the kidney plays a key role in the regulation of blood pressure by contributing to the Na+ reabsorption in renal tubules. Thus, we investigated the effect of quercetin, a flavonoid, on ENaC mRNA expression in the kidney of hypertensive Dahl salt-sensitive rats. Dahl salt-sensitive rats of 8 weeks were acclimated for 1 week in a metabolic cage and were subsequently kept for 4 weeks under four different conditions: (1) normal salt diet (0.3% NaCl), (2) normal salt diet with quercetin (10 mg/kg/day), (3) high-salt diet (8% NaCl), and (4) high-salt diet with quercetin. Quercetin diminished the alphaENaC mRNA expression in the kidney associated with reduction of the systolic blood pressure elevated by high-salt diet, suggesting that one of the mechanisms of the flavonoid's antihypertensive effect on salt-sensitive hypertension would be mediated through downregulation of ENaC expression in the kidney.     

Prostanoid EP4 receptor is involved in suppression of 3T3-L1 adipocyte differentiation

Prostaglandins (PGs) have been shown to play various roles in adipogenesis. In this study, we investigated on which PGE receptor subtypes are involved in the inhibition of 3T3-L1 preadipocyte differentiation. The triglyceride content of cells, used as an index of differentiation, was decreased when PGE(2), the FP-agonist fluprostenol or dibutyryl cAMP, was exogenously added to differentiation cocktails. 3T3-L1 preadipocyte cells express mRNAs for the prostanoid EP4, FP, and IP receptors. PGE(2) and the EP4 agonist AE1-329 increased cAMP levels in preadipocytes in a dose-dependent manner. AE1-329 suppressed the expression induction of differentiation marker genes such as resistin and peroxisome proliferator-activated receptor-gamma. The inhibitory effect of PGE(2) but not that of fluprostenol was reversed by the addition of the EP4 antagonist AE3-208. AE3-208 mimicked the differentiation-promoting effects of indomethacin. These results suggest that the EP4 receptor mediates the suppressive action of PGE(2) in 3T3-L1 adipocyte differentiation.