Expression and function of class B scavenger receptor type I on both apical and basolateral sides of the plasma membrane of polarized testicular Sertoli cells of the rat

Class B scavenger receptor type I (SR-BI), a multiligand membrane protein, exists in various organs and cell types. In the testis, SR-BI is expressed in two somatic cell types: Leydig cells and Sertoli cells. Unlike interstitially localized Leydig cells, Sertoli cells present within the seminiferous tubules keep contact with spermatogenic cells and form the tight junction to divide the seminiferous epithelium into the basal and adluminal compartments. In this study, the expression and function of SR-BI in rat Sertoli cells were examined with respect to dependency on the spermatogenic cycle, the plasma membrane polarity, and the pituitary hormone follicle-stimulating hormone (FSH). When the expression of SR-BI was histochemically examined with testis sections, both protein and mRNA were already present in Sertoli cells during the first-round spermatogenesis and continued to be detectable thereafter. The level of SR-BI mRNA expression in Sertoli cells was lower at spermatogenic stages I-VI than at other stages. SR-BI was present and functional (in mediating cellular incorporation of lipids of high density lipoprotein) at both the apical and basolateral surfaces of polarized Sertoli cells. Finally, SR-BI expression at both the protein and mRNA levels was stimulated by FSH in cultured Sertoli cells. These results indicate that SR-BI functions on both the apical and basolateral plasma membranes of Sertoli cells, and that SR-BI expression in Sertoli cells changes during the spermatogenic cycle and is stimulated, at least in cultures, by FSH.     

Hydrolysis of third-generation cephalosporins by class C beta-lactamases. Structures of a transition state analog of cefotoxamine in wild-type and extended spectrum enzymes

Bacterial resistance to the third-generation cephalosporins is an issue of great concern in current antibiotic therapeutics. An important source of this resistance is from production of extended-spectrum (ES) beta-lactamases by bacteria. The Enterobacter cloacae GC1 enzyme is an example of a class C ES beta-lactamase. Unlike wild-type (WT) forms, such as the E. cloacae P99 and Citrobacter freundii enzymes, the ES GC1 beta-lactamase is able to rapidly hydrolyze third-generation cephalosporins such as cefotaxime and ceftazidime. To understand the basis for this ES activity, m-nitrophenyl 2-(2-aminothiazol-4-yl)-2-[(Z)-methoxyimino]acetylaminomethyl phosphonate has been synthesized and characterized. This phosphonate was designed to generate a transition state analog for turnover of cefotaxime. The crystal structures of complexes of the phosphonate with both ES GC1 and WT C. freundii GN346 beta-lactamases have been determined to high resolution (1.4-1.5 Angstroms). The serine-bound analog of the tetrahedral transition state for deacylation exhibits a very different binding geometry in each enzyme. In the WT beta-lactamase the cefotaxime-like side chain is crowded against the Omega loop and must protrude from the binding site with its methyloxime branch exposed. In the ES enzyme, a mutated Omega loop adopts an alternate conformation allowing the side chain to be much more buried. During the binding and turnover of the cefotaxime substrate by this ES enzyme, it is proposed that ligand-protein contacts and intra-ligand contacts are considerably relieved relative to WT, facilitating positioning and activation of the hydrolytic water molecule. The ES beta-lactamase is thus able to efficiently inactivate third-generation cephalosporins.     

Selection and Characterization of Nickel-Tolerant Tobacco Cells

Tobacco (Nicotiana tabacum L. cv. BY-2) cell lines tolerant to 700 M Ni in which unselected cells can not grow, were selected. The Ni-tolerant cells were also more tolerant to Co, but not to Cd than unselected cells. Ni concentrations in Ni-tolerant cells were always higher than those in medium. Since buthionine sulfoximine did not affect their Ni-tolerance, it is suggested that phytochelatins are not involved in Ni-tolerance of Ni-tolerant cells. On the other hand, histidine contents in Ni-tolerant and unselected cells, which were treated with Ni, were higher that those treated without Ni, and the degree of the elevation of histidine contents by Ni-treatment was higher in Ni-tolerant cells than in unselected cells. Additionally, exogenous histidine reduced the inhibitory effect of Ni on the growth of unselected cells. In addition, the cells that were tolerant to histidine-analogue, had higher contents of histidine and Ni-tolerance. These results suggest that histidine is involved in Ni-tolerance and the detoxification of Ni in symplast in Ni-tolerant cells.     

Cooperation of the N-terminal Helicase and C-terminal endonuclease activities of Archaeal Hef protein in processing stalled replication forks

Blockage of replication fork progression often occurs during DNA replication, and repairing and restarting stalled replication forks are essential events in all organisms for the maintenance of genome integrity. The repair system employs processing enzymes to restore the stalled fork. In Archaea Hef is a well conserved protein that specifically cleaves nicked, flapped, and fork-structured DNAs. This enzyme contains two distinct domains that are similar to the DEAH helicase family and XPF nuclease superfamily proteins. Analyses of truncated mutant proteins consisting of each domain revealed that the C-terminal nuclease domain independently recognized and incised fork-structured DNA. The N-terminal helicase domain also specifically unwound fork-structured DNA and Holliday junction DNA in the presence of ATP. Moreover, the endonuclease activity of the whole Hef protein was clearly stimulated by ATP hydrolysis catalyzed by the N-terminal domain. These enzymatic properties suggest that Hef efficiently resolves stalled replication forks by two steps, which are branch point transfer to the 5'-end of the nascent lagging strand by the N-terminal helicase followed by template strand incision for leading strand synthesis by the C-terminal endonuclease.     

Inhibition of hepatocyte growth factor induction in human dermal fibroblasts by interleukin-1 and its prevention by interferon-gamma

Hepatocyte growth factor (HGF) is one of the vital factors for liver regeneration. HGF production is induced by the activation of protein kinase A and protein kinase C-mediated pathways, interleukin (IL)-1, tumor necrosis factor (TNF)-alpha, and epidermal growth factor (EGF) in mesenchymal cells. We here report that IL-1 and TNF-alpha, hitherto regarded as HGF inducers, potently inhibited HGF production stimulated by other HGF inducers. IL-1alpha, IL-1beta, and TNF-alpha alone had minimal stimulating effects on HGF production in human dermal fibroblasts, but they strongly inhibited production of HGF induced by cholera toxin, 8-bromo-cAMP, EGF, and phorbol 12-myristate 13-acetate (PMA). Moreover, although the high level of HGF production in MRC-5 cells was enhanced by PMA and less markedly by IL-1beta, HGF production in MRC-5 cells treated with PMA plus IL-1beta was less than that in the cells treated with PMA alone. In the presence of interferon (IFN)-gamma, however, cholera toxin- and 8-bromo-cAMP-induced HGF production was not inhibited by IL-1beta. Pretreatment of cells with IL-1beta suppressed the phosphorylation of cAMP-responsive element-binding protein induced by cholera toxin but not that induced by 8-bromo-cAMP. Taken together, our results indicate that IL-1 inhibited HGF production stimulated by various inducers, including protein kinase A-activating agents, and that IFN-gamma overcame this inhibition of induction of HGF production.     

Major and minor capsid proteins of human polyomavirus JC cooperatively accumulate to nuclear domain 10 for assembly into virions

The human polyomavirus JC (JCV) replicates in the nuclei of infected cells. Here we report that JCV virions are efficiently assembled at nuclear domain 10 (ND10), which is also known as promyelocytic leukemia (PML) nuclear bodies. The major capsid protein VP1, the minor capsid proteins VP2 and VP3, and a regulatory protein called agnoprotein were coexpressed from a polycistronic expression vector in COS-7 cells. We found that VP1 accumulated to distinct subnuclear domains in the presence of VP2/VP3 and agnoprotein, while VP1 expressed alone was distributed both in the cytoplasm and in the nucleus. Mutation analysis revealed that discrete intranuclear accumulation of VP1 requires the presence of either VP2 or VP3. However, VP2 or VP3 expressed in the absence of VP1 showed diffuse, not discrete, nuclear localization. The C-terminal sequence of VP2/VP3 contains two basic regions, GPNKKKRRK (cluster 1) and KRRSRSSRS (cluster 2). The deletion of cluster 2 abolished the accumulation of VP1 to distinct subnuclear domains. Deletion of the C-terminal 34 residues of VP2/VP3, including both cluster 1 and cluster 2, caused VP1 to localize both in the cytoplasm and in the nucleus. Using immunoelectron microscopy of cells that coexpressed VP1, VP2/VP3, and agnoprotein, we detected the assembly of virus-like particles in discrete locations along the inner nuclear periphery. Both in oligodendrocytes of the human brain and in transfected cells, discrete nuclear domains for VP1 accumulation were identified as ND10, which contains the PML protein. These results indicate that major and minor capsid proteins cooperatively accumulate in ND10, where they are efficiently assembled into virions.     

DHEAS improves learning and memory in aged SAMP8 mice but not in diabetic mice

Dehydroepiandrosterone sulfate (DHEAS) has been reported to improve memory in aged animals and suggested as a treatment for age-related dementias. The SAMP8 mouse, a model of Alzheimer's disease, has an age-related impairment in learning and memory and an increase in brain levels of amyloid precursor protein (APP) and amyloid beta protein (Abeta). Male SAMP8 mice also have a decrease in testosterone, to which DHEA is a precursor. Diabetes has been suggested as a model of aging and to be linked to Alzheimer's disease. Diabetics can have memory deficits and lower DHEAS levels. Here, we examined the effects of chronic oral DHEAS on acquisition and retention for T-maze footshock avoidance in 12 mo male SAMP8 mice and in CD-1 mice with streptozocin-induced diabetes. Learning and memory were improved in aged SAMP8 mice, but not in CD-1 mice with streptozocin-induced diabetes. These findings suggest that DHEAS is more effective in reversing the cognitive impairments associated with overexpression of Abeta than with diabetes.     

Upregulation of P2Y2 receptors by retinoids in normal human epidermal keratinocytes

Retinoids, vitamin A derivatives, are important regulators of the growth and differentiation of skin cells. Although retinoids are therapeutically used for several skin ailments, little is known about their effects on P2 receptors, known to be involved in various functions in the skin. DNA array analysis showed that treatment of normal human epidermal keratinocytes (NHEKs) with all-trans-retinoic acid (ATRA), an agonist to RAR (retinoic acid receptor), enhanced the expression of mRNA for the P2Y2 receptor, a metabotropic P2 receptor that is known to be involved in the proliferation of the epidermis. The expression of other P2 receptors in NHEKs was not affected by ATRA. ATRA increased the mRNA for the P2Y2 receptor in a concentration-dependent fashion (1 nM to 1 μM). Am80, a synthesized agonist to RAR, showed a similar enhancement, whereas 9-cis-retinoic acid (9-cisRA), an agonist to RXR (retinoid X receptor), enhanced P2Y2 gene expression to a lesser extent. Ca²⁺ imaging analysis showed that ATRA also increased the function of P2Y2 receptors in NHEKs. Retinoids are known to enhance the turnover of the epidermis by increasing both proliferation and terminal differentiation. The DNA microarray analysis also revealed that ATRA upregulates various genes involved in the differentiation of NHEKs. Our present results suggest that retinoids, at least in part, exert their proliferative effects by upregulating P2Y2 receptors in NHEKs. This effect of retinoids may be closely related to their therapeutic effect against various ailments or aging events in skins such as over-keratinization, pigmentation and re-modeling.     

Evaluation of mutation effects on UGT1A1 activity toward 17beta-estradiol using liquid chromatography-tandem mass spectrometry

Mutations in the gene encoding UDP-glucuronosyltransferase 1A1 (UGT1A1) may reduce the glucuronidation of estradiol, bilirubin, etc. In the present study, we used a liquid chromatography-tandem mass spectrometry (LC/MS/MS) method to assay the activities of recombinant mutated UGT1A1 toward 17beta-estradiol (E2), by determining its glucuronide (E2G) content. Direct evidence for glucuronide formation was provided by E2G-specific ion peaks. The UGT1A1 activities of G71R (exon 1), F83L (exon 1), I322V (exon 2) and G493R (exon 5) mutants were 24, 30, 18 and 0.6% of the normal UGT1A1 activity, respectively. In conclusion, our study showed that LC/MS/MS enabled accurate evaluation of the effects of mutations on recombinant UGT1A1 activity towards E2.     

The Middle Region of an HP1-binding Protein, HP1-BP74, Associates with Linker DNA at the Entry/Exit Site of Nucleosomal DNA

In higher eukaryotic cells, DNA molecules are present as chromatin fibers, complexes of DNA with various types of proteins; chromatin fibers are highly condensed in metaphase chromosomes during mitosis. Although the formation of the metaphase chromosome structure is essential for the equal segregation of replicated chromosomal DNA into the daughter cells, the mechanism involved in the organization of metaphase chromosomes is poorly understood. To identify proteins involved in the formation and/or maintenance of metaphase chromosomes, we examined proteins that dissociated from isolated human metaphase chromosomes by 0.4 m NaCl treatment; this treatment led to significant chromosome decondensation, but the structure retained the core histones. One of the proteins identified, HP1-BP74 (heterochromatin protein 1-binding protein 74), composed of 553 amino acid residues, was further characterized. HP1-BP74 middle region (BP74Md), composed of 178 amino acid residues (Lys⁹⁷–Lys²⁷⁴), formed a chromatosome-like structure with reconstituted mononucleosomes and protected the linker DNA from micrococcal nuclease digestion by ∼25 bp. The solution structure determined by NMR revealed that the globular domain (Met¹⁵³–Thr²³⁷) located within BP74Md possesses a structure similar to that of the globular domain of linker histones, which underlies its nucleosome binding properties. Moreover, we confirmed that BP74Md and full-length HP1-BP74 directly binds to HP1 (heterochromatin protein 1) and identified the exact sites responsible for this interaction. Thus, we discovered that HP1-BP74 directly binds to HP1, and its middle region associates with linker DNA at the entry/exit site of nucleosomal DNA in vitro.