Evidence that an Arg-Gly-Asp adhesion sequence plays a role in mammalian fertilization

The Arg-Gly-Asp (RGD) sequence is known to play a role in many recognition systems involved in cell-to-cell and cell-to-matrix adhesion. In our experiments we demonstrated that an RGD-dependent recognition is involved in sperm-oolemmal adhesion and egg penetration. Following coincubation of RGD-containing oligopeptides in a heterologous system (human sperm and zona-free hamster eggs), a significant decrease in the number of oolemma-adherent sperm was noted at 15 microM RGDV (Arg-Gly-Asp-Val) and at 5 microM GRGDTP (Gly-Arg-Gly-Asp-Thr-Pro), and fertilization was completely inhibited at 250 microM RGDV and 30 microM GRGDTP. In a homologous system (hamster sperm and zona-free hamster eggs), a concentration-dependent decrease in oolemmal adhesion and egg penetration was also noted, with complete inhibition of fertilization at 200 microM GRGDTP. The specificity of the receptor was confirmed by the fact that small changes in aminoacid composition impaired the peptide's effectiveness and that peptide-dependent inhibition of fertilization was partially reversible in competition studies. The presence of a molecule on the oolemma capable of binding the RGD sequence was demonstrated by using immunobeads coupled with an RGD-containing hexapeptide (GRGDTP), which rosetted over the egg surface in a manner reversible by the addition of free GRGDTP in the medium.     

Characterization of an essential Orc2p-associated factor that plays a role in DNA replication.

The Saccharomyces cerevisiae Orc2 protein is a subunit of the origin recognition complex, ORC, which binds in a sequence-specific manner to yeast origins of DNA replication. With screens for orc2-1 synthetic lethal mutations and Orc2p two-hybrid interactors, a novel Orc2p-associated factor (Oaf1p) was identified. OAF1 is essential, its gene product is localized to the nucleus, and an oaf1 temperature-sensitive mutant arrests as large budded cells with a single nucleus. The mutant oaf1-2, isolated in the synthetic lethal screen, loses plasmids containing a single origin of DNA replication at a high rate, but it maintains plasmids carrying multiple potential origins of DNA replication. In addition, the OAF1 gene product tagged with the hemagglutinin antigen epitope binds to a DNA affinity column containing covalently linked tandem repeats of an essential origin element. These results suggest a role for OAFI in the initiation of DNA replication. Mutant alleles of cdc7 and cdc14 were also isolated in the orc2-1 synthetic lethal screen. Cdc7p, like Oaf1p, also interacts with Orc2p in two-hybrid assays.     

A haloarchaeal ferredoxin electron donor that plays an essential role in nitrate assimilation

In the absence of ammonium, many organisms, including the halophilic archaeon Haloferax volcanii DS2 (DM3757), may assimilate inorganic nitrogen from nitrate or nitrite, using a ferredoxin-dependent assimilatory NO??/NO?? reductase pathway. The small acidic ferredoxin Hv-Fd plays an essential role in the electron transfer cascade required for assimilatory nitrate and nitrite reduction by the cytoplasmic NarB- and NirA-type reductases respectively. UV-visible absorbance and EPR spectroscopic characterization of purified Hv-Fd demonstrate that this protein binds a single [2Fe-2S] cluster, and potentiometric titration reveals that the cluster shares similar redox properties with those present in plant-type ferredoxins.     

Sucrose may play an additional role to that of an osmolyte in Synechocystis sp. PCC 6803 salt-shocked cells.

The role of sucrose in cyanobacteria is still not fully understood. It is generally considered a salt-response molecule, and particularly, in Synechocystis sp. strain PCC 6803, it is referred as a secondary osmolyte. We showed that sucrose accumulates transiently in Synechocystis cells at early stages of a salt shock, which could be ascribed to salt activation of sucrose-phosphate synthase (SPS, UDP-glucose: D-fructose-6-phosphate 2-alpha-D-glucosyltransferase; EC 2.4.1.14), the key enzyme in sucrose synthesis pathway, and to an increase of the expression of the SPS encoding gene. Experiments with a mutant strain impaired in sucrose biosynthesis showed that sucrose is essential in stationary phase cells to overcome a later salt stress. Taken together, these results led us to suggest a more intricate function for sucrose than to be an osmoprotectant compound.     

A structural protein that plays an enzymatic role in the eggshell of Drosophila melanogaster

E.S.P. is responsible for the hardening process of the egg-shell at the end of oogenesis (stage 14B) and constitutes a structural component. By immunoblotting, using polyclonal rabbit anti-HRP antibody and anti-rabbit IgG-HRP or Protein A-1251 as second antibody, one major band with MW 38KD on nitrocellulose filter showed positive reaction. We conclude that the E.S.P. is identical to the S38 chorionic protein. Morphological immunogold staining, using pre-embedding procedure, revealed positive reaction in the innermost chorionic layer (ICL) and the endochorion of the eggshell. In addition, electron probe X-ray microanalysis revealed the existence of 37% calcium (explained since the enzyme is Ca2(+)-activated) and 5% iron (explained due to the fact that it is a haemoprotein).     

Autophagy dysregulation caused by ApoM deficiency plays an important role in liver lipid metabolic disorder

Autophagy is thought to be a key mechanism in maintaining the balance of liver lipid metabolism. However, the relationship between apolipoprotein M (ApoM) and autophagy has not been reported, and the role of ApoM in triglyceride metabolism is still unclear. In this study, we investigated the correlation between ApoM and autophagy and liver triglyceride metabolism in ApoM-knockout animal and cellular models. First, we observed that spontaneous hepatic steatosis developed in the liver of adult ApoM^?/? mice, which was presented as the accumulation of large quantities of lipid droplets in hepatocytes under electron microscopy; Oil Red O staining showed significant accumulation of triglycerides. At the molecular level, the expression of lipid synthesis-associated proteins (primarily triglyceride synthesis) as well as acetyl-CoA carboxylase alpha (ACACA), fatty acid synthase (FASN) and sterol regulatory element-binding protein 1 (SREBP1) was upregulated. Moreover, lipid metabolic disorder and accumulation were accompanied by dysfunction in autophagy, which displayed predominantly as inhibition of the degradation pathway; for example, P62 protein accumulated and key proteins involved in the initiation of autophagy including ATG7, ATG5-12, Beclin1 and the LC3BII/LC3BI ratio were upregulated as a feedback response. When the autophagy dysfunction was ameliorated by the activation of autophagy pathways induced by starvation, the lipid metabolic disorder was corrected to a certain extent. This suggests that the autophagy dysfunction caused by the deficiency of ApoM is an important factor in hepatic steatosis (triglyceride accumulation). ApoM plays a key role in normal autophagy activity in the liver and thereby further regulates the metabolism of liver lipids, particularly triglycerides.     

GDI1 encodes a GDP dissociation inhibitor that plays an essential role in the yeast secretory pathway.

GTP binding proteins of the Sec4/Ypt/rab family regulate distinct vesicular traffic events in eukaryotic cells. We have cloned GDI1, an essential homolog of bovine rab GDI (GDP dissociation inhibitor) from the yeast Saccharomyces cerevisiae. Analogous to the bovine protein, purified Gdi1p slows the dissociation of GDP from Sec4p and releases the GDP-bound form from yeast membranes. Depletion of Gdi1p in vivo leads to loss of the soluble pool of Sec4p and inhibition of protein transport at multiple stages of the secretory pathway. Complementation analysis indicates that GDI1 is allelic to sec19-1. These results establish that Gdi1p plays an essential function in membrane traffic and are consistent with a role for Gdi1p in the recycling of proteins of the Sec4/Ypt/rab family from their target membranes back to their vesicular pools.     

A novel transmembrane MSP-containing protein that plays a role in right ventricle development

We have identified and characterized a gene, Mospd3 on mouse chromosome 5 using gene trapping in ES cells. MOSPD3 is part of a family of proteins, including MOSPD1, which is defined by the presence of a major sperm protein (MSP) domain and two transmembrane domains. Interestingly Mospd3 is mammalian specific and highly conserved between mouse and man. Insertion of the gene trap vector at the Mospd3 locus is mutagenic and breeding to homozygosity results in a characteristic right ventricle defect and neonatal lethality in 50% of mice. The phenotypic defect is dependent on the genetic background, indicating the presence of genetic modifier loci. We speculate that the further characterization of Mospd3 will shed light on the complex genetic interactions involved in cardiac development and disease.     

GPNMB plays a protective role against obesity-related metabolic disorders by reducing macrophage inflammatory capacity

Obesity is a global health problem that is often related to cardiovascular and metabolic diseases. Chronic low-grade inflammation in white adipose tissue (WAT) is a hallmark of obesity. Previously, during a search for differentially expressed genes in WAT of obese mice, we identified glycoprotein nonmetastatic melanoma protein B (GPNMB), of which expression was robustly induced in pathologically expanded WAT. Here, we investigated the role of GPNMB in obesity-related metabolic disorders utilizing GPNMB-deficient mice. When fed a high-fat diet (HFD), GPNMB-deficient mice showed body weight and adiposity similar to those of wild-type (WT) mice. Nonetheless, insulin and glucose tolerance tests revealed significant obesity-related metabolic disorders in GPNMB-KO mice compared with WT mice fed with HFD. Chronic WAT inflammation was remarkably worsened in HFD-fed GPNMB-KO mice, accompanied by a striking increase in crown-like structures, typical hallmarks for diseased WAT. Macrophages isolated from GPNMB-KO mice were observed to produce more inflammatory cytokines than those of WT mice, a difference abolished by supplementation with recombinant soluble GPNMB extracellular domain. We demonstrated that GPNMB reduced the inflammatory capacity of macrophages by inhibiting NF-κB signaling largely through binding to CD44. Finally, we showed that macrophage depletion by addition of clodronate liposomes abolished the worsened WAT inflammation and abrogated the exacerbation of metabolic disorders in GPNMB-deficient mice fed on HFD. Our data reveal that GPNMB negatively regulates macrophage inflammatory capacities and ameliorates the WAT inflammation in obesity; therefore we conclude that GPNMB is a promising therapeutic target for the treatment of metabolic disorders associated with obesity.     

AMP-activated protein kinase plays a role in initiating metabolic rate suppression in goldfish hepatocytes

In the present study, we test the hypothesis that AMP-activated protein kinase (AMPK) initiates metabolic rate suppression in isolated goldfish hepatocytes. To accomplish this, we attempted to pharmacologically activate AMPK in goldfish hepatocytes with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) and the thienopyridone, A769662, to examine the effects of AMPK activation on eukaryotic elongation factor-2 (eEF2), protein synthesis, and cellular oxygen consumption rate ( [(M)\dot]_{\textO 2} \dot{M}_{{{\text{O}}_{ 2} }} ). Goldfish hepatocytes treated with 1 mM AICAR under normoxic conditions (>200 μM O₂) showed a modest but significant 1.1-fold increase in AMPK phosphorylation, a 7.5-fold increase in AMPK activity, a 1.4-fold increase in eEF2 phosphorylation, and a 24% decrease in [(M)\dot]_{\textO 2} \dot{M}_{{{\text{O}}_{ 2} }} . At physiologically relevant [O₂] (<40 μM O₂), the addition of 1 mM AICAR resulted in only a 13% decrease in cellular [(M)\dot]_{\textO 2} \dot{M}_{{{\text{O}}_{ 2} }} with no change in sensitivity to [O₂] as assessed by estimates of cellular P₅₀ and P₉₀ values. The addition of compound C, a general protein kinase inhibitor, after AICAR incubation did not reverse the effects of AICAR on [(M)\dot]_{\textO 2} \dot{M}_{{{\text{O}}_{ 2} }} in normoxia. Treatment of hepatocytes with ≤200 μM A769662 did not affect AMPK activity, AMPK phosphorylation, eEF2 phosphorylation, or cellular [(M)\dot]_{\textO 2} \dot{M}_{{{\text{O}}_{ 2} }} . These data suggest that A769662 is not an activator of AMPK in goldfish hepatocytes. Although our study provides support for the hypothesis that AMPK plays a role in initiating metabolic rate suppression in goldfish hepatocytes, this support must be viewed cautiously because of the known off-target effects of the pharmacological agents used.     

The source of early IFN-gamma that plays a role in Th1 priming

When naive CD4 T cells are primed, they rapidly differentiate into polarized Th1 and/or Th2 phenotypes. A major factor in producing such polarization is the early production of cytokines (IL-12 and IFN-gamma in the case of Th1 cells and IL-4 in the case of Th2 cells). One issue that remains unresolved is the source of the early IFN-gamma that synergizes with IL-12 to fully polarize CD4 T cells into Th1 cells. We have examined this question by injecting mice with anti-CD3 and examining cells from normal and various MHC-knockout mice. We found that IFN-gamma is induced rapidly in a small subset of CD8 T cells. This subset is absent in mice that lack beta2-microglobulin, but not in K(b)D(b)-double-knockout mice, indicating that these CD8 T cells are dependent on nonclassical MHC class Ib molecules. The early burst of IFN-gamma polarizes CD4 T cells toward Th1 cells, in part by stimulating the release of IL-12 from APC. We also use TAP- and CD1-knockout mice to show that such cells are not CD1-restricted NK T cells, nor are they dependent on TAP-1 transport for surface expression of the relevant MHC class Ib molecule. Therefore, they arise on MHC class Ib molecules that do not depend on TAP-1 transporters.     

Studies of embryotoxic mechanisms of niridazole: evidence that oxygen depletion plays a role in dysmorphogenicity

Previous study has shown that niridazole (NDZ) is dysmorphogenic to rat embryos between days 10 and 11 under culture conditions including 5% oxygen. Other studies have found that reductive embryonic biotransformation is required but that covalent binding is not a major basis of this embryotoxicity. In research presented here, NDZ exposure of homogenates prepared from day 10 rat embryos resulted in stimulation of oxygen uptake from incubation media. Further studies showed that a large percentage of this increased oxygen uptake was associated with the generation of superoxide anion radical and hydrogen peroxide. These findings led us to hypothesize that redox cycling forms the basis of the in vitro dysmorphogenicity of NDZ. The basic premise of this hypothesis is that as a result of redox cycling, oxygen is depleted from the sensitive tissues of embryos. In order to investigate it, we devised a technique for carefully controlling and monitoring oxygen tensions in embryo cultures. We found that when oxygen concentrations of 4% were established, a highly significant incidence of asymmetric defects resulted. These defects appeared analogous to those induced by NDZ exposure, consisting of asymmetric necrosis of mesenchymal tissue near the cephalic end of the neural tube and thinning of the neuroepithelium on the right. We concluded that the hypoxia induced by redox cycling of NDZ and related nitroheterocycles represents a major embryotoxic principle of action.     

Mammalian sperm-egg fusion: evidence that epididymal protein DE plays a role in mouse gamete fusion

Rat epididymal protein DE associates with the sperm surface during epididymal maturation and is a candidate molecule for mediating gamete membrane fusion in the rat. Here, we provide evidence supporting a role for DE in mouse sperm-egg fusion. Western blot studies indicated that the antibody against rat protein DE can recognize the mouse homologue in both epididymal tissue and sperm extracts. Indirect immunofluorescence studies using this antibody localized the protein on the dorsal region of the acrosome. Experiments in which zona-free mouse eggs were coincubated with mouse capacitated sperm in the presence of DE showed a significant and concentration-dependent inhibition in the percentage of penetrated eggs, with no effect on either the percentage of oocytes with bound sperm or the number of sperm bound per egg. Immunofluorescence experiments revealed specific DE-binding sites on the fusogenic region of mouse eggs. Because mouse sperm can penetrate zona-free rat eggs, the participation of DE in this interaction was also investigated. The presence of the protein during gamete coincubation produced a significant reduction in the percentage of penetrated eggs, without affecting the binding of sperm to the oolemma. These observations support the involvement of DE in an event subsequent to sperm-egg binding and leading to fusion in both homologous (mouse-mouse) and heterologous (mouse-rat) sperm-egg interaction. The lack of disintegrin domains in DE indicates that the protein interacts with its egg-binding sites through a novel mechanism that does not involve the reported disintegrin-integrin interaction.     

PAR-1 kinase plays an initiator role in a temporally ordered phosphorylation process that confers tau toxicity in Drosophila

Multisite hyperphosphorylation of tau has been implicated in the pathogenesis of neurodegenerative diseases including Alzheimer's disease (AD). However, the phosphorylation events critical for tau toxicity and mechanisms regulating these events are largely unknown. Here we show that Drosophila PAR-1 kinase initiates tau toxicity by triggering a temporally ordered phosphorylation process. PAR-1 directly phosphorylates tau at S262 and S356. This phosphorylation event is a prerequisite for the action of downstream kinases, including glycogen synthase kinase 3 (GSK-3) and cyclin-dependent kinase-5 (Cdk5), to phosphorylate several other sites and generate disease-associated phospho-epitopes. The initiator role of PAR-1 is further underscored by the fact that mutating PAR-1 phosphorylation sites causes a much greater reduction of overall tau phosphorylation and toxicity than mutating S202, one of the downstream sites whose phosphorylation depends on prior PAR-1 action. These findings begin to differentiate the effects of various phosphorylation events on tau toxicity and provide potential therapeutic targets.     

mRNA localization studies suggest that murine FGF-5 plays a role in gastrulation.

During gastrulation in the mouse, the pluripotent embryonic ectoderm cells form the three primary germ layers, ectoderm, mesoderm and endoderm. Little is known about the mechanisms responsible for these processes, but evidence from previous studies in amphibians, as well as expression studies in mammals, suggest that signalling molecules of the Fibroblast Growth Factor (FGF) family may play a role in gastrulation. To determine whether this might be the case for FGF-5 in the mouse embryo, we carried out RNA in situ hybridization studies to determine when and where in the early postimplantation embryo the Fgf-5 gene is expressed. We chose to study this particular member of the FGF gene family because we had previously observed that its pattern of expression in cultures of teratocarcinoma cell aggregates is consistent with the proposal that Fgf-5 plays a role in gastrulation in vivo. The results reported here show that Fgf-5 expression increases dramatically in the pluripotent embryonic ectoderm just prior to gastrulation, is restricted to the cells forming the three primary germ layers during gastrulation, and is not detectable in any cells in the embryo once formation of the primary germ layers is virtually complete. Based on this provocative expression pattern and in light of what is known about the functions in vitro of other members of the FGF family, we hypothesize that in the mouse embryo Fgf-5 functions in an autocrine manner to stimulate the mobility of the cells that contribute to the embryonic germ layers or to render them competent to respond to other inductive or positional signals.     

Identification of the region that plays an important role in determining antibacterial activity of bovine seminalplasmin

Seminalplasmin (SPLN) is a 47-residue protein isolated from bovine seminal plasma having potent antimicrobial activity against a broad spectrum of microorganisms. SPLN, also known as caltrin, acts as a calcium transport regulator in bovine sperms. Analysis of the sequence of SPLN reveals a 27-residue stretch with the sequence SLSRYAKLANRLANPKLLETFLSKWIG more hydrophobic than the rest of the protein. It is demonstrated that a synthetic peptide corresponding to this 27-residue segment has antimicrobial activity comparable to that of SPLN. It does not exhibit hemolytic activity at concentrations where antibacterial activity is observed. Since P27 can be conveniently obtained in large amounts by chemical synthesis, it could serve not only as a starting compound to obtain peptides with improved antibacterial activity but also to understand the role of SPLN in reproductive physiology.     

Upregulated miR-106a plays an oncogenic role in pancreatic cancer

Carcinogenesis is a complex process during which cells undergo genetic and epigenetic alterations. MicroRNAs control gene expression by negatively regulating protein-coding mRNAs. Several reports demonstrated that miR-106a is up-regulated in gastric and colorectal cancers and promotes tumor progression. In contrast, in glioma miR-106a plays the role of a tumor suppressor gene rather than an oncogene. Here we demonstrate that a high level of miR-106a expression is present in pancreatic cancer. Furthermore, our investigation shows that miR-106a has an oncogenic role in pancreatic tumorigenesis by promoting cancer cell proliferation, epithelial–mesenchymal transition and invasion by targeting tissue inhibitors of metalloproteinase 2 (TIMP-2). MiR-106a could be a critical therapeutic target in pancreatic cancer.     

RNA-mediated interference indicates that SEL1L plays a role in pancreatic beta-cell growth

The specificity of SEL1L expression and promoter activity for the pancreatic cell population, its chromosomal location, as well as its similarities to the yeast Hrd3p protein, a component of HRD complex which is responsible for endoplasmic reticulum (ER)-associated degradation of numerous ER-resident proteins, prompted us to study its effects on beta cell function. In this study we show that lowering SEL1L expression, by using the short interfering RNAs technology as well as antisense transfection, resulted in severe perturbation of betaTC-3 growth and metabolic activity. We hypothesize that SEL1L may exert its function by protecting the cells from ER stress and could counteract immune responses.     

RLIP76, a glutathione-conjugate transporter, plays a major role in the pathogenesis of metabolic syndrome

Purpose

Characteristic hypoglycemia, hypotriglyceridemia, hypocholesterolemia, lower body mass, and fat as well as pronounced insulin-sensitivity of RLIP76^−/− mice suggested to us the possibility that elevation of RLIP76 in response to stress could itself elicit metabolic syndrome (MSy). Indeed, if it were required for MSy, drugs used to treat MSy should have no effect on RLIP76^−/− mice.

Research Design and Methods

Blood glucose (BG) and lipid measurements were performed in RLIP76^+/+ and RLIP76^−/− mice, using Ascensia Elite Glucometer® for glucose and ID Labs kits for cholesterol and triglycerides assays. The ultimate effectors of gluconeogenesis are the three enzymes: PEPCK, F-1,6-BPase, and G6Pase, and their expression is regulated by PPARγ and AMPK. The activity of these enzymes was tested by protocols standardized by us. Expressions of RLIP76, PPARα, PPARγ, HMGCR, pJNK, pAkt, and AMPK were performed by Western-blot and tissue staining.

Results

The concomitant activation of AMPK and PPARγ by inhibiting transport activity of RLIP76, despite inhibited activity of key glucocorticoid-regulated hepatic gluconeogenic enzymes like PEPCK, G6Pase and F-1,6-BP in RLIP76^−/− mice, is a salient finding of our studies. The decrease in RLIP76 protein expression by rosiglitazone and metformin is associated with an up-regulation of PPARγ and AMPK.

Conclusions/Significance

All four drugs, rosiglitazone, metformin, gemfibrozil and atorvastatin failed to affect glucose and lipid metabolism in RLIP76^−/− mice. Studies confirmed a model in which RLIP76 plays a central role in the pathogenesis of MSy and RLIP76 loss causes profound and global alterations of MSy signaling functions. RLIP76 is a novel target for single-molecule therapeutics for metabolic syndrome.     

Adenovirus 2 Ip+ locus codes for a 19 kd tumor antigen that plays an essential role in cell transformation

Adenovirus 2 large plaque (Ip) mutants produce large clear plaques on human KB cells. These mutants are shown to be defective in inducing transformation of the established rat embryo cell line 3Y1. The Ip mutation was localized within one of the two transforming early gene blocks, E1b (map position 4.5 to 11.2) which codes for two major T antigens of 53 kd and 19 kd by marker transfer. The mutational defects in mutants Ip3 and Ip5 were analyzed by DNA sequence analysis and by analysis of viral E1 proteins. These results reveal that Ip3 and Ip5 mutations map within the 19 kd tumor antigen coding region. Mutant Ip3 has a single base pair change at the N terminus of 19 kd polypeptide, resulting in the substitution of valine for alanine. Mutant Ip5 has two mutational changes, one of which results in the substitution of tyrosine for aspartic acid near the N-terminal region. The second mutation changes the termination codon into a leucine codon, increasing the size of the 19 kd tumor antigen. These results provide direct genetic evidence for an essential role of the 19 kd tumor antigen in cell transformation and indicate that the N-terminal region of the 19 kd tumor antigen is an essential function domain for the induction of cell transformation.