Simultaneous regulation of lactate production and fatty acid metabolism by Resveratrol in rat Sertoli cells.

Sertoli cells provide structural and nutritional support for germ cell development. They actively metabolize glucose and convert it into lactate, which is an important source of energy for germ cells. They also oxidize fatty acids (FA), stored as triacylglycerides (TAGs) within lipid droplets (LD), to fulfill their own energy requirements. So, the combined regulation of lactate production and FA metabolism may be relevant to the physiology of seminiferous tubules. Resveratrol (RSV) is a nutritional supplement found primarily in red grape skin that exhibits multiple beneficial health effects: it is cardioprotective, anti-inflammatory, anticancer, and antiaging. The aim of this study was to evaluate the effect of RSV in Sertoli cells lactate production and lipid metabolism. Sertoli cell cultures obtained from 20-day-old rats were incubated for different times with 10 or 50 µM RSV. RSV treatment increased lactate production and glucose consumption. These increments were accompanied by a rise in GLUT1 expression, which is the main glucose transporter in Sertoli cells. On the other hand, RSV decreased LD content and TAG levels. In addition, an increase in ATGL and FAT/CD36 mRNA levels was observed, which suggests augmented cytoplasmatic FA availability. RSV treatment also increased P-ACC levels, which might indicate that RSV promotes FA transport into the mitochondria to be oxidized. An enhanced expression of LCAD and MCAD, enzymes that participate in the oxidation of FA, was also observed. Altogether, these results suggest that RSV simultaneously regulates Sertoli cells lactate production and lipid metabolism, ensuring an adequate energetic balance both in germ and Sertoli cells.

Bora phosphorylation substitutes in trans for T-loop phosphorylation in Aurora A to promote mitotic entry.

Polo-like kinase 1 (Plk1) is instrumental for mitotic entry and progression. Plk1 is activated by phosphorylation on a conserved residue Thr210 in its activation segment by the Aurora A kinase (AURKA), a reaction that critically requires the co-factor Bora phosphorylated by a CyclinA/B-Cdk1 kinase. Here we show that phospho-Bora is a direct activator of AURKA kinase activity. We localize the key determinants of phospho-Bora function to a 100 amino acid region encompassing two short Tpx2-like motifs and a phosphoSerine-Proline motif at Serine 112, through which Bora binds AURKA. The latter substitutes in trans for the Thr288 phospho-regulatory site of AURKA, which is essential for an active conformation of the kinase domain. We demonstrate the importance of these determinants for Bora function in mitotic entry both in Xenopus egg extracts and in human cells. Our findings unveil the activation mechanism of AURKA that is critical for mitotic entry.

Adaptation and evaluation of polymerase chain reaction for Brucella ovis detection in semen, urine and organs of rams experimentally infected / Adaptação e avaliação da reação em cadeia da polimerase para detecção de Brucella ovis em sêmen, urina e órgãos de carneiros infectados experimentalmente

O objetivo do estudo foi adaptar e avaliar a PCR para detecção de Brucella ovis e comparar os resultados com aqueles obtidos por cultivo microbiológico do sêmen, urina e dos órgãos de carneiros infectados experimentalmente. Dos 31 animais infectados experimentalmente, amostras de PCR do sêmen apresentaram maior sensibilidade (21,6 por cento) do que o cultivo (8,0 por cento). Em amostras de urina, a sensibilidade das técnicas foi semelhante (10,1 por cento para a cultivo e 12,7 por cento para PCR). PCR detectou a presença do agente em 21,5 por cento das amostras testadas, enquanto os órgãos de cultivo detectaram em apenas 3,3 por cento das amostras. PCR detectou um maior número de amostras positivas do que o cultivo microbiológico.

A comparison of two agar gel immunodiffusion methods and a complement fixation test for serologic diagnosis of Brucella ovis infection in experimentally infected rams / Comparação entre dois métodos de imunodifusão em gel de Agar e um método de fixação de complemento para o diagnóstico sorológico da infecção por Brucella ovis em carneiros experimentalmente infectados

A infecção por Brucella ovis é considerada uma das principais causas de epididimite e infertilidade em carneiros, resultando em falhas reprodutivas e perdas econômicas significativas em rebanhos ovinos ao redor do mundo. O estudo teve o objetivo de avaliar três testes sorológicos disponíveis para o diagnóstico da brucelose ovina por B. ovis, utilizando 181 soros ovinos. Amostras de soro provenientes de carneiros experimentalmente infectados foram coletadas ao longo de 192 dias pós-infecção (n=117) e durante o período pré-infecção (n=9). Adicionalmente, amostras de soro foram obtidas de ovinos provenientes de um rebanho livre para B. ovis (n=55). As técnicas de imunodifusão em gel de agar (IDGA), utilizando dois antígenos disponíveis comercialmente, e de fixação de complemento foram comparadas (FC). Foram obtidos resultados de sensibilidade especificidade semelhantes para ambos os métodos de IDGA e ainda, a técnica de IDGA foi mais eficiente do que a da FC para o diagnóstico sorológico da infecção por B. ovis.

[Assembly of correct kinetochore architecture in Xenopus laevis egg extract requires transition of sperm DNA through interphase].

Xenopus egg extracts provide a powerful tool for studying formation and function of chromosomes. Two alternative protocols are generally used to obtain mitotic chromosomes. The first one employs direct assembly of chromatin from sperm nuclei in CSF-arrested meiotic extracts, while the second is based on transition of sperm DNA through a replication step, followed by re-establishing of CSF arrest. In this study we show that general kinetochore structure is disrupted in chromosomes assembled directly in CSF egg extracts: the amounts of outer kinetochore proteins such as Bub1, BubR1 and Dynactin subunit p150glued are reduced and the components of the inner centromeric region (Aurora B kinase and Survivin) show compromised recruitment to centromeres. In contrast, kinetochores on chromosomes assembled according to the second protocol closely resemble those in somatic cells. Our results argue that transition of sperm nuclei through interphase is an essential step for proper kinetochore assembly.

Ran at kinetochores.

The Ran GTPase controls many cellular functions, including nucleocytoplasmic trafficking, spindle assembly, nuclear assembly and cell-cycle progression. Considerable evidence suggests that diffusible Ran-GTP near mitotic chromatin facilitates the release of critical factors from nuclear transport receptors, thereby promoting organization of mitotic spindles with respect to chromosomes. In addition to this role of soluble Ran-GTP, Ran has two important but less understood roles at mitotic kinetochores. Namely, it is essential for regulation of the spindle assembly checkpoint and for assembly of microtubule fibres that attach kinetochores to spindle poles. Here, I will briefly summarize evidence for these kinetochore-associated functions and mention some of the issues that remain to be addressed regarding them.

The promyelocytic leukemia protein stimulates SUMO conjugation in yeast.

The promyelocytic leukemia gene was first identified through its fusion to the gene encoding the retinoic acid receptor alpha (RARalpha) in acute promyelocytic leukemia (APL) patients. The promyelocytic leukemia gene product (PML) becomes conjugated in vivo to the small ubiquitin-like protein SUMO-1, altering its behavior and capacity to recruit other proteins to PML nuclear bodies (PML-NBs). In the NB4 cell line, which was derived from an APL patient and expresses PML:RARalpha, we observed a retinoic acid-dependent change in the modification of specific proteins by SUMO-1. To dissect the interaction of PML with the SUMO-1 modification pathway, we used the budding yeast Saccharomyces cerevisiae as a model system through expression of PML and human SUMO-1 (hSUMO-1). We found that PML stimulated hSUMO-1 modification in yeast, in a manner that was dependent upon PML's RING-finger domain. PML:RARalpha also stimulated hSUMO-1 conjugation in yeast. Interestingly, however, PML and PML:RARalpha differentially complemented yeast Smt3p conjugation pathway mutants. These findings point toward a potential function of PML and PML:RARalpha as SUMO E3 enzymes or E3 regulators, and suggest that fusion of RARalpha to PML may affect this activity.

Partial characterization of a human submandibular/sublingual salivary adhesion-promoting protein.

Human submandibular/sublingual saliva contains a protein that promotes adhesion of Streptococcus mutans JBP serotype-c to spheroidal hydroxyapatite in vitro. A high molecular-weight (250,000-300,000 Da) adhesion-promoting protein (APP) was purified by Trisacryl 2000 M gel-filtration chromatography and gel electroelution before it was partially characterized. Lectin blotting identified that the terminal carbohydrates include N-acetyl glucosamine-beta 1-4-N-acetylglucosamine, galactose and galactose-beta 1-3-N-acetyl galactosamine. Antibodies to APP demonstrated no difference in the immunoreactive pattern of APP from saliva of caries-active or caries-resistant individuals belonging to four different ethnic groups: Asian, African-American, Hispanic or Caucasian. No immunological similarities to salivary mucins or parotid agglutinins were detected by Western blotting using immuno-cross-reactivity as a criterion. APP appears to be a unique protein found in submandibular/sublingual saliva. Understanding such a protein could help prevent S. mutans attachment to the enamel surface.

Bitter taste transduced by PLC-beta(2)-dependent rise in IP(3) and alpha-gustducin-dependent fall in cyclic nucleotides.

Current evidence points to the existence of multiple processes for bitter taste transduction. Previous work demonstrated involvement of the polyphosphoinositide system and an alpha-gustducin (Galpha(gust))-mediated stimulation of phosphodiesterase in bitter taste transduction. Additionally, a taste-enriched G protein gamma-subunit, Ggamma(13), colocalizes with Galpha(gust) and mediates the denatonium-stimulated production of inositol 1,4,5-trisphosphate (IP(3)). Using quench-flow techniques, we show here that the bitter stimuli, denatonium and strychnine, induce rapid (50-100 ms) and transient reductions in cAMP and cGMP and increases in IP(3) in murine taste tissue. This decrease of cyclic nucleotides is inhibited by Galpha(gust) antibodies, whereas the increase in IP(3) is not affected by antibodies to Galpha(gust). IP(3) production is inhibited by antibodies specific to phospholipase C-beta(2) (PLC-beta(2)), a PLC isoform known to be activated by Gbetagamma-subunits. Antibodies to PLC-beta(3) or to PLC-beta(4) were without effect. These data suggest a transduction mechanism for bitter taste involving the rapid and transient metabolism of dual second messenger systems, both mediated through a taste cell G protein, likely composed of Galpha(gust)/beta/gamma(13), with both systems being simultaneously activated in the same bitter-sensitive taste receptor cell.

The role of Ran in nuclear function.

There have been three major advances in the understanding of the Ran pathway during the past two years: first, a general model for Ran's function in nuclear transport has been proposed and extensively tested. Second, crystal structures for many proteins that regulate or interact with Ran have been reported, which provide molecular details of how Ran works. Third, it has been documented that Ran regulates mitotic spindle assembly in a transport-independent fashion.

The ran decathlon: multiple roles of Ran.

The Ran GTPase system affects many cellular processes, including the regulation of cell cycle progression, nuclear envelope structure and function, and nucleocytoplasmic transport. The biochemical basis for the involvement of Ran in nuclear import and export has been well documented, but the direct targets of Ran in other cellular processes have not yet been identified. There is, however, mounting evidence that Ran directly affects at least some of these other cellular processes by mechanisms independent of its role in transport. In this Commentary we discuss evidence linking Ran to different aspects of cell function, and how these multiple facets of Ran's activity may relate to each other.

The ran GTPase regulates mitotic spindle assembly.

Ran is an abundant nuclear GTPase with a clear role in nuclear transport during interphase but with roles in mitotic regulation that are less well understood. The nucleotide-binding state of Ran is regulated by a GTPase activating protein, RanGAP1, and by a guanine nucleotide exchange factor, RCC1. Ran also interacts with a guanine nucleotide dissociation inhibitor, RanBP1. RanBP1 has a high affinity for GTP-bound Ran, and it acts as a cofactor for RanGAP1, increasing the rate of GAP-mediated GTP hydrolysis on Ran approximately tenfold. RanBP1 levels oscillate during the cell cycle [4], and increased concentrations of RanBP1 prolong mitosis in mammalian cells and in Xenopus egg extracts (our unpublished observations). We investigated how increased concentrations of RanBP1 disturb mitosis. We found that spindle assembly is dramatically disrupted when exogenous RanBP1 is added to M phase Xenopus egg extracts. We present evidence that the role of Ran in spindle assembly is independent of nuclear transport and is probably mediated through changes in microtubule dynamics.

Special-interest subgroups at the ASCB: Are there multiple roles for the Ran GTPase?

The annual meeting of the American Society for Cell Biology (ASCB) is a large and diverse gathering. At last year's meeting**The American Society for Cell Biology 38th Annual Meeting, San Francisco, USA; 12-16 December, 1998. Program chair: Jennifer Lippincott-Schwartz., there were over 8000 attendees, and the topics discussed covered many areas of cell biology. It would be impossible to cover the entire meeting within a trends in CELL BIOLOGY report, so instead we are focusing on an aspect of it that provided some of the most interesting and fruitful discussions. On Saturday afternoon, before the main symposia began, there were 11 special-interest subgroup meetings. The atmosphere at these meetings was informal, and they encouraged open and frank discussion of data and issues. This report provides a brief summary of the discussions at seven of the special-interest subgroup meetings.

Possible novel mechanism for bitter taste mediated through cGMP.

Taste is the least understood among sensory systems, and bitter taste mechanisms pose a special challenge because they are elicited by a large variety of compounds. We studied bitter taste signal transduction with the quench-flow method and monitored the rapid kinetics of the second messenger guanosine 3',5'-cyclic monophosphate (cGMP) production and degradation in mouse taste tissue. In response to the bitter stimulants, caffeine and theophylline but not strychnine or denatonium cGMP levels demonstrated a rapid and transient increase that peaked at 50 ms and gradually declined throughout the following 4.5 s. The theophylline- and caffeine-induced effect was rapid, transient, concentration dependent and gustatory tissue-specific. The effect could be partially suppressed in the presence of the soluble guanylyl cyclase (GC) inhibitor 10 microM ODQ and 30 microM methylene blue but not 50 microM LY 83583 and boosted by nitric oxide donors 25 microM NOR-3 or 100 microM sodium nitroprusside. The proposed mechanism for this novel cGMP-mediated bitter taste signal transduction is cGMP production partially by the soluble GC and caffeine-induced inhibition of one or several phosphodiesterases.

The hCSE1/CAS protein is phosphorylated by HeLa extracts and MEK-1: MEK-1 phosphorylation may modulate the intracellular localization of CAS.

hCSE1/CAS (CAS), the human homologue of the yeast chromosome segregation gene CSE1, is a nuclear transport factor that plays a role in proliferation and apoptosis. A MEK-1 phosphorylation sequence in CAS raises the possibility that MEK-phosphorylation regulates the function of CAS. CAS protein from cell extracts shows covalent charge modifications; one of these charge variants contains phosphotyrosine. CAS protein can be captured from cell extracts by immobilized anti-phosphotyrosine antibodies. We have produced recombinant protein fragments containing the N-terminal or central portion of CAS and found that the N-terminal fragment, which contains a putative MEK phosphorylation site, is phosphorylated by the HeLa extracts and MEK-1. Treatment of cells with an inhibitor of MEK-1 phosphorylation in vivo changes the intracellular localization of CAS from predominantly cytoplasmic to nuclear. This suggests that a function of CAS in nuclear transport may be regulated by phosphorylation.

The disruption of ND10 during herpes simplex virus infection correlates with the Vmw110- and proteasome-dependent loss of several PML isoforms.

The small nuclear structures known as ND10 or PML nuclear bodies have been implicated in a variety of cellular processes including response to stress and interferons, oncogenesis, and viral infection, but little is known about their biochemical properties. Recently, a ubiquitin-specific protease enzyme (named HAUSP) and a ubiquitin-homology family protein (PIC1) have been found associated with ND10. HAUSP binds strongly to Vmw110, a herpesvirus regulatory protein which has the ability to disrupt ND10, while PIC1 was identified as a protein which interacts with PML, the prototype ND10 protein. We have investigated the role of ubiquitin-related pathways in the mechanism of ND10 disruption by Vmw110 and the effect of virus infection on PML stability. The results show that the disruption of ND10 during virus infection correlates with the loss of several PML isoforms and this process is dependent on active proteasomes. The PML isoforms that are most sensitive to virus infection correspond closely to those which have recently been identified as being covalently conjugated to PIC1. In addition, a large number of PIC1-protein conjugates can be detected following transfection of a PIC1 expression plasmid, and many of these are also eliminated in a Vmw110-dependent manner during virus infection. These observations provide a biochemical mechanism to explain the observed effects of Vmw110 on ND10 and suggest a simple yet powerful mechanism by which Vmw110 might function during virus infection.

Ubc9p and the conjugation of SUMO-1 to RanGAP1 and RanBP2.

The yeast UBC9 gene encodes a protein with homology to the E2 ubiquitin-conjugating enzymes that mediate the attachment of ubiquitin to substrate proteins [1]. Depletion of Ubc9p arrests cells in G2 or early M phase and stabilizes B-type cyclins [1]. p18(Ubc9), the Xenopus homolog of Ubc9p, associates specifically with p88(RanGAP1) and p340(RanBP2) [2]. Ran-binding protein 2 (p340(RanBP2)) is a nuclear pore protein [3] [4], and p88(RanGAP1) is a modified form of RanGAP1, a GTPase-activating protein for the small GTPase Ran [2]. It has recently been shown that mammalian RanGAP1 can be conjugated with SUMO-1, a small ubiquitin-related modifier [5-7], and that SUMO-1 conjugation promotes RanGAP1's interaction with RanBP2 [2,5,6]. Here we show that p18(Ubc9) acts as an E2-like enzyme for SUMO-1 conjugation, but not for ubiquitin conjugation. This suggests that the SUMO-1 conjugation pathway is biochemically similar to the ubiquitin conjugation pathway but uses a distinct set of enzymes and regulatory mechanisms. We also show that p18(Ubc9) interacts specifically with the internal repeat domain of RanBP2, which is a substrate for SUMO-1 conjugation in Xenopus egg extracts.