Paracrine effects of oocyte secreted factors and stem cell factor on porcine granulosa and theca cells <Emphasis Type="Italic">in vitro</Emphasis>

Oocyte control of granulosa and theca cell function may be mediated by several growth factors via a local feedback loop(s) between these cell types. This study examined both the role of oocyte-secreted factors on granulosa and thecal cells, cultured independently and in co-culture, and the effect of stem cell factor (SCF); a granulosa cell derived peptide that appears to have multiple roles in follicle development. Granulosa and theca cells were isolated from 2–6 mm healthy follicles of mature porcine ovaries and cultured under serum-free conditions, supplemented with: 100 ng/ml LR3 IGF-1, 10 ng/ml insulin, 100 ng/ml testosterone, 0–10 ng/ml SCF, 1 ng/ml FSH (granulosa), 0.01 ng/ml LH (theca) or 1 ng/ml FSH and 0.01 ng/ml LH (co-culture) and with/without oocyte conditioned medium (OCM) or 5 oocytes. Cells were cultured in 96 well plates for 144 h, after which viable cell numbers were determined. Medium was replaced every 48 h and spent medium analysed for steroids.     

The cell shape proteins MreB and MreC control cell morphogenesis by positioning cell wall synthetic complexes

MreB, the bacterial actin homologue, is thought to function in spatially co-ordinating cell morphogenesis in conjunction with MreC, a protein that wraps around the outside of the cell within the periplasmic space. In Caulobacter crescentus, MreC physically associates with penicillin-binding proteins (PBPs) which catalyse the insertion of intracellularly synthesized precursors into the peptidoglycan cell wall. Here we show that MreC is required for the spatial organization of components of the peptidoglycan-synthesizing holoenzyme in the periplasm and MreB directs the localization of a peptidoglycan precursor synthesis protein in the cytosol. Additionally, fluorescent vancomycin (Van-FL) labelling revealed that the bacterial cytoskeletal proteins MreB and FtsZ, as well as MreC and RodA, were required for peptidoglycan synthetic activity. MreB and FtsZ were found to be required for morphogenesis of the polar stalk. FtsZ was required for a cell cycle-regulated burst of peptidoglycan synthesis early in the cell cycle resulting in the synthesis of cross-band structures, whereas MreB was required for lengthening of the stalk. Thus, the bacterial cytoskeleton and cell shape-determining proteins such as MreC, function in concert to orchestrate the localization of cell wall synthetic complexes resulting in spatially co-ordinated and efficient peptidoglycan synthetic activity.     

Phylogenetic analysis of the tenascin gene family: evidence of origin early in the chordate lineage

Background  

Tenascins are a family of glycoproteins found primarily in the extracellular matrix of embryos where they help to regulate cell proliferation, adhesion and migration. In order to learn more about their origins and relationships to each other, as well as to clarify the nomenclature used to describe them, the tenascin genes of the urochordate Ciona intestinalis, the pufferfish Tetraodon nigroviridis and Takifugu rubripes and the frog Xenopus tropicalis were identified and their gene organization and predicted protein products compared with the previously characterized tenascins of amniotes.     

Dissociation of intracellular lysosomal rupture from the cell death caused by silica

The relationship between intracellular lysosomal rupture and cell death caused by silica was studied in P388d(1) macrophages. After 3 h of exposure to 150 μg silica in medium containing 1.8 mM Ca(2+), 60 percent of the cells were unable to exclude trypan blue. In the absence of extracellular Ca(2+), however, all of the cells remained viable. Phagocytosis of silica particles occurred to the same extent in the presence or absence of Ca(2+). The percentage of P388D(1) cells killed by silica depended on the dose and the concentration of Ca(2+) in the medium. Intracellular lyosomal rupture after exposure to silica was measured by acridine orange fluorescence or histochemical assay of horseradish peroxidase. With either assay, 60 percent of the cells exposed to 150 μg silica for 3 h in the presence of Ca(2+) showed intracellular lysosomal rupture, was not associated with measureable degradation of total DNA, RNA, protein, or phospholipids or accelerated turnover of exogenous horseradish peroxidase. Pretreatment with promethazine (20 μg/ml) protected 80 percent of P388D(1) macrophages against silica toxicity although lysosomal rupture occurred in 60-70 percent of the cells. Intracellular lysosomal rupture was prevented in 80 percent of the cells by pretreatment with indomethacin (5 x 10(-5)M), yet 40-50 percent of the cells died after 3 h of exposure to 150 μg silica in 1.8 mM extracellular Ca(2+). The calcium ionophore A23187 also caused intracellular lysosomal rupture in 90-98 percent of the cells treated for 1 h in either the presence or absence of extracellular Ca(2+). With the addition of 1.8 mM Ca(2+), 80 percent of the cells was killed after 3 h, whereas all of the cells remained viable in the absence of Ca(2+). These experiments suggest that intracellular lysosomal rupture is not causally related to the cell death cause by silica or {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Cell death is dependent on extracellular Ca(2+) and may be mediated by an influx of these ions across the plasma membrane permeability barrier damaged directly by exposure to these toxins.     

Epidemiology of training injuries in amateur taekwondo athletes: a retrospective cohort study

The objectives of this study were to estimate the incidence and describe the pattern and severity of training injuries in taekwondo, and to compare pattern and severity of training injuries with competition injuries. One hundred and fifty-two active Australian amateur taekwondo athletes, aged 12 years or over, completed an online survey comprising questions on training exposure and injury history over the preceding 12 months. The main outcome measures were: overall injury incidence rate per athlete-year; training injury incidence rate per athlete-year, per 1000 athlete-training-sessions, and per 1000 athlete-hours of training; injury severity; and injury proportions by anatomical region and by type of injury. Injury incidence rates were calculated with 95% confidence intervals using standard methods, while injury proportions were compared using Fisher''s exact test. The vast majority (81.5%) of taekwondo injuries in an average athlete-year occurred during training. The training injury incidence rate was estimated to be 1.6 (95% CI: 1.4, 1.9) per athlete-year, 11.8 (95% CI: 10.4, 13.4) per 1000 athlete-training-sessions, and 7.0 (95% CI: 6.1, 7.9) per 1000 athlete-hours of training. Among athletes with five or fewer injuries, the severity and injury pattern of training injuries were, by and large, the same as for competition injuries. Approximately sixty percent (60.3%) of training injuries required treatment by a health professional. Considering the burden of training injuries exceeds that of competition injuries, taekwondo governing bodies and stakeholders are encouraged to devote more efforts towards the identification of risk factors for, and prevention of, training injuries in the sport of taekwondo.     

Theiler's Murine Encephalomyelitis Virus L* Amino Acid Position 93 Is Important for Virus Persistence and Virus-Induced Demyelination

The DA strain and other members of the TO subgroup of Theiler''s murine encephalomyelitis virus (TMEV) induce a persistent central nervous system infection associated with an inflammatory white matter demyelinating disease. TO subgroup strains synthesize an 18-kDa protein, L*, out of frame with the polyprotein from an initiation codon 13 nucleotides downstream from the polyprotein''s AUG codon. We previously generated a mutant virus from our infectious DA full-length clone that has a change of the L* AUG codon to ACG (with no change in the polyprotein''s amino acid sequence). Studies of this mutant virus showed that L* was key to the TO subgroup phenotype because the mutant had a decreased ability to persist and demyelinate. This work was initially called into question because a similar mutant derived from a different full-length DA infectious clone persisted and demyelinated similarly to wild-type DA virus (O. van Eyll and T. Michiels, J. Virol. 74:9071-9077, 2000). We now report that (i) the sequence of the L* coding region differs in the two infectious clones, resulting in a Ser or Leu as the predicted amino acid at position 93 of L* (with no change in the polyprotein''s amino acid sequence), (ii) the difference in this amino acid is key to the phenotypic differences between the two mutants, and (iii) the change in amino acid 93 may affect L* phosphorylation. It is of interest that this amino acid only appears critical in determining the virus phenotype when L* is present in a significantly reduced amount (i.e., following translation from an ACG initiating codon).The DA strain and other members of the TO subgroup of Theiler''s murine encephalomyelitis virus (TMEV) induce an acute subclinical gray matter central nervous system (CNS) infection in SJL mice that is followed by a chronic inflammatory white matter demyelinating disease; virus persists in the CNS for the life of the mouse (for a review, see reference 16). The DA virus demyelinating disease serves as a model of multiple sclerosis since the two are similar in white matter pathology and both disease processes appear to be immune mediated. In contrast, the GDVII strain and other members of the GDVII subgroup of TMEV cause an acute fatal neuronal infection and fail to persist.The study of the TMEV model system is an especially attractive one for molecular pathogenesis studies because TMEV is a relatively simple virus with only four structural proteins in the virion; the nucleotide sequence and deduced amino acid sequence are known for several TMEV strains; the three-dimensional crystal structure of two TMEV strains has been solved; infectious TMEV clones are available, easing the preparation of recombinant or mutated viruses; TMEV-neutralizing monoclonal antibody sites and T-cell epitopes are known; and the experimental and natural host for TMEV, the mouse, is easily manipulated and is well studied immunologically and genetically. An understanding of the determinants of the TMEV disease phenotypes (neurovirulence, virus persistence, restricted infection, demyelination) may be valuable in clarifying the pathogenesis of picornaviral diseases, as well as and non-virus-induced CNS diseases such as amyotrophic lateral sclerosis and multiple sclerosis.The genome of all picornaviruses contains a long open reading frame that is translated into a polyprotein. One remarkable feature of the TO subgroup strains is that an 18-kDa protein, L*, is synthesized out of frame with the polyprotein from an initiation codon that is 13 nucleotides (nt) downstream from the polyprotein''s AUG codon (8) (Fig. ​(Fig.1A1A).Open in a separate windowFIG. 1.The DA virus genome (A) and parental wild-type and L* mutant viruses (B). (A) The DA virus genome with a 5′ untranslated region (5′ UTR), polyprotein (LP1P2P3), and 3′ UTR, as well as L*, which is out of frame with the polyprotein''s reading frame; the regions of the genome are not drawn to size. The polyprotein''s initiating AUG codon is at nt 1066, while the L* AUG codon is at nt 1079. (B) Parts of the nucleotide sequences of the L* proteins of two wild-type parental DA viruses (DA, TMDA) and the mutant L* proteins derived from one or the two parental wild-type infectious clones, with a replacement of the initiating AUG codon with an ACG codon, as well as a C or U at nt 1359 coding for a Ser or Leu at amino acid 93.We previously engineered a mutant virus, called DAL*-1, which had a change of the L* AUG codon to ACG (but with no change in the amino acid sequence of the polyprotein) (4) from our infectious clone pDAFL3 (17) (Fig. ​(Fig.1B).1B). The DAL*-1 virus had a decreased ability to persist and demyelinate, suggesting that L* is key to the distinctive TO subgroup phenotype. Subsequent studies showed that L* interfered with virus clearance by CD4⁺ T cells, allowing the virus to persist (10). This work was initially called into question by Michiels and colleagues (21) because a mutant DA virus, OV48, with the same mutation as DAL*-1 (as well as an additional replacement of an AUG codon, in the fifth codon of the L* reading frame, to ACG) had no effect on the ability of the DA virus to persist or demyelinate; however, this mutant was engineered from a different full-length DA clone, pTMDA1 (11). In a subsequent study, they further investigated the importance of L* in virus persistence and demyelination by demonstrating that an L* mutant that contained a stop codon in the L* sequence (with no change in the polyprotein''s amino acid sequence) had a significant decrease in virus persistence and demyelination (22). We suspected that the contrasting disease phenotypes induced by the DAL*-1 and OV48 viruses might have resulted from a sequence difference(s) in L*. In the present study, we demonstrate that a change in L* of nucleotide (nt) 1356/amino acid 93 led to the change in phenotype of DAL*-1 versus OV48.     

Assessment of the Correlation Between Two Defining Criteria for Bidirectional Isthmic Block in the Ablation of Typical Atrial Flutter

Background

A complete, bidirectional conduction block in the cavotricuspid isthmus (CTI) represents the end-point of the typical atrial flutter ablation. We investigated the correlation between two criteria for successful ablation, one based on the atrial bipolar electrogram morphology before and after complete CTI conduction block, compared to the standard criteria of differential pacing and reversal in the right atrial depolarization sequence during coronary sinus (CS) pacing.

Method

We conducted a retrospective study in 111 patients (81 males, average age 62±10 years) who underwent an atrial flutter ablation during September 2007 - July 2009 in the Cardiology - Rehabilitation Hospital, UMF Cluj-Napoca. We assessed the presence of a bidirectional block at the end of the procedure using the standard criteria. We then analyzed the morphology of the bipolar atrial electrograms adjacent to the ablation line, before and after CTI conduction block.

Results

A change from a qRs morphology to a rSr'' morphology when pacing from the coronary sinus and from a rsr'' morphology to a QRS morphology when pacing from the low-lateral right atrium was associated with a CTI conduction block. Sensitivity (Se), specificity(Sp), positive predictive value (PPV), negative predictive value (NPV) were 96%, 89%, 99% and 67% respectively.

Conclusion

Our study suggests that the analysis of the atrial bipolar electrogram next to the ablation line before and after CTI ablation may be used as a reliable criterion to validate CTI conduction block due to its high sensitivity, specificity and positive predictive value.     

Inverting character of alpha-glucuronidase A from Aspergillus tubingensis

Alpha-glucuronidase A from Aspergillus tubingensis was found to be capable of liberating 4-O-methyl-D-glucuronic acid (MeGlcA) only from those beechwood glucuronoxylan fragments in which the acid is attached to the non-reducing terminal xylopyranosyl residue. Reduced aldotetrauronic acid, 4-O-methyl-D-glucuronosyl-alpha-1,2-D-xylopyranosyl-beta-1,4-xylopyranosyl-beta-1,4-xylitol, was found to be a suitable substrate to follow the stereochemical course of the hydrolytic reaction catalyzed by the purified enzyme. The configuration of the liberated MeGlcA was followed in a D(2)O reaction mixture by (1)H-NMR spectroscopy. It was unambiguously established that MeGlcA was released from the substrate as its beta-anomer from which the alpha-anomer was formed on mutarotation. This result represents the first experimental evidence for the inverting character of a microbial alpha-glucuronidase, a member of glycosyl hydrolase family 67 (EC 3.1.1.139).     

高等植物中的磷酸烯醇式丙酮酸羧激酶

简要介绍了近年来有关高等植物中磷酸烯醇式丙酮酸羧激酶（ＰＥＰＣＫ）的研究进展,并讨论了此酶的结构、功能和调节等方面的问题。