Identification and functional characterization of the sex-determining gene <Emphasis Type="Italic">doublesex</Emphasis> in the sawfly, <Emphasis Type="Italic">Athalia rosae</Emphasis> (Hymenoptera: Tenthredinidae)

Sexual fate of the sawfly, Athalia rosae (Hymenoptera: Tenthredinidae) is determined by the complementary sex determination (CSD) mechanism as is the case in honeybees. However, to date, genes involved in sex determination have not been identified in this species. In this study, we attempted to identify orthologs of complementary sex-determiner (csd), feminizer (fem), and doublesex (dsx) from the A. rosae genome, all of which are crucial components of the sex determination cascade in the honeybee. As a result, we identified a sawfly ortholog of dsx (designated as Ardsx). Rapid amplification of cDNA ends (RACE) using total RNA extracted from male and female larvae identified three male-specific variants and three female-specific variants. Comparison between the full-length Ardsx cDNAs and the genomic sequence revealed that exon 5 was differentially spliced between the male- and female-specific variants. RT-PCR analysis demonstrated that Ardsx pre-mRNA was spliced alternatively in a sex-dependent manner at almost all the developmental stages. RNAi-mediated knockdown of Ardsx in males caused severe defects in the reproductive organs and, notably, induced development of the ovipository apparatus containing the dorsal pair of blades and the sheath. These males also showed abnormalities in testes and seminal vesicles and lacked mature sperm. The present study provides the first direct evidence that dsx is essential for sexual development in hymenopteran species.     

Does MAX open up a new avenue for meiotic research?

Meiosis is a central event of sexual reproduction. Like somatic cells, germ cells conduct mitosis to increase their cell number, but unlike somatic cells, germ cells switch their cell division mode from mitosis to meiosis at a certain point in gametogenesis. However, the molecular basis of this switch remains elusive. In this review article, we give an overview of the onset of mammalian meiosis, including our recent finding that MYC Associated Factor X (MAX) prevents ectopic and precocious meiosis in embryonic stem cells (ESCs) and germ cells, respectively. We present a hypothetical model of a MAX‐centered molecular network that regulates meiotic entry in mammals and propose that inducible Max knockout ESCs provide an excellent platform for exploring the molecular mechanisms of meiosis initiation, while excluding other aspects of gametogenesis.     

Zinc inhibition of pyruvate kinase of M-type isozyme

Inhibitory effect of Zn on the pyruvate kinase of M (muscle)-type isozyme was analyzed for the purpose of elucidating the cytotoxicity of Zn. Zn inhibited pyruvate kinase uncompetitively with respect to the substrate PEP, and competitively with respect to ADP. Quotient velocity plot calculated from the Zn-inhibition curves showed that Zn²⁺ as a ZnADP complex acted as competitive and uncompetitive inhibitors of the enzyme with respect to the substrate ADP and PEP, respectively: Zn²⁺ forms a ZnADP complex, which may bind to the ADP-binding site of the free enzyme with the K_i value of 1.4 μM causing competitive inhibition, or to the ADP-site of the enzyme-PEP complex with 2.6 μM resulting in uncompetitive inhibition. The inhibition of pyruvate kinase by Zn²⁺ may be responsible for the cytotoxicity of this metal by decreasing glycolytic flux.     

Cyclic strain stimulates isoform-specific PKC activation and translocation in cultured human keratinocytes

Previous studies have demonstrated that cyclic strain induces keratinocyte proliferative and morphological changes. Since protein kinase C (PKC) is known to play an important role in the regulation of keratinocyte growth and differentiation, the objective of this study was to determine the role of the PKC signaling pathway as a mediator of strain modulation of the keratinocyte phenotype. In particular, we tested the following specific hypotheses: (1) cyclic strain stimulates PKC activity and translocation, (2) cyclic strain activates PKC in an isoform-specific manner, and (3) PKC mediates the strain activated proliferative and morphological response in cultured human keratinocytes. To test these hypotheses, keratinocytes were subjected to vacuum-generated cyclic strain (10% average strain), followed by measurement of PKC activity, PKC isoform distribution by Western blot analysis and confocal microscopy, and examination of the effect of PKC inhibitors (calphostin C and staurosporine) on strain induced proliferative and morphological changes. We observed stimulation of PKC activity (62.3 ± 5.1% increase) coupled with translocation of PKC from the cytosolic to the membrane fraction in keratinocytes subjected to acute cyclic strain. Cyclic strain also caused translocation of PKC α and δ, but not ζ isoforms, from the cytosolic to the membrane fraction as demonstrated by both Western blot analysis and confocal microscopy. PKC β was not detected in these cells. PKC inhibitors, calphostin C (10 nM), and staurosporine (5 nM), inhibited strain-induced PKC activation and keratinocyte proliferation, but did not block the effects of strain on cellular morphology or alignment. We conclude that these data support our hypothesis that cyclic strain stimulates PKC activity and translocation in an isoform-specific manner in cultured human keratinocytes. Moreover, our studies with PKC inhibitors support the hypothesis that strain-induced changes in the keratinocyte phenotype may be selectively modulated by PKC. J. Cell. Biochem. 67:327–337, 1997. © 1997 Wiley-Liss, Inc.     

A Particulate Phospholipid Synthesizing System from Escherichia coli B

The phosphatidate formed by glycerol 3-phosphate acyltransferase present in a particulate fraction from E. coli remained associated with the particulate material and was converted to phosphatidylethanolamine and phosphatidylglycerol by other enzymes contained in the particulate fraction. Those enzymes required a heat-labile factor for the synthesis of phosphatidylethanolamine and a heat-stable factor for the synthesis of phosphatidylglycerol. Both of these phospholipids like their precursor remained bound to the particulate fraction.