A negative feedback mechanism revealed by functional analysis of the alternative isoforms of the Drosophila splicing regulator transformer-2

The Drosophila sex determination gene transformer-2 (tra-2) is a splicing regulator that affects the sex-specific processing of several distinct pre-mRNAs. While the tra-2 gene itself is known to produce alternative mRNAs that together encode three different TRA-2 protein isoforms, the respective roles of these isoforms in affecting individual pre-mRNA targets has remained unclear. We have generated transgenic fly strains with mutations affecting specific TRA-2 isoforms to investigate their individual roles in regulating the alternative processing of doublesex, exuperantia and tra-2 pre-mRNA. Our results indicate that in somatic tissues two different isoforms function redundantly to direct female differentiation and female-specific doublesex pre-mRNA splicing. In the male germline, where tra-2 has an essential role in spermatogenesis, a single isoform was found to uniquely perform all necessary functions. This isoform appears to regulate its own synthesis during spermatogenesis through a negative feedback mechanism involving intron retention.     

Identification of alternative splicing form of Stat2

Three cases of pulmonary leiomyosarcoma were presented. The characteristic clinical features were described with review of literature. In comparison with bronchogenic carcinoma, the leiomyosarcoma has some characteristics: 1) On chest X-ray, it usually appears as a sharply demarcated, even density round mass, growing rapidly within the lung, it rarely accompanies with hilar or mediastinal lymph node metastasis. 2) The preoperative cytological or pathological diagnosis is difficult either by sputum smear or by bronchoscopic biopsy or by fine needle percutaneous aspiration biopsy. 3) Pathological differential diagnosis of leiomyosarcoma of lung from anaplastic lung cancer is difficult. In conclusion, the primary pulmonary leiomyosarcoma is a rare malignant tumor, detecting the present illness seriously, paying attention to the chest X-ray films characterize, early surgical resection is the only way to get diagnosis and effective treatment method.     

Drosophila Cyclin B3 is required for female fertility and is dispensable for mitosis like Cyclin B

Cyclin B3 has been conserved during higher eukaryote evolution as evidenced by its identification in chicken, nematodes, and insects. We demonstrate that Cyclin B3 is present in addition to Cyclins A and B in mitotically proliferating cells and not detectable in endoreduplicating tissues of Drosophila embryos. Cyclin B3 is coimmunoprecipitated with Cdk1(Cdc2) but not with Cdk2(Cdc2c). It is degraded abruptly during mitosis like Cyclins A and B. In contrast to these latter cyclins, which accumulate predominantly in the cytoplasm during interphase, Cyclin B3 is a nuclear protein. Genetic analyses indicate functional redundancies. Double and triple mutant analyses demonstrate that Cyclins A, B, and B3 cooperate to regulate mitosis, but surprisingly single mutants reveal that neither Cyclin B3 nor Cyclin B is required for mitosis. However, both are required for female fertility and Cyclin B also for male fertility.     

Heterosis for viability, fecundity, and male fertility in Drosophila melanogaster: comparison of mutational and standing variation

If genetic variation for fitness traits in natural populations ("standing" variation) is maintained by recurrent mutation, then quantitative-genetic properties of standing variation should resemble those of newly arisen mutations. One well-known property of standing variation for fitness traits is inbreeding depression, with its converse of heterosis or hybrid vigor. We measured heterosis for three fitness traits, pre-adult viability, female fecundity, and male fertility, among a set of inbred Drosophilia melanogaster lines recently derived from the wild, and also among a set of lines that had been allowed to accumulate spontaneous mutations for over 200 generations. The inbred lines but not the mutation-accumulation (MA) lines showed heterosis for pre-adult viability. Both sets of lines showed heterosis for female fecundity, but heterosis for male fertility was weak or absent. Crosses among a subset of the MA lines showed that they were strongly differentiated for male fertility, with the differences inherited in autosomal fashion; the absence of heterosis for male fertility among the MA lines was therefore not caused by an absence of mutations affecting this trait. Crosses among the inbred lines also gave some, albeit equivocal, evidence for male fertility variation. The contrast between the results for female fecundity and those for male fertility suggests that mutations affecting different fitness traits may differ in their average dominance properties, and that such differences may be reflected in properties of standing variation. The strong differentiation among the MA lines in male fertility further suggests that mutations affecting this trait occur at a high rate.     

The gene fl(2)d is needed for the sex-specific splicing of transformer pre-mRNA but not for double-sex pre-mRNA in Drosophila melanogaster

In Drosophila melanogaster, regulation of the sex determination genes throughout development occurs by sex-specific splicing of their products. The first gene is Sex-lethal(Sxl). The downstream target of Sxl is the gene transformer (tra): the Sxl protein controls the female-specific splicing of the Tra pre-mRNA. The downstream target of the gene tra is the gene double-sex (dsx): the Tra protein of females, controls the female-specific splicing of the Dsx pre-mRNA. We have identified a gene, female-lethal-2-d fl(2) d, whose function is required for the female-specific splicing of Sxl pre-mRNA. In this report we analyze whether the gene fl(2)d is also required for the sex-specific splicing of both Tra and Dsx pre-mRNAs. We found that the Sxl protein is not sufficient for the female-specific splicing of Tra pre-mRNA, the fl(2)d function also being necessary. This gene, however, is not required for the female-specific splicing of Dsx pre-mRNA.     

Delayed male maturity is a cost of producing large sperm in Drosophila

Among fruit-fly species of the genus Drosophila there is remarkable variation in sperm length, with some species producing gigantic sperm (e.g., > 10 times total male body length). These flies are also unusual in that males of some species exhibit a prolonged adult nonreproductive phase. We document sperm length, body size, and sex-specific ages of reproductive maturity for 42 species of Drosophila and, after controlling for phylogeny, test hypotheses to explain the variation in rates of sexual maturation. Results suggest that delayed male maturity is a cost of producing long sperm. A possible physiological mechanism to explain the observed relationship is discussed.     

Accumulation of acetylcholine receptors is a necessary condition for normal accumulation of acetylcholinesterase during in vitro neuromuscular synaptogenesis

To study a step of the very complex processes of the formation of the neuromuscular junction (NMJ), we have analysed the clustering of acetylcholine receptors (AChR) and acetylcholinesterase (AChE) in myotubes cultured in various conditions. On the surface of rat myotubes cultured in the presence of spinal cord cells from embryonic rat, numerous AChE clusters appeared. Such clusters are always co-localized with AChR clusters, but the reverse is not true: the number of AChR clusters largely exceeds that of AChE clusters. Very few AChE clusters formed when such co-cultures were treated with monoclonal antibodies (mAbs) against the main immunogenic region (MIR) of the AChR, which provoke internalization and degradation of the AChRs of the muscular membrane. The total levels of AChE and proportions of molecular forms were unaffected. We also used non-innervated myotubes in which addition of agrin, a protein normally synthesized by motoneurons, transported to nerve terminals and inserted into the synaptic basal lamina, induces the formation of small clusters of AChE. When added to rat myotubes devoid of membrane AChR, agrin-induced AChE clusters did not form. Finally, we analysed the capacity of the variant of the C2 mouse muscle cell line deficient in AChR (1R-) to form clusters of AChE in co-cultures with spinal cord cells from rat: no formation of AChE clusters could be observed. In all these different systems of cultures, the conditions which prevented clustering of AChR (anti-AChR antibodies, deficiency of the variant C2 cell line) also suppressed AChE clustering. We concluded that clustering of AChR is a prerequisite for clustering of AChE, so that NMJ formation implies the sequential accumulation of these two components.     

The glutamine-rich domain of the Drosophila GAGA factor is necessary for amyloid fibre formation in vitro, but not for chromatin remodelling

The Drosophila GAGA factor binds specifically to the sequence GAGAG, and synergises with nucleosome remodelling factor to remodel chromatin in vitro. It consists of an N-terminal domain (POZ/BTB) which mediates protein-protein interactions, a central region which contains the DNA-binding domain, and a C-terminal glutamine-rich region. It is shown that the glutamine-rich region is responsible for the formation of fibres in vitro which, on the basis of their tinctorial properties and CD spectra, may be classified as amyloid fibres. A large structural change, probably resulting in beta-sheet structure, is observed upon fibre formation. Mutants containing the central region, either alone or together with the glutamine-rich region, are largely lacking in secondary structure but they bind specifically to the cognate DNA and are able to remodel chromatin in vitro. Consequently, neither the N-terminal domain nor the C-terminal glutamine-rich regions of the GAGA factor are necessary for chromatin remodelling in vitro.     

The Drosophila bifocal gene encodes a novel protein which colocalizes with actin and is necessary for photoreceptor morphogenesis

Photoreceptor cells of the Drosophila compound eye begin to develop specialized membrane foldings at the apical surface in midpupation. The microvillar structure ultimately forms the rhabdomere, an actin-rich light-gathering organelle with a characteristic shape and morphology. In a P-element transposition screen, we isolated mutations in a gene, bifocal (bif), which is required for the development of normal rhabdomeres. The morphological defects seen in bif mutant animals, in which the distinct contact domains established by the newly formed rhabdomeres are abnormal, first become apparent during midpupal development. The later defects seen in the mutant adult R cells are more dramatic, with the rhabdomeres enlarged, elongated, and frequently split. bif encodes a novel putative protein of 1063 amino acids which is expressed in the embryo and the larval eye imaginal disc in a pattern identical to that of F actin. During pupal development, Bif localizes to the base of the filamentous actin associated with the forming rhabdomeres along one side of the differentiating R cells. On the basis of its subcellular localization and loss-of-function phenotype, we discuss possible roles of Bif in photoreceptor morphogenesis.     

Effect of cyclosporine on fertility in male rats

The effect of cyclosporine (CsA) on fertility has assumed greater importance with the increasing numbers of pediatric transplantations being performed all over the world. Conflicting reports on the effects of CsA on sex hormones are available. This experimental animal study was designed to examine the effect of CsA on testicular weight, sperm counts, seminiferous tubular diameter (STD), testicular morphology, DNA flowcytometry, sex hormone levels, and fertility in male rats. Those rats who received CsA (20 mg/kg per day) showed significant reductions in testicular weight (P < 0.05), sperm count (P < 0.01), Johnsen score (P < 0.05), STD (P < 0.01), serum testosterone levels (P < 0.05), haploid cell population (P < 0. 001) in the testis, and fertility (P < 0.001) compared to those receiving CsA 10 mg/kg per day and control rats. These findings will have an important bearing for children receiving cyclosporine for long periods to guide the physician in optimally adjusting long-term treatment.     

Alternative splicing of GTBP in normal human tissues

The relationship between serum hepatitis Be antigen (HBeAg) and serum hepatitis B virus (HBV) DNA determined by two commercially available assays was examined in 345 Chinese patients with chronic HBV infection. HBV DNA was detected by these commercial assays in 85% of the HBeAg-positive patients. Discrepancies between test results were found to occur when serum HBV DNA levels were low (< 5 pg ml-1 for the Abbott Genostics and < 100 MEq ml-1 for Chiron Quantiplex assays). An equation for the conversion between results generated by these two assays was derived, which was found to be very similar to the equation recently described by Kapke et al.     

The presence of wild-type TP53 is necessary for the radioprotective effect of the Bowman-Birk proteinase inhibitor in normal fibroblasts

This paper uses definitions of a consensus conference (ACCP/CCM) describing the epidemiology of SIRS, sepsis and septic shock in surgical ICU patients. During a period of 2 years a total of 656 patients were prospectively enrolled into the study. 335 patients (51.1% of the total population) developed SIRS (systemic inflammatory response syndrome); in 65 of these patients infection could be documented, i.e. they met the criteria of sepsis, 47 of these 65 septic patients developed septic shock, with mortality of 53.2%. SIRS is associated with a high sensitivity but a low specificity in predicting the outcome of ICU patients. Moreover, SIRS and sepsis appear to be of minor clinical relevance. On the contrary, septic shock describes a very high risk group of patients which should be characterized more closely in future studies.     

Autoregulation of the pTF-FC2 proteic poison-antidote plasmid addiction system (pas) is essential for plasmid stabilization

The stabilization of a test plasmid by the proteic, poison-antidote plasmid addiction system (pas) of plasmid pTF-FC2 was host strain dependent, with a 100-fold increase in stability in Escherichia coli CSH50, a 2.5-fold increase in E. coli JM105, and no detectable stabilization in E. coli strains JM107 and JM109. The lethality of the PasB toxin was far higher in the E. coli strains in which the pas was most effective. Models for the way in which poison-antidote systems stabilize plasmids require that the antidote have a much higher rate of turnover than that of the toxin. A decrease in host cell death following plasmid loss from an E. coli lon mutant and a decrease in plasmid stability suggested that the Lon protease plays a role in the rate of turnover of PasA antidote.     

Identification of trans-acting genes necessary for centromere function in Drosophila melanogaster using centromere-defective minichromosomes

Deletions in the Drosophila minichromosome Dp1187 were used to investigate the genetic interactions of trans-acting genes with the centromere. Mutations in several genes known to have a role in chromosome inheritance were shown to have dominant effects on the stability of minichromosomes with partially defective centromeres. Heterozygous mutations in the ncd and klp3A kinesin-like protein genes strongly reduced the transmission of minichromosomes missing portions of the genetically defined centromere but had little effect on the transmission of minichromosomes with intact centromeres. Using this approach, ncd and klp3A were shown to require only the centromeric region of the chromosome for their roles in chromosome segregation. Increased gene dosage also affected minichromosome transmission and was used to demonstrate that the nod kinesin-like protein gene interacts genetically with the centro mere, in addition to interacting with extracentromeric regions as demonstrated previously. The results presented in this study strongly suggest that dominant genetic interactions between mutations and centromere-defective minichromosomes could be used effectively to identify novel genes necessary for centromere function.