[Modification factors associated with maternally inherited non-syndromic hearing loss].

Mutations in the mitochondrial DNA have been certified to be one of the most important causes of maternally inherited sensorineural hearing loss. Among these, mitochondrial 12S rRNA1555A>G, 1494C>T and other mutations are associated with both nonsyndromic and drug induced hearing loss caused by aminoglycosides. Individuals carrying 1555A>G or 1494C>T mutation have a variety of clinical manifestations, which implies that the 1555A>G or 1494C>T mutation is a chief factor underlying the development of deafness but insufficient to produce the clinical phenotype. Therefore other modifier factors, such as aminoglycosides, mitochondrial haplotypes, secondary mutation or nuclear modifier genes, may play an important role in the phenotypic expression of the deafness-associated mitochondrial 12S rRNA1555A>G or 1494C>T mutation. In this review, the modifier factors for the phenotypic expression of deafness-associated mitochondrial 12S rRNA1555A>G or 1494C>T mutations were summarized and proposed the pathogenesis of maternally inherited deafness.

Bortezomib is synergistic with rituximab and cyclophosphamide in inducing apoptosis of mantle cell lymphoma cells in vitro and in vivo.

Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma with poor clinical outcome. Although front therapy induces a high rate of complete remission (CR), relapse is inevitable and new regimens are much needed for relapsed MCL. The proteasome inhibitor bortezomib (BTZ) induces apoptosis and sensitizes MCL cells to chemotherapy in relapsed MCL, but CR rates are low, with a short duration of response and severe toxicity. Here we evaluated whether BTZ is additive or synergistic with cyclophosphamide (CTX) and rituximab (RTX). Increasing doses of BTZ with a fixed dose of RTX and CTX (BRC regimen) resulted in markedly synergistic growth inhibition of MCL cells. BRC significantly enhanced apoptosis in MCL cell lines and primary tumor cells compared with single-agent treatment. Furthermore, western blotting analysis indicated that BRC induces apoptosis earlier via activation and cleavage of caspases-8, -9 and -3, and poly (ADP-ribose) polymerase, than single-agent treatment. The pan-caspase inhibitor completely blocked apoptosis induced by BRC. In vivo studies showed that BRC eradicated subcutaneous tumors in MCL-bearing SCID mice and significantly prolonged the long-term event-free survival in 70% of the mice. Hence, our study demonstrates that cytoreductive chemotherapy with both BTZ and anti-CD20 antibody may offer a better therapeutic modality for relapsed MCL.

Detection and analysis of alternative splicing in the silkworm by aligning expressed sequence tags with the genomic sequence.

We identified 277 alternative splice forms in silkworm genes based on aligning expressed sequence tags with genomic sequences, using a transcript assembly program. A large fraction (74%) of these alternative splices are located in protein-coding regions and alter protein products, whereas only 26% are in untranslated regions. From the alternative splices located in protein-coding regions, some (43%) affect protein domains that bind various biological molecules. The vast majority of the detected alternative forms in this study appear to be novel, and potentially affect biologically meaningful control of function in silkworm genes. Our results indicate that alternative splicing in silkworm largely produces protein diversity and functional diversity, and is a widely used mechanism for regulating gene expression.

Distribution of paclitaxel and its congeners in Taxus mairei.

Concentrations of paclitaxel and its congeners 10-deacetylpaclitaxel, cephalomannine, 10-deacetylcephalomannine, baccatin III and 10-deacetylbaccatin III in Taxus mairei were determined by HPLC. It was found that paclitaxel is abundant in roots and stem bark. The concentrations of paclitaxel and its congeners in the plants growing in one area may vary to a great extent from those growing in another area.

Effects of hydrostatic pressure on the structure and biological activity of infectious bursal disease virus.

The effects of high hydrostatic pressure on the structure and biological activity of infectious bursal disease virus (IBDV), a commercially important pathogen of chickens, were investigated. IBDV was completely dissociated into subunits at a pressure of 240 MPa and 0 degrees C revealed by the change in intrinsic fluorescence spectrum and light scattering. The dissociation of IBDV showed abnormal concentration dependence as observed for some other viruses. Electron microscopy study showed that morphology of IBDV had an obvious change after pressure treatment at 0 degrees C. It was found that elevating pressure destroyed the infectivity of IBDV, and a completely pressure-inactivated IBDV could be obtained under proper conditions. The pressure-inactivated IBDV retained the original immunogenic properties and could elicit high titers of virus neutralizing antibodies. These results indicate that hydrostatic pressure provides a potential physical means to prepare antiviral vaccine.

Central nervous system lesions induced experimentally by a very virulent strain of Marek's disease virus in Marek's disease-resistant chickens.

The histological lesions of central nervous system (CNS) in Marek's disease-resistant chickens inoculated with a very virulent Marek's disease virus strain (vvMDV), Md/5, were examined. This vvMDV induced high incidences of CNS lesions as well as visceral and peripheral nerve lesions. The principal histopatho-logical changes of CNS consisted of non-suppurative meningoencephalomyelitis and lymphomatous lesion, which were categorized into two types, non-necrotizing and necrotizing. The main changes in the former type were perivascular cuffing of lymphoid cells of variable thickness. Although diffuse lymphomatous lesions were sometimes observed, gliosis, neuronal and axonal degeneration, and satellitosis as well as demyelination in the myelinated fibre paths, were infrequent. The most significant change of the latter type was necrotizing lymphomatous and, sometimes, non-suppurative inflammatory lesions (malacia). In the malacic foci, all the cells, including infiltrating lymphoid cells, neurons and glial cells, were necrotic or degenerated. The malacic lesions were frequently accompanied by fibrinoid necrosis of blood vessels. The present results indicate that necrotizing vasculitis associated with vvMDV may lead to ischaemic damage which in turn induces necrotizing lymphomatous and sometimes, meningoencephalomyelitis lesions. In addition, it is considered that vvMDV can induce a high incidence not only of CNS lesions, but also of visceral and nerve lesions in Marek's disease-resistant chickens.

Angiotensinogen gene expression is stimulated by the cAMP-responsive element binding protein in opossum kidney cells.

It has been reported previously that the addition of isoproterenol or forskolin stimulates the expression of the angiotensinogen (ANG) gene in opossum kidney (OK) 27 cells, an OK cell line with a fusion gene containing the 5'-flanking regulatory sequence of the rat ANG gene fused with a human growth hormone (hGH) gene as a reporter, pOGH (ANG N-1498/+18), permanently integrated into their genomes. To investigate whether the effect of isoproterenol or forskolin on the expression of the ANG gene is mediated via the nuclear 43-kD cAMP-responsive element binding protein (CREB), OK 27 cells were transiently transfected with an expression plasmid containing the cDNA for the 43-kD CREB (pRSV/CREB). The level of expression of the pOGH (ANG N-1498/+18) in OK 27 cells was estimated by the amount of immunoreactive hGH secreted into the culture medium. Transfection of pRSV/CREB alone stimulated the expression of pOGH (ANG N-1498/+18). The addition of isoproterenol or forskolin further enhanced the stimulatory effect of pRSV/ CREB on the expression of pOGH (ANG N-1498/+18). The enhancing effect of isoproterenol was inhibited by the presence of propranolol (an inhibitor of beta-adrenoceptors) and (R)-p-adenosine 3'5'-cyclic monophospho-orthioate (Rp)-cAMP (an inhibitor of cAMP-dependent protein kinase A I and II). Transfection of pRSV/CREB had no effect on the expression of thymidine kinase growth hormone in OK 13 cells, an OK cell line with a fusion gene containing the promoter/enhancer DNA sequence of the viral thymidine-kinase gene fused with an hGH gene as a reporter, thymidine kinase growth hormone, permanently integrated into their genomes. These studies demonstrate that isoproterenol stimulates the expression of ANG gene via the cAMP-dependent protein kinase A and probably via the interaction of the 43-kD CREB with the 5'-flanking region of the ANG gene. Our data indicate that the nuclear 43-kD CREB may have a modulatory role on the expression of the ANG gene in OK cells.

Up-regulated P21CIP1 expression is part of the regulation quantitatively controlling serum deprivation-induced apoptosis.

Among the many genes which have been suggested to be required by the molecular mechanism dictating apoptotic death, some have been shown to function as pacemakers to pave the way for cells either to liver or to die. Previously we have shown that immediate early gene expression associated with the G1 phase of cell cycle traverse are candidates for this function. Here we report that well-known key regulator for halting cell cycling at the G1/S border, the p21 protein known as WAF1, Cip1, Pic1, or Sdi1, is also involved in the execution of cells' suicidal death. p21 up-regulation is seen in quiescent mouse 3T3 fibroblasts stimulated to die by serum deprivation, at both message and protein levels, evidenced by increased protein presence in its targeted functional site, the nucleus. In addition, we show that this up-regulation of p21 is functionally related to the operational efficiency of the apoptotic process, in that when cells are stably transfected with an antisense construct to repress the endogenous p21-protein level, death is delayed. Quantitative protection from apoptosis with antisense p21 transfection is relatively proportional to the repressed level of this protein in the cells. Taken together, our results suggest that the apoptotic-dependent additional increase of p21 beyond the base level, seen in serum-deprived quiescent cells, may be involved in the molecular events precipitating a rapid program of cell demise, and that repression of this increase may obstruct the operation of this program and postpone the eventual death.

Human transforming growth factor alpha: sequence analysis of the 4.5-kb and 1.6-kb mRNA species.

Two transforming growth factor alpha (TGF alpha) mRNA species with the apparent sizes of 4.5 and 1.6 kb were identified in all human cell lines analysed. The cDNA corresponding to the 4.5-kb species was entirely sequenced, revealing the presence of a 3'-untranslated region (3'-UTR) of 3571 nucleotides, which contained several potential polyadenylation signals. Our results indicate that the 1.6-kb species is derived from the same precursor by alternative polyadenylation. In addition, we present evidence suggesting that TGF alpha-specific mRNAs could be initiated from transcription start points (tsp) located upstream from the tsp previously identified by Jakobovitz et al. [Mol. Cell. Biol. 8 (1988) 5549-5554].

Genetic variation in transforming growth factor alpha: possible association of BamHI polymorphism with bilateral sporadic cleft lip and palate.

Non-syndromic cleft lip with or without cleft palate (CL/P) is one of the most common birth defects affecting 1/1000 Caucasians. Genetic factors are thought to contribute to the development of this disorder. A significant association between two restriction fragment length polymorphisms, the TGF alpha TaqI 2.7-kb allele and the TGF alpha BamHI 40-kb allele, at the transforming growth factor alpha (TGF alpha) locus and the occurrence of clefting has previously been reported. A total of 98 Caucasian patients of Alsacian ancestry was recruited from our registry of congenital malformations. These patients had isolated CL/P but no other anomalies. In addition 57 patients with cleft palate, but without cleft lip, were studied. A control group comprised 99 unrelated healthy Caucasians of the same Alsacian ancestry. TaqI and BamHI identify two-allele polymorphisms. The TGFA Taq and BamHI alleles showed no significant association with the presence of clefting, the only exception being that the BamHI 10.0-kb allele was significantly more frequent in patients with bilateral CL/P.

Transforming growth factor-alpha: characterization of the BamHI, RsaI, and TaqI polymorphic regions.

We have characterized the nature of structural alleles of the transforming growth factor-alpha (TGF alpha) locus by restriction-enzyme digestion with BamHI, RsaI, and TaqI. The BamHI polymorphic site is located within exon VI, which codes for the 3' untranslated region. The two BamHI alleles differ by a single point mutation at the restriction site. The RsaI and TaqI polymorphic sites are located within intron V. The two alleles differ at the restriction site, either by a point mutation (RsaI) or by a 4-bp deletion (TaqI). This analysis permits us to devise a PCR method coupled with restriction digestions to directly identify the TGF alpha polymorphisms. Analysis of 99 Caucasian controls has revealed a highly significant (P < .001) association between the RsaI and the BamHI genotype. The frequency of the rare BamHI allele was significantly higher (P < .001) in transformed cell lines (.30) than in controls (.076).

Human breast cancer: identification of populations with a high risk of early relapse in relation to both oestrogen receptor status and c-erbB-2 overexpression.

We recently defined a new early prognostic factor, the ER+(R) status, which permits the discrimination of a group presenting a high risk of early relapse among the ER+ patients. This group was referred to as ER+(R2) in contrast to ER+(R1) which corresponded to the group of ER+ patients having a lower risk of early relapse. Taking into account the whole population including the ER- and inflammatory tumours, we have extended this view and showed that ER+(R) status is a significant predictor of disease-free survival. Determination of c-erbB-2 mRNA levels in the same series of tumours showed that high expression of c-erbB-2 mRNA is significantly correlated with ER-, inflammatory tumours and with lymph nodes involvement. Moreover, a multivariate analysis showed that c-erbB-2 mRNA overexpression was a significant predictor of early relapse (P = 0.02), as significant as ER negativity and ER+(R2). For ER+ patients a high level of c-erbB-2 mRNA constitutes a higher risk of relapse for both ER+(R1) and ER+(R2) patients. However, in the case of ER- patients, early relapses were strongly correlated with c-erbB-2 overexpression. The counterpart of this observation is that ER- patients with no overexpression of c-erbB-2 constitute a group with a relatively good prognosis.