A proteomic approach to the identification and characterisation of protein composition in wheat germ

Proteome analyses were carried out on commercial wheat germ of mature grain from the biscuit-making wheat cultivar, Rosella. Wheat germ protein extracts were fractionated by two-dimensional gel electrophoresis across two different immobilised pH gradients: pH 4.0–7.0 and 6.0–9.0. A total of 612 individual protein spots were excised from the gels and characterised by peptide mass fingerprinting. From these analyses, 347 individual proteins were identified from protein sequence database interrogation, and 301 different types of protein were catalogued according to protein function. The remaining 265 protein spots gave poor or no matches to proteins in the databases and were not identified in this study. Six different classes of enzymes were identified in the germ, many of them having roles in the mobilisation of energy reserves for germination. Abundantly expressed enzyme classes include the oxidoreductases, transferases and hydrolases. A comparison was also made between the major protein classes expressed in the germ and protein classes expressed in the endosperm from previous proteomic work. This study contributes significantly to our knowledge of protein expression and heterogeneity in the germ of wheat grain and forms the basis for future studies in regard to the characterisation of proteins during the initial stages of germination.     

Beta1-integrin orients epithelial polarity via Rac1 and laminin

Epithelial cells polarize and orient polarity in response to cell-cell and cell-matrix adhesion. Although there has been much recent progress in understanding the general polarizing machinery of epithelia, it is largely unclear how this machinery is controlled by the extracellular environment. To explore the signals from cell-matrix interactions that control orientation of cell polarity, we have used three-dimensional culture systems in which Madin-Darby canine kidney (MDCK) cells form polarized, lumen-containing structures. We show that interaction of collagen I with apical beta1-integrins after collagen overlay of a polarized MDCK monolayer induces activation of Rac1, which is required for collagen overlay-induced tubulocyst formation. Cysts, comprised of a monolayer enclosing a central lumen, form after embedding single cells in collagen. In those cultures, addition of a beta1-integrin function-blocking antibody to the collagen matrix gives rise to cysts that have defects in the organization of laminin into the basement membrane and have inverted polarity. Normal polarity is restored by either expression of activated Rac1, or the inclusion of excess laminin-1 (LN-1). Together, our results suggest a signaling pathway in which the activation of beta1-integrins orients the apical pole of polarized cysts via a mechanism that requires Rac1 activation and laminin organization into the basement membrane.     

Rearrangement of antigen receptor genes is defective in mice with severe combined immune deficiency

A process unique to lymphocyte differentiation is the rearrangement of genes encoding antigen-specific receptors on B and T cells. A mouse mutant (C.B-17scid) with severe combined immune deficiency, i.e., that lacks functional B and T cells, shows no evidence of such gene rearrangements. However, rearrangements were detected in Abelson murine leukemia virus-transformed bone marrow cells and in spontaneous thymic lymphomas from C.B-17scid mice. Most of these rearrangements were abnormal: approximately 80% of Igh rearrangements deleted the entire Jh region, and approximately 60% of TCR beta rearrangements deleted the entire J beta 2 region. The deletions appeared to result from faulty D-to-J recombination. No such abnormal rearrangements were detected in transformed tissues from control mice. The scid mutation may adversely affect the recombinase system catalyzing the assembly of antigen receptor genes in developing B and T lymphocytes.     

Sulfide ameliorates metal toxicity for deep-sea hydrothermal vent archaea

The chemical stress factors for microbial life at deep-sea hydrothermal vents include high concentrations of heavy metals and sulfide. Three hyperthermophilic vent archaea, the sulfur-reducing heterotrophs Thermococcus fumicolans and Pyrococcus strain GB-D and the chemolithoautotrophic methanogen Methanocaldococcus jannaschii, were tested for survival tolerance to heavy metals (Zn, Co, and Cu) and sulfide. The sulfide addition consistently ameliorated the high toxicity of free metal cations by the formation of dissolved metal-sulfide complexes as well as solid precipitates. Thus, chemical speciation of heavy metals with sulfide allows hydrothermal vent archaea to tolerate otherwise toxic metal concentrations in their natural environment.     

Spawning periodicity of three species of Nodilittorina in Hong Kong

The spawning periodicity of Nodilittorina trochoides, N. radiata and N. vidua from two rocky shores of different wave exposure, Big Wave Bay (more exposed site) and Cape d'Aguilar, in Hong Kong was monitored for one year (November 1994–November 1995). The breeding season was similar for the three species, mainly during the summer (June–September). The spawning duration was 9 months for N. vidua and 7 months for the other two littorinids. Spawning was initiated at different times for the two sites. At Big Wave Bay, spawning occurred between May–November for N. trochoides; March–September for N. radiata and March–November for N. vidua. At Cape d'Aguilar, the spawning period of N. trochoides and N. radiata was similar (April–October), whilst for N. vidua, spawning extended up to December. A decrease in monthly fecundity was recorded during July and August at Big Wave Bay and Cape d'Aguilar respectively. A weak relationship was found between the size of reproductive females and egg production. The seasonal fecundity of N. trochoides and N. vidua was similar at both sites and estimated as 10 000 and 7500 eggs per female respectively. Fecundity of N. radiata varied between sites, with 15 000 eggs and 9000 eggs per female being recorded from Cape d'Aguilar and Big Wave Bay respectively.     

Mortality and smoking in Hong Kong: case-control study of all adult deaths in 1998

ObjectiveTo assess the mortality currently associated with smoking in Hong Kong, and, since cigarette consumption reached its peak 20 years earlier in Hong Kong than in mainland China, to predict mortality in China 20 years hence.DesignCase-control study. Past smoking habits of all Chinese adults in Hong Kong who died in 1998 (cases) were sought from those registering the death.SettingAll the death registries in Hong Kong.Participants27 507 dead cases (81% of all registered deaths) and 13 054 live controls aged ⩾35 years.ResultsIn men aged 35-69 the adjusted risk ratios (and 95% confidence intervals) comparing smokers with non-smokers were 1.92 (1.70 to 2.16) for all deaths, 2.22 (1.94 to 2.55) for neoplastic deaths, 2.60 (2.10 to 3.21) for respiratory deaths (including tuberculosis, risk ratio 2.54), and 1.68 (1.43 to 1.97) for vascular deaths (each P<0.0001). In women aged 35-69 the corresponding risk ratios were 1.62 (1.40 to 1.88) for all deaths, 1.60 (1.33 to 1.93) for neoplastic deaths, 3.13 (2.21 to 4.44) for respiratory deaths, and 1.55 (1.20 to 1.99) for vascular deaths (each P<0.001). If these associations with smoking are largely or wholly causal then, among all registered deaths at ages 35-69 in 1998, tobacco caused about 33% (2534/7588) of all male deaths and 5% (169/3341) of all female deaths (hence 25% of all deaths at these ages). At older ages tobacco seemed to be the cause of 15% (3017/20 420) of all deaths.ConclusionsAmong middle aged men the proportion of deaths caused by smoking is more than twice as big in Hong Kong now (33%) as in mainland China 10 years earlier. This supports predictions of a large increase in tobacco attributable mortality in China as a whole.

What is already known on this topic

China, with 20% of the world''s population, smokes 30% of the world''s cigarettes. Men smoke most, and the proportion of male deaths at ages 35-69 attributable to tobacco has been predicted to rise over the next few decades from 13% (in 1988) to about 33%In Hong Kong cigarette consumption reached its peak 20 years earlier than in mainland China, so the epidemic of male deaths from tobacco should now be at a more advanced stage

What this study adds

In the general population of Hong Kong in 1998 tobacco caused about 33% of all male deaths at ages 35-69 plus 5% of all female deaths, and hence 25% of all deaths at these agesIn the male smokers tobacco caused about half of all deaths at ages 35-69The hazards now seen in Hong Kong foreshadow a substantial increase in tobacco deaths among middle aged men in mainland China over the next few decades if current smoking patterns persist     

Molecular cloning and biochemical characterisation of proteases from Staphylococcus epidermidis.

We report the complete coding sequence and the partial amino acid sequence (determined by chemical sequencing) of Staphylococcus epidermidis extracellular cysteine (Ecp) and serine (Esp) proteases. The first enzyme shows an extended sequence similarity to Staphylococcus aureus cysteine protease (staphopain) and the second one resembles the serine protease produced by that species. The region directly upstream of the sequence coding for the mature protein in both enzymes displays significant homology to the profragments encoded by sspB and sspA, respectively, thus suggesting that the characterised enzymes may also be produced as proproteins. Furthermore, we report some biological properties of the cysteine protease, contributing to a better understanding of its role as a possible virulence factor. The proteolytic activity of this enzyme was rapidly and efficiently inhibited by human alpha-2-macroglobulin; however, human kininogen as well as cystatins (A, C and D) were not inhibitory. Moreover, the protease was capable of inactivating, by limited proteolysis, both alpha-1-antitrypsin and HMW-kininogen, but neither alpha-1-antichymotrypsin nor antithrombin III.     

Indian hedgehog signals independently of PTHrP to promote chondrocyte hypertrophy

Chondrocyte hypertrophy is an essential process required for endochondral bone formation. Proper regulation of chondrocyte hypertrophy is also required in postnatal cartilage homeostasis. Indian hedgehog (Ihh) and PTHrP signaling play crucial roles in regulating the onset of chondrocyte hypertrophy by forming a negative feedback loop, in which Ihh signaling regulates chondrocyte hypertrophy by controlling PTHrP expression. To understand whether there is a PTHrP-independent role of Ihh signaling in regulating chondrocyte hypertrophy, we have both activated and inactivated Ihh signaling in the absence of PTHrP during endochondral skeletal development. We found that upregulating Ihh signaling in the developing cartilage by treating PTHrP(-/-) limb explants with sonic hedgehog (Shh) protein in vitro, or overexpressing Ihh in the cartilage of PTHrP(-/-) embryos or inactivating patched 1 (Ptch1), a negative regulator of hedgehog (Hh) signaling, accelerated chondrocyte hypertrophy in the PTHrP(-/-) embryos. Conversely, when Hh signaling was blocked by cyclopamine or by removing Smoothened (Smo), a positive regulator of Hh signaling, chondrocyte hypertrophy was delayed in the PTHrP(-/-) embryo. Furthermore, we show that upregulated Hh signaling in the postnatal cartilage led to accelerated chondrocyte hypertrophy during secondary ossification, which in turn caused reduction of joint cartilage. Our results revealed a novel role of Ihh signaling in promoting chondrocyte hypertrophy independently of PTHrP, which is particularly important in postnatal cartilage development and homeostasis. In addition, we found that bone morphogenetic protein (Bmp) and Wnt/beta-catenin signaling in the cartilage may both mediate the effect of upregulated Ihh signaling in promoting chondrocyte hypertrophy.     

The tyrosine kinase p56lck is essential in coxsackievirus B3-mediated heart disease

Infections are thought to be important in the pathogenesis of many heart diseases. Coxsackievirus B3 (CVB3) has been linked to chronic dilated cardiomyopathy, a common cause of progressive heart disease, heart failure and sudden death. We show here that the sarcoma (Src) family kinase Lck (p56lck) is required for efficient CVB3 replication in T-cell lines and for viral replication and persistence in vivo. Whereas infection of wild-type mice with human pathogenic CVB3 caused acute and very severe myocarditis, meningitis, hepatitis, pancreatitis and dilated cardiomyopathy, mice lacking the p56lck gene were completely protected from CVB3-induced acute pathogenicity and chronic heart disease. These data identify a previously unknown function of Src family kinases and indicate that p56lck is the essential host factor that controls the replication and pathogenicity of CVB3.