The iron-responsive regulator Fur of Campylobacter jejuni is expressed from two separate promoters

A lacZ-based reporter gene system was used to identify the promoter of the Campylobacter jejuni iron-responsive gene regulator Fur. In other Gram-negative bacteria, the fur promoter is usually located directly upstream of the fur gene and is often autoregulated in response to iron. In this study we demonstrate that expression of the C. jejuni fur gene is controlled from two promoters located in front of the first and second open reading frames upstream of fur. Neither of these promoters was iron-regulated, and the presence of both promoters in front of fur gives higher expression of the lacZ reporter than with either promoter alone. Expression from two distal promoters might be a mechanism for regulating the level of the C. jejuni Fur protein in response to unknown stimuli.     

Chemosensory proteins from the proboscis of mamestra brassicae

Soluble, low molecular weight proteins were immunodetected in proboscis extracts of Mamestra brassicae males by Western blot, using antibodies raised against the general odorant-binding protein of the moth Antheraea polyphemus. The same antibodies weakly labelled the sensillum lymph and subcuticular space of sensilla styloconica on ultrathin sections of the proboscis. The morphology of sensilla styloconica is described. The immunodetected proteins yielded several N-terminal sequences, three of which showed strong affinity for tritiated analogues of pheromonal compounds of M. brassicae in binding assays. The cDNAs coding for these sequences were cloned and it was shown that the new proteins are related to the OS-D protein of DROSOPHILA: They are named chemosensory proteins (CSP-MBRA:A1-CSP-MBRA:A5 and CSP-MBRA:B1 and CSP-MBRA:B2) and may have an odorant-binding protein-like function. A common localization in both olfaction and taste organs suggests a physiological role depending on the cellular environment.     

Multiple acquisition of methanogenic archaeal symbionts by anaerobic ciliates

Anaerobic heterotrichous ciliates (Armophoridae and Clevelandellidae) possess hydrogenosomes that generate molecular hydrogen and ATP. This intracellular source of hydrogen provides the basis for a stable endosymbiotic association with methanogenic archaea. We analyzed the SSU rRNA genes of 18 heterotrichous anaerobic ciliates and their methanogenic endosymbionts in order to unravel the evolution of this mutualistic association. Here, we show that the anaerobic heterotrichous ciliates constitute at least three evolutionary lines. One group consists predominantly of gut-dwelling ciliates, and two to three, potentially four, additional clades comprise ciliates that thrive in freshwater sediments. Their methanogenic endosymbionts belong to only two different taxa that are closely related to free-living methanogenic archaea from the particular ecological niches. The close phylogenetic relationships between the endosymbionts and free-living methanogenic archaea argue for multiple acquisitions from environmental sources, notwithstanding the strictly vertical transmission of the endosymbionts. Since phylogenetic analysis of the small-subunit (SSU) rRNA genes of the hydrogenosomes of these ciliates indicates a descent from the mitochondria of aerobic ciliates, it is likely that anaerobic heterotrichous ciliates hosted endosymbiotic methanogens prior to their radiation. Therefore, our data strongly suggest multiple acquisitions and replacements of endosymbiotic methanogenic archaea during their host's adaptation to the various ecological niches.     

Changes in cytoskeletal organization in polyoma middle T antigen-transformed fibroblasts: involvement of protein phosphatase 2A and src tyrosine kinases

The major transforming activity of polyomavirus, middle T antigen, targets several cellular regulatory effectors including protein phosphatase 2A and src tyrosine kinases. Although transformed cells exhibit profound morphological changes, little is known about how middle T antigen-induced changes in the cellular regulatory environment specifically affect the cytoskeleton. We have investigated these changes in 10T(1/2) mouse fibroblasts transformed with polyoma middle T antigen. Immunofluorescence microscopy revealed that expression of middle T antigen (Pym T cells) depleted the stable (acetylated) microtubule array and increased the sensitivity of dynamic (tyrosinated) microtubules to nocodazole-induced disassembly. These effects were associated with a modest but statistically significant (P相似文献   

Regulation of tissue inhibitor of metalloproteinases-1 in rat Sertoli cells: induction by germ cell residual bodies, interleukin-1alpha, and second messengers

In the testis, FSH has been shown to induce the expression and secretion of tissue inhibitor of metalloproteinases-1 (TIMP-1) from Sertoli cells in vitro. This study was performed to elucidate further the cellular origin of testicular TIMP-1 and its expression by hormonal and paracrine factors. This is the first report on the expression of testicular TIMP-1 in vivo. TIMP-1 mRNA in whole testis was decreased after hypophysectomy and strongly increased by the injection of FSH-S17 to hypophysectomized rats. Primary cultures of both peritubular and Sertoli cells showed basal expression of TIMP-1 mRNA. In contrast, we were unable to detect TIMP-1 mRNA in Leydig cells, freshly isolated immature germ cells (primary spermatocytes and spermatids), or residual bodies. We further show that treatment of Sertoli cells with 8-(4-chlorophenyl)thio-cAMP (8-CPTcAMP) in combination with 12-O-tetradecanoylphorbol 13-acetate (TPA) or Ca(2+) inducers (calcium ionophore A23187 or thapsigargin) had additive (TPA) and synergistic effects (Ca(2+)) on the level of TIMP-1 mRNA and secreted protein. We also show that both the level of TIMP-1 mRNA and secreted protein from Sertoli cells were strongly increased by residual bodies, as well as by the cytokine interleukin-1alpha. TIMP-1 was not up-regulated by either 8-CPTcAMP or interleukin-1alpha in peritubular cells. In contrast to the regulated secretory fraction of TIMP-1, we also detected constitutively expressed immunoreactive TIMP-1 in the nucleus of Sertoli cells, suggesting a role of nuclear TIMP-1 in these cells. In conclusion, our data show that secretion of TIMP-1 from Sertoli cells is highly regulated by hormonal and local processes in the testis, indicating that TIMP-1 is of physiological importance during both testicular development and spermatogenesis.     

The cytotoxic lipid peroxidation product, 4-hydroxy-2-nonenal, specifically inhibits decarboxylating dehydrogenases in the matrix of plant mitochondria

4-Hydroxy-2-nonenal (HNE), a cytotoxic product of lipid peroxidation, inhibits O(2) consumption by potato tuber mitochondria. 2-Oxoglutarate dehydrogenase (OGDC), pyruvate dehydrogenase complex (PDC) (both 80% inhibited) and NAD-malic enzyme (50% inhibited) are its major targets. Mitochondrial proteins identified by reaction with antibodies raised to lipoic acid lost this antigenicity following HNE treatment. These proteins were identified as acetyltransferases of PDC (78 kDa and 55 kDa), succinyltransferases of OGDC (50 kDa and 48 kDa) and glycine decarboxylase H protein (17 kDa). The significance of the effect of these inhibitions on the impact of lipid peroxidation and plant respiratory functions is discussed.     

Proteoglycan metabolism during repair of the ruptured medial collateral ligament in skeletally mature rabbits

The metabolism of the chondroitin/dermatan sulfate (CS/DS) proteoglycans (PGs) decorin and biglycan is markedly altered during short-term (3-6 weeks) and long-term (40 weeks-2 years) repair of surgically ruptured medial collateral ligaments from mature rabbits. A PG-rich extracellular matrix accumulates in injury gaps by 3 weeks postsurgery and extends into tissue regions containing the original ligaments, and elevated PG levels remain apparent up to 2 years postinjury. CS/DS PGs were prepared from such ligaments and identified after SDS-polyacrylamide gel electrophoresis by Alcian blue staining or immunoblotting. In normal ligaments, decorin is the most abundant proteoglycan (accounting for approximately 80% of the total); the remainder is biglycan and a large PG, possibly versican. In repairing ligaments, decorin is barely detected, but instead a large proteoglycan and abundant amounts of biglycan accumulate. Biglycan is present in two forms in repairing ligaments, and they can be separated on SDS-PAGE into 200- and 140-kDa forms. The slower migrating species is absent in normal ligaments and may represent a different glycoform (containing either a single or two short chondroitin/dermatan sulfate chains) of biglycan. Alteration in PG expression and posttranslational processing during medial collateral ligament repair are similar to those reported for repair and scar formation of other connective tissues. The accumulation of biglycan observed here may interfere with proper collagen network remodeling and may lead to persistent inflammatory and matrix turnover processes, thus preventing restoration of a long-term functional ligament tissue.     

Identification of two focal adhesion targeting sequences in the adapter molecule p130(Cas)

The adapter molecule CAS is localized primarily within focal adhesions in fibroblasts. Because many of the cellular functions attributed to CAS are likely to be dependent on its presence in focal adhesions, this study was undertaken to identify regions of the protein that are involved in its localization. The SH3 domain of CAS, when expressed in isolation from the rest of the protein, was able to target to focal adhesions, whereas a variant containing a point mutation that rendered the SH3 domain unable to associate with FAK remained cytoplasmic. However, in the context of full-length CAS, this mutation did not prevent CAS localization to focal adhesions. Two other variants of CAS that contained deletions of either the SH3 domain alone, or the SH3 domain together with an adjoining proline-rich region, also retained the capacity to localize to focal adhesions. A second focal adhesion targeting region was mapped to the extreme carboxy terminus of CAS. The identification of this second focal adhesion targeting domain in CAS ascribes a previously unknown function to the highly conserved C terminus of CAS. The regulated targeting of CAS to focal adhesions by two independent domains may reflect the important role of CAS within this subcellular compartment.     

Comparative mapping of quantitative trait loci sculpting the curd of Brassica oleracea

The enlarged inflorescence (curd) of cauliflower and broccoli provide not only a popular vegetable for human consumption, but also a unique opportunity for scientists who seek to understand the genetic basis of plant growth and development. By the comparison of quantitative trait loci (QTL) maps constructed from three different F(2) populations, we identified a total of 86 QTL that control eight curd-related traits in Brassica oleracea. The 86 QTL may reflect allelic variation in as few as 67 different genetic loci and 54 ancestral genes. Although the locations of QTL affecting a trait occasionally corresponded between different populations or between different homeologous Brassica chromosomes, our data supported other molecular and morphological data in suggesting that the Brassica genus is rapidly evolving. Comparative data enabled us to identify a number of candidate genes from Arabidopsis that warrant further investigation to determine if some of them might account for Brassica QTL. The Arabidopsis/Brassica system is an important example of both the challenges and opportunities associated with extrapolation of genomic information from facile models to large-genome taxa including major crops.