Identification of a new ribosomal protection type of tetracycline resistance gene,tet(36), from swine manure pits

Previously, only one ribosome protection type of a tetracycline resistance gene, tetQ, had been identified in Bacteroides spp. During an investigation of anaerobic bacteria present in swine feces and manure storage pits, a tetracycline-resistant Bacteroides strain was isolated. Subsequent analysis showed that this new Bacteroides strain, Bacteroides sp. strain 139, did not contain tetQ but contained a previously unidentified tetracycline resistance gene. Sequence analysis showed that the tetracycline resistance gene from Bacteroides sp. strain 139 encoded a protein (designated Tet 36) that defines a new class of ribosome protection types of tetracycline resistance. Tet 36 has 60% amino acid identity over 640 aa to TetQ and between 31 and 49% amino acid identity to the nine other ribosome protection types of tetracycline resistance genes. The tet(36) region was not observed to transfer from Bacteroides sp. strain 139 to another Bacteroides sp. under laboratory conditions. Yet tet(36) was found in other genera of bacteria isolated from the same swine manure pits and from swine feces. Phylogenetic analysis of the tet(36)-containing isolates indicated that tet(36) was present not only in the Cytophaga-Flavobacter-Bacteroides group to which Bacteroides sp. strain 139 belongs but also in gram-positive genera and gram-negative proteobacteria, indicating that horizontal transfer of tet(36) is occurring between these divergent phylogenetic groups in the farm environment.     

A new Bacteroides conjugative transposon that carries an ermB gene

The erythromycin resistance gene ermB has been found in a variety of gram-positive bacteria. This gene has also been found in Bacteroides species but only in six recently isolated strains; thus, the gene seems to have entered this genus only recently. One of the six Bacteroides ermB-containing isolates, WH207, could transfer ermB to Bacteroides thetaiotaomicron strain BT4001 by conjugation. WH207 was identified as a Bacteroides uniformis strain based on the sequence of its 16S rRNA gene. Results of pulsed-field gel electrophoresis experiments demonstrated that the transferring element was normally integrated into the Bacteroides chromosome. The element was estimated from pulsed-field gel data to be about 100 kb in size. Since the element appeared to be a conjugative transposon (CTn), it was designated CTnBST. CTnBST was able to mobilize coresident plasmids and the circular form of the mobilizable transposon NBU1 to Bacteroides and Escherichia coli recipients. A 13-kb segment that contained ermB was cloned and sequenced. Most of the open reading frames in this region had little similarity at the amino acid sequence level to any proteins in the sequence databases, but a 1,723-bp DNA segment that included a 950-bp segment downstream of ermB had a DNA sequence that was virtually identical to that of a segment of DNA found previously in a Clostridium perfringens strain. This finding, together with the finding that ermB is located on a CTn, supports the hypothesis that CTnBST could have entered Bacteroides from some other genus, possibly from gram-positive bacteria. Moreover, this finding supports the hypothesis that many transmissible antibiotic resistance genes in Bacteroides are carried on CTns.     

Leaf extracts of Carlowrightia cordifolia induce macrophage nitric oxide production

Carlowrightia cordifolia (Acanthaceae) is a medicinal plant used in northeastern Mexico as a traditional remedy against inflammation. As tissue release of nitric oxide (NO) has been correlated with both inflammatory and anti-inflammatory processes, the aim of this study was to determine the effect of C. cordifolia leaf extracts on macrophage NO production. Lipopolysaccharide (LPS)-stimulated and non-LPS-stimulated mouse peritoneal macrophages were incubated with aqueous, ethanol, methanol and hexane extracts of C. cordifolia leaves. All extracts inhibited NO release from LPS-stimulated macrophages, with methanol and hexane extracts showing the greatest inhibition. On the other hand, macrophage cultures treated with extracts without LPS-stimulation produced high releases of NO. These unexpected results suggest two different ways by which leaf extracts may act, depending on cell status. On the other hand, data on NO activity in relation to inflammatory/anti-inflammatory auto-regulatory feedback and high concentrations of NO release by non-stimulated macrophages agreed with the hypothesis that NO may have an inhibitory effect in vascular inflammation.     

Steroidogenesis and apoptosis in the mammalian ovary

Ovarian cell death is an essential process for the homeostasis of ovarian function in human and other mammalian species. It ensures the selection of the dominant follicle and the demise of excess follicles. In turn, this process minimizes the possibility of multiple embryo development during pregnancy and assures the development of few, but healthy embryos. Degeneration of the old corpora lutea in each estrous/menstrual cycle by programmed cell death is essential to maintain the normal cyclicity of ovarian steroidogenesis. Although there are multiple pathways that can determine cell death or survival, crosstalk among endocrine, paracrine and autocrine factors, as well as among protooncogenes, tumor suppressor genes, survival genes and death genes, plays an important role in determining the fate of ovarian somatic and germ cells. The establishment of immortalized rat and human steroidogenic granulosa cell lines and the investigation of pure populations of primary granulosa cells allows systematic studies of the mechanisms that control steroidogenesis and apoptosis in granulosa cells. We have discovered that during initial stages of granulosa cell apoptosis progesterone production does not decrease. In contrast, we found that it is elevated up to 24h following the onset of the apoptotic stimuli exerted by starvation, cAMP, p53 or TNF-alpha stimulation, before total cell collapse. These observations raise the possibility for an alternative unique apoptotic pathway, one not involving mitochondrial Cyt C release associated with the destruction of mitochondrial structure and steroidogenic function. Using mRNA from apoptotic cells and affymetrix DNA microarray technology we discovered that granzyme B, a protease that normally resides in T cytotoxic lymphocytes and natural killer cells of the immune system is expressed and activated in granulosa cells. Thus, the apoptotic signals could bypass mitochondrial signals for apoptosis, which can preserve their steroidogenic activity until complete cell destruction. This unique apoptotic pathway assures cyclicity of estradiol and progesterone release in the estrous/menstruous cycle even during the initial stages of apoptosis.     

Fitness of Cuscuta salina (Convolvulaceae) parasitizing Beta vulgaris (Chenopodiaceae) grown under different salinity regimes

The distribution of saltmarsh dodder (Cuscuta salina) worldwide is restricted to areas of high salinity, where it parasitizes a variety of salt-tolerant plants. Because dodders do not maintain root connections to the soil, this pattern of parasitization may be related to the effects of salt stress on the host that increase the ability of attached dodders to more easily transfer host contents. This study explored whether a saline host environment is required for successful infection and whether stem contents of potential hosts become more concentrated in response to salinity. Fecundity of dodder was highest when hosts (Beta vulgaris) were grown either without salt or at high (250 mmol/L) salinity; it produced significantly fewer flowers and fruit at intermediate salinities. Stem constituents of two unparasitized host species had high conductivity and elevated levels of dissolved sugars at increasing salinities. Nitrate content of stems was also increased by salinity, but declined at the extreme salinity (400 mmol/L). Salinity effects on host suitability may partially explain differences in growth and vigor of C. salina in certain areas of salt marsh, but information on nonrandom dispersal of C. salina seeds may be needed to fully explain this parasite's distribution.     

Characterization of genes involved in modulation of conjugal transfer of the Bacteroides conjugative transposon CTnDOT

In previous studies we identified an 18-kb region of the Bacteroides conjugative transposon CTnDOT that was sufficient for mobilization of coresident plasmids and unlinked integrated elements, as well as self-transfer from Bacteroides to Escherichia coli. When this 18-kb region was cloned on a plasmid (pLYL72), the plasmid transferred itself constitutively in the absence of a coresident conjugative transposon. However, when this plasmid was present in a Bacteroides strain containing a coresident conjugative transposon, conjugal transfer was repressed in the absence of tetracycline and enhanced in the presence of tetracycline. These results suggested that a negative and a positive regulator of conjugal transfer were encoded outside the transfer region of the CTnDOT element. In this work, a minimal and inducible transfer system was constructed and used in transfer and Western blot analyses to identify the differentially regulated genes from CTnDOT responsible for the enhancement and repression of pLYL72 conjugal transfer. Both of these regulatory functions have been localized to a region of the CTnDOT element that is essential for CTn excision. In the presence of tetracycline, the regulatory protein RteC activates the expression of a putative topoisomerase gene, exc, which in turn results in an increase in transfer protein expression and a concomitant 100- to 1,000-fold increase in the frequency of pLYL72 transfer. Our results also suggest that since exc alone cannot result in enhancement of transfer, other factors encoded upstream of exc are also required. Conversely, in the absence of tetracycline, a gene located near the 3' end of exc is responsible for the repression of transfer protein expression and also results in a 100- to 1,000-fold decrease in the frequency of pLYL72 transfer.     

Memory CD8+ T cells vary in differentiation phenotype in different persistent virus infections

The viruses HIV-1, Epstein-Barr virus (EBV), cytomegalovirus (CMV) and hepatitis C virus (HCV) are characterized by the establishment of lifelong infection in the human host, where their replication is thought to be tightly controlled by virus-specific CD8+ T cells. Here we present detailed studies of the differentiation phenotype of these cells, which can be separated into three distinct subsets based on expression of the costimulatory receptors CD28 and CD27. Whereas CD8+ T cells specific for HIV, EBV and HCV exhibit similar characteristics during primary infection, there are significant enrichments at different stages of cellular differentiation in the chronic phase of persistent infection according to the viral specificity, which suggests that distinct memory T-cell populations are established in different virus infections. These findings challenge the current definitions of memory and effector subsets in humans, and suggest that ascribing effector and memory functions to subsets with different differentiation phenotypes is no longer appropriate.