C/EBPß LIP and c-Jun synergize to regulate expression of the murine progesterone receptor.

CCAAT/enhancer binding protein ß (C/EBPß) is required for murine mammary ductal morphogenesis and alveologenesis. Progesterone is critical for proliferation and alveologenesis in adult mammary glands, and there is a similar requirement for progesterone receptor isoform B (PRB) in alveologenesis. We examined C/EBPß regulation of PR expression. All three C/EBPß isoforms, including typically inhibitory LIP, transactivated the PR promoter. LIP, particularly, strongly synergized with c-Jun to drive PR transcription. Endogenous C/EBPß and c-Jun stimulated a PR promoter-reporter and these two factors showed promoter occupancy on the endogenous PR gene. Additionally, LIP overexpression elevated endogenous PR protein expression. In pregnancy, both PRB and the relative abundance of LIP among C/EBPß isoforms increase. Consistent with a role in PRB expression, in vivo C/EBPß and PR isoform A expression showed mutually exclusive localization in mammary epithelium, while C/EBPß and PRB largely co-localized. We suggest a critical role for C/EBPß, particularly LIP, in PRB expression.

Antibiotic Resistance of Escherichia coli Serotypes from Cochin Estuary.

This study aimed at detecting the prevalence of antibiotic-resistant serotypes of Escherichia coli in Cochin estuary, India. E. coli strains were isolated during the period January 2010-December 2011 from five different stations set at Cochin estuary. Water samples from five different stations in Cochin estuary were collected on a monthly basis for a period of two years. Isolates were serotyped, antibiogram-phenotyped for twelve antimicrobial agents, and genotyped by polymerase chain reaction for uid gene that codes for ß-D-glucuronidase. These E. coli strains from Cochin estuary were tested against twelve antibiotics to determine the prevalence of multiple antibiotic resistance among them. The results revealed that more than 53.33% of the isolates were multiple antibiotic resistant. Thirteen isolates showed resistance to sulphonamides and two of them contained the sul 1 gene. Class 1 integrons were detected in two E. coli strains which were resistant to more than seven antibiotics. In the present study, O serotyping, antibiotic sensitivity, and polymerase chain reaction were employed with the purpose of establishing the present distribution of multiple antibiotic-resistant serotypes, associated with E. coli isolated from different parts of Cochin estuary.

Propofol's effects on phagocytosis, proliferation, nitrate production, and cytokine secretion in pressure-stimulated microglial cells.

BACKGROUND: Intracranial hypertension complicates severe traumatic brain injury frequently and might be associated with poor outcomes. Traumatic brain injury induces a neuroinflammatory response by microglial activation and upregulation of proinflammatory cytokines, such as interleukin-1ß, tumor necrosis factor alpha, and interleukin-6. To elucidate the effect of increased intracranial pressure on microglial function, we studied the effects of increased extracellular pressure on primary human microglial cell phagocytosis, proliferation, cytokine secretion, and total nitrate production. In addition, because many patients receive propofol during anesthesia or intensive care unit sedation, we evaluated whether propofol alters the effects of pressure. METHODS: Human microglial cells were pretreated with (2.5-20 µg/mL) propofol or Intralipid as a vehicle control were incubated at ambient atmospheric pressure or at 15 or 30 mm Hg increased pressure for 2 h for phagocytosis assays or 24 h for proliferation, cytokine secretion, and total nitrate production studies. Phagocytosis was determined by incorporation of intracellular fluorescent latex beads. Tumor necrosis factor alpha, interleukin-1ß, and interleukin-6 were assayed by sandwich enzyme-linked immunosorbent assay and total nitrate by Greiss reagent. RESULTS: Increased extracellular pressure stimulated phagocytosis versus untreated microglial cells or cells treated with an Intralipid vehicle control. Propofol also stimulated microglial phagocytosis at ambient pressure. Increased pressure, however, decreased phagocytosis in the presence of propofol. Pressure also increased microglial tumor necrosis factor-α and interleukin-1ß secretion and propofol pretreatment blocked the pressure-stimulated effect. Interleukin-6 production was not altered either by pressure or by propofol. Pressure also induced total nitrate secretion, and propofol pretreatment decreased basal as well as pressure-induced microglial nitrate production. CONCLUSION: Extracellular pressures consistent with increased intracranial pressure after a head injury activate inflammatory signals in human primary microglial cells in vitro, stimulating phagocytosis, proliferation, tumor necrosis factor-α, interleukin-1ß, and total nitrate secretion but not affecting interleukin-6. Such inflammatory events may contribute to the worsened prognosis of traumatic brain injury after increased intracranial pressure. Because propofol alleviated these potentially proinflammatory effects, these results suggest that the inflammatory cascade activated by intracranial pressure might be targeted by propofol in patients with increased intracranial pressure after traumatic brain injury.

Progesterone receptor A-regulated gene expression in mammary organoid cultures.

Progesterone, through the progesterone receptor (PR), promotes development of the normal mammary gland and is implicated in the etiology of breast cancer. We identified PRA-regulated genes by microarray analysis of cultured epithelial organoids derived from pubertal and adult mouse mammary glands, developmental stages with differing progesterone responsiveness. Microarray analysis showed significant progestin (R5020)-regulation of 162 genes in pubertal organoids and 104 genes in adult organoids, with 68 genes regulated at both developmental stages. Greater induction of receptor activator of NFkappaB ligand and calcitonin expression was observed in adult organoids, suggesting possible roles in the differential progesterone responsiveness of the adult and pubertal mammary glands. Analysis of the R5020-responsive transcriptome revealed several enriched biological processes including cell adhesion, immune response, and survival. R5020 both induced Agtr1 and potentiated angiotensin II-stimulated proliferation, highlighting the functional significance of the latter process. Striking up-regulation of genes involved in innate immunity processes included the leukocyte chemoattractants serum amyloid A1, 2 and 3 (Saa1, 2, 3). In vivo analysis revealed that progesterone treatment increased SAA1 protein expression and leukocyte density in mammary gland regions undergoing epithelial expansion. These studies reveal novel targets of PRA in mammary epithelial cells and novel linkages of progesterone action during mammary gland development.

Strain-specific differences in the mechanisms of progesterone regulation of murine mammary gland development.

Progesterone (P) is required for normal mammary gland development, and is implicated in the etiology of mammary cancer in rodents and humans. We analyzed mammary gland developmental responses to P and estrogen (E) in two strains of mice (BALB/c and C57BL/6) that exhibit differences in ductal development at sexual maturity and alveologenesis during pregnancy. C57BL/6 mice exhibited reduced proliferative and morphological responses to P. Analysis of known mediators of sidebranching and alveologenesis revealed that reduced P-induced expression of P receptor isoform B and receptor activator of nuclear factor-kappaB ligand (RANKL), as well as altered expression and regulation of cyclin D1, CCAAT/enhancer binding protein beta, and the downstream effectors of RANKL, nuclear Id2 and p21, contribute significantly to the reduced P responsiveness of the C57BL/6 mammary gland. In contrast, E responsiveness was greater in C57BL/6 than in BALB/c glands. E may play a compensatory role in C57BL/6 alveologenesis through its effect on the induction and activation of signal transducer and activator of transcription 5a, a known regulator of RANKL. These observations suggest that in human populations with heterogeneous genetic backgrounds, individuals may respond differentially to the same hormone. Thus, genetic diversity may have a role in determining the effects of P in normal mammary development and tumorigenesis.

C/EBPbeta serine 64, a phosphoacceptor site, has a critical role in LPS-induced IL-6 and MCP-1 transcription.

C/EBPbeta is a member of the CCAAT/enhancer binding protein family of transcription factors and has been shown to be a critical transcriptional regulator of various proinflammatory genes, including IL-6 and MCP-1. Serine 64 in the transactivation domain of C/EBPbeta has recently been identified as a Ras-induced phosphoacceptor site. The integrity of serine 64 along with threonine 189 is important for the Ha-ras(V12)-induced transformation of NIH3T3 cells, however no target genes dependent upon serine 64 for their expression have been reported. In order to evaluate a potential role of serine 64 in C/EBPbeta-regulated cytokine expression, we expressed a form of C/EBPbeta with an alanine substitution at serine 64 (C/EBPbeta(S64A)) in P388 murine B lymphoblasts, which lack endogenous C/EBPbeta expression and are normally unresponsive to LPS for expression of IL-6 and MCP-1. In comparison to wild type C/EBPbeta, which robustly supports the LPS-induced expression of IL-6 and MCP-1, C/EBPbeta(S64A) was severely impaired in its ability to support the LPS-induced transcription of IL-6 and MCP-1. Furthermore, LPS stimulation increased the level of phosphorylation detected at serine 64. Thus, serine 64, probably through its phosphorylation, is a critical determinant of C/EBPbeta activity in the transcription of IL-6 and MCP-1.