Distinct expression and localization of serine protease HtrA1 in human endometrium and first-trimester placenta.

Mammalian embryos cannot survive without the placenta. Development of the human placenta requires trophoblast proliferation, differentiation, and invasion as well as highly coordinated modulation of the maternal uterus. HtrA1 is a member of the recently identified mammalian HtrA (high temperature requirement factor A) serine protease family with a high level of expression in the placenta. In this study, we examined whether HtrA1 expression (mRNA and protein) is associated with placental development in the human. HtrA1 is up-regulated in both endometrial glands and decidual cells during endometrial preparation for embryo implantation and during first-trimester pregnancy at placentation. HtrA1 expression was also detected in certain trophoblast subtypes during early pregnancy. The villous syncytiotrophoblast and cytotrophoblast showed the strongest expression while the interstitial extravillous trophoblast showed the lowest or no expression of HtrA1. The distinct distribution of HtrA1 at the maternal-trophoblast interface suggests that HtrA1 may play a role in placental development.     

A2 adenosine receptors inhibit calcium influx through L-type calcium channels in rod photoreceptors of the salamander retina.

Presynaptic inhibition is a major mechanism for regulating synaptic transmission in the CNS and adenosine inhibits Ca(2+) currents (I(Ca)) to reduce transmitter release at several synapses. Rod photoreceptors possess L-type Ca(2+) channels that regulate the release of L-glutamate. In the retina, adenosine is released in the dark when L-glutamate release is maximal. We tested whether adenosine inhibits I(Ca) and intracellular Ca(2+) increases in rod photoreceptors in retinal slice and isolated cell preparations. Adenosine inhibited both I(Ca) and the [Ca(2+)]i increase evoked by depolarization in a dose-dependent manner with approximately 25% inhibition at 50 microM. An A2-selective agonist, (N(6)-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)-ethyl]adenosine) (DPMA), but not the A1- or A3-selective agonists, (R)-N(6)-(1-methyl-2-phenylethyl)adenosine and N(6)-2-(4-aminophenyl)ethyladenosine, also inhibited I(Ca) and depolarization-induced [Ca(2+)]i increases. An inhibitor of protein kinase A (PKA), Rp-cAMPS, blocked the effects of DPMA on both I(Ca) and the depolarization-evoked [Ca(2+)]i increase in rods. The results suggest that activation of A2 receptors stimulates PKA to inhibit L-type Ca(2+) channels in rods resulting in a decreased Ca(2+) influx that should suppress glutamate release.     

Non-invasive and quantitative near-infrared haemoglobin spectrometry in the piglet brain during hypoxic stress, using a frequency-domain multidistance instrument

The frequency-domain multiple-distance (FDMD) method is capable of measuring the absolute absorption and reduced scattering coefficients of optically turbid media. Absolute measurement of absorption at two near-infrared (NIR) wavelengths makes possible the quantitation of tissue haemoglobin concentration and tissue haemoglobin oxygen-saturation (StO2). However, errors are introduced by the uncertainties of background absorption and the dissimilarities between real tissues and the simplified mathematical model on which these measurements are based. An FDMD-based tissue instrument has been used for the monitoring of tissue haemoglobin concentration and oxygenation in the brain of newborn piglets during periods of hypoxia and hyperoxia. These tissue haemoglobin saturation values were compared with arterial saturation (SaO2) and venous saturation (SvO2) measured by blood gas analyses. A linear correlation was observed between StO2 and the average of SaO2 and SvO2. However, StO2 is not equal to any fixed weighted average of SaO2 and SvO2 unless we introduce an effective background tissue absorption. The magnitude of the background absorption was about 0.08 cm(-1) at 758 nm and 0.06 cm(-1) at 830 nm, and it was nearly consistent between piglets. The origin of this 'effective' background absorption may be real, an artefact caused by the application of a simplified model to a complex sample, or a combination of factors.     

CXCR4 and CCR5 expression on CD4+ T cells in vivo and HIV-1 antigen beta-chemokine production in vitro after treatment with HIV-1 immunogen (REMUNE)

BACKGROUND: The chemokine receptors CXCR4 and CCR5 have been identified as the major coreceptors for HIV-1 on CD4+ cells and macrophages. The natural ligands for these receptors are SDF-1 and the beta-chemokines (MIP-1alpha, MIP-1beta, RANTES), respectively, and are the products of a variety of immune cells, including CD8+ T lymphocytes. STUDY DESIGN/METHODS: We hypothesized that the ability to stimulate the natural ligands for these receptors using an immune based therapy might influence in vivo chemokine receptor expression. RESULTS: In vivo CXCR4 expression remained stable after treatment with an HIV-1 Immunogen (REMUNE), whereas CCR5 expression on CD4+ T cells decreased (p < .05). Furthermore, HIV-1 antigen-specific production of beta-chemokines in vitro was also augmented (P < .05). CONCLUSIONS: These preliminary results suggest that this HIV-1-specific immune-based therapy can stimulate antigen-specific beta-chemokine production in vitro and downregulate CCR5 receptor expression on CD4 cells in vivo.     

Cellular dysfunction in the diabetic fibroblast: impairment in migration,vascular endothelial growth factor production,and response to hypoxia

Although it is known that systemic diseases such as diabetes result in impaired wound healing, the mechanism for this impairment is not understood. Because fibroblasts are essential for wound repair, we compared the in vitro behavior of fibroblasts cultured from diabetic, leptin receptor-deficient (db/db) mice with wild-type fibroblasts from mice of the same genetic background in processes important during tissue repair. Adult diabetic mouse fibroblast migration exhibited a 75% reduction in migration compared to normal fibroblasts (P < 0.001) and was not significantly stimulated by hypoxia (1% O(2)), whereas wild-type fibroblast migration was up-regulated nearly twofold in hypoxic conditions (P < 0.05). Diabetic fibroblasts produced twice the amount of pro-matrix metalloproteinase-9 as normal fibroblasts, as measured by both gelatin zymography and enzyme-linked immunosorbent assay (P < 0.05). Adult diabetic fibroblasts exhibited a sevenfold impairment in vascular endothelial growth factor (VEGF) production (4.5 +/- 1.3 pg/ml versus 34.8 +/- 3.3 pg/ml, P < 0.001) compared to wild-type fibroblasts. Moreover, wild-type fibroblast production of VEGF increased threefold in response to hypoxia, whereas diabetic fibroblast production of VEGF was not up-regulated in hypoxic conditions (P < 0.001). To address the question whether these differences resulted from chronic hyperglycemia or absence of the leptin receptor, fibroblasts were harvested from newborn db/db mice before the onset of diabetes (4 to 5 weeks old). These fibroblasts showed no impairments in VEGF production under basal or hypoxic conditions, confirming that the results from db/db fibroblasts in mature mice resulted from the diabetic state and were not because of alterations in the leptin-leptin receptor axis. Markers of cellular viability including proliferation and senescence were not significantly different between diabetic and wild-type fibroblasts. We conclude that, in vitro, diabetic fibroblasts show selective impairments in discrete cellular processes critical for tissue repair including cellular migration, VEGF production, and the response to hypoxia. The VEGF abnormalities developed concurrently with the onset of hyperglycemia and were not seen in normoglycemic, leptin receptor-deficient db/db mice. These observations support a role for fibroblast dysfunction in the impaired wound healing observed in human diabetics, and also suggest a mechanism for the poor clinical outcomes that occur after ischemic injury in diabetic patients.     

In-vitro recombination in rad and rnc mutants of Saccharomyces cerevisiae

Summary Extracts of S. cerevisiae cells can catalyze homologous recombination between plasmids in vitro. Extracts prepared from rad50, rad52 or rad54 disruption mutants all have reduced recombinational activity compared to wild-type. The rad52 and rad54 extracts are more impaired in the recombination of plasmids containing double-strand breaks than of intact plasmids, whereas rad50 extracts are deficient equally for both types of substrate. The nuclease RhoNuc (previously designated yNucR), encoded by the RNC1 (previously designated NUC2) gene and regulated by the RAD52 gene, is not required for recombination when one substrate is single-stranded but is essential for the majority of recombination events when both substrates are double-stranded. Furthermore, elimination of this nuclease restores recombination in rad52 extracts to levels comparable to those in wild-type extracts.     

Teenagers' health concerns: implications for primary health care professionals.

Four hundred and eighty five students, aged 13-15 years, at nine comprehensive schools in the London borough of Brent completed a questionnaire about health-related behaviours and health concerns. Among general health concerns, most prominent were weight, acne, nutrition and exercise. There appeared to be a considerable unmet need to discuss sexual development, sexually transmitted diseases and contraception. A substantial proportion (16% of the girls and 3% of the boys) reported sexual abuse, but few wished to discuss this with a doctor or nurse. Although a relatively high percentage of the students smoked and a smaller percentage used alcohol or drugs regularly, there was little concern or interest in discussing these matters with a health professional. Most of the schools did not have a formal health education programme, and in none of them were health professionals available for discussion of the issues under study. There appears to be a need for more comprehensive health education in schools and for primary health care professionals, particularly general practitioners to raise these issues opportunistically with their teenage patients.     

Disk diffusion testing with polymyxin and amikacin for differentiation of Mycobacterium fortuitum and Mycobacterium chelonei.

Disk diffusion is one method of susceptibility testing of the Mycobacterium fortuitum complex to antibacterial agents. We utilized disks of polymyxin B (300 U), amikacin, and kanamycin to determine whether they could also be used for species identification when compared with standard biochemical methods. With the polymyxin disk, 100% of 75 M. fortuitum strains produced zones of inhibition, whereas none (0%) of 58 Mycobacterium chelonei subspecies abscessus and chelonei strains had any zone of inhibition. With the amikacin disk, 99% of M. fortuitum biovariant fortuitum had zones of greater than or equal to 30 mm compared with 6% of M. chelonei. The rare M. chelonei-like organisms gave variable results, and 42% of the unnamed "third group" biovariant of M. fortuitum exhibited an unusual but diagnostic pattern of small zone sizes to amikacin and no zone to kanamycin. The kanamycin disk was otherwise not helpful, although it resulted in larger zone sizes for M. chelonei than did amikacin. Thus, disk diffusion susceptibilities which include these disks (especially polymyxin) will provide presumptive evidence of species as well as susceptibility data.