Environmental modulation of microcystin and ß-N-methylamino-L-alanine as a function of nitrogen availability.

The most significant modulators of the cyanotoxins microcystin and ß-N-methylamino-L-alanine in laboratory cyanobacterial cultures are the concentration of growth-medium combined nitrogen and nitrogen uptake rate. The lack of field studies that support these observations led us to investigate the cellular content of these cyanotoxins in cyanobacterial bloom material isolated from a freshwater impoundment and to compare these to the combined nitrogen availability. We established that these toxins typically occur in an inverse relationship in nature and that their presence is mainly dependent on the environmental combined nitrogen concentration, with cellular microcystin present at exogenous combined nitrogen concentrations of 29 µM and higher and cellular BMAA correlating negatively with exogenous nitrogen at concentrations below 40 µM. Furthermore, opposing nutrient and light gradients that form in dense cyanobacterial blooms may result in both microcystin and BMAA being present at a single sampling site.

Ageing and long-term CD4 cell count trends in HIV-positive patients with 5 years or more combination antiretroviral therapy experience.

OBJECTIVES: The aim of this study was to describe the long-term changes in CD4 cell counts beyond 5 years of combination antiretroviral therapy (cART). If natural ageing leads to a long-term decline in the immune system via low-grade chronic immune activation/inflammation, then one might expect to see a greater or earlier decline in CD4 counts in older HIV-positive patients with increasing duration of cART. METHODS: Retrospective and prospective data were examined from long-term virologically stable HIV-positive adults from the Australian HIV Observational Database. We estimated mean CD4 cell count changes following the completion of 5 years of cART using linear mixed models. RESULTS: A total of 37 916 CD4 measurements were observed for 892 patients over a combined total of 9753 patient-years. Older patients (> 50 years old) at cART initiation had estimated mean (95% confidence interval) changes in CD4 counts by year-5 CD4 count strata (< 500, 500-750 and > 750 cells/µL) of 14 (7 to 21), 3 (-5 to 11) and -6 (-17 to 4) cells/µL/year. Of the CD4 cell count rates of change estimated, none were indicative of long-term declines in CD4 cell counts. CONCLUSIONS: Our results suggest that duration of cART and increasing age do not result in decreasing mean changes in CD4 cell counts for long-term virologically suppressed patients, indicating that the level of immune recovery achieved during the first 5 years of treatment is sustained through long-term cART.

Systematic analysis of cell cycle effects of common drugs leads to the discovery of a suppressive interaction between gemfibrozil and fluoxetine.

Screening chemical libraries to identify compounds that affect overall cell proliferation is common. However, in most cases, it is not known whether the compounds tested alter the timing of particular cell cycle transitions. Here, we evaluated an FDA-approved drug library to identify pharmaceuticals that alter cell cycle progression in yeast, using DNA content measurements by flow cytometry. This approach revealed strong cell cycle effects of several commonly used pharmaceuticals. We show that the antilipemic gemfibrozil delays initiation of DNA replication, while cells treated with the antidepressant fluoxetine severely delay progression through mitosis. Based on their effects on cell cycle progression, we also examined cell proliferation in the presence of both compounds. We discovered a strong suppressive interaction between gemfibrozil and fluoxetine. Combinations of interest among diverse pharmaceuticals are difficult to identify, due to the daunting number of possible combinations that must be evaluated. The novel interaction between gemfibrozil and fluoxetine suggests that identifying and combining drugs that show cell cycle effects might streamline identification of drug combinations with a pronounced impact on cell proliferation.

A systematic analysis of cell cycle regulators in yeast reveals that most factors act independently of cell size to control initiation of division.

Upstream events that trigger initiation of cell division, at a point called START in yeast, determine the overall rates of cell proliferation. The identity and complete sequence of those events remain unknown. Previous studies relied mainly on cell size changes to identify systematically genes required for the timely completion of START. Here, we evaluated panels of non-essential single gene deletion strains for altered DNA content by flow cytometry. This analysis revealed that most gene deletions that altered cell cycle progression did not change cell size. Our results highlight a strong requirement for ribosomal biogenesis and protein synthesis for initiation of cell division. We also identified numerous factors that have not been previously implicated in cell cycle control mechanisms. We found that CBS, which catalyzes the synthesis of cystathionine from serine and homocysteine, advances START in two ways: by promoting cell growth, which requires CBS's catalytic activity, and by a separate function, which does not require CBS's catalytic activity. CBS defects cause disease in humans, and in animals CBS has vital, non-catalytic, unknown roles. Hence, our results may be relevant for human biology. Taken together, these findings significantly expand the range of factors required for the timely initiation of cell division. The systematic identification of non-essential regulators of cell division we describe will be a valuable resource for analysis of cell cycle progression in yeast and other organisms.

Nitrogen starvation of cyanobacteria results in the production of ß-N-methylamino-L-alanine.

ß-N-Methylamino-L-alanine, an unusual amino acid implicated in neurodegenerative disease, has been detected in cultures of nearly all genera of environmentally ubiquitous cyanobacteria tested. The compound is present within cyanobacterial cells in free and protein-associated forms, with large variations occurring in the concentration of these pools between species as well as within single strains. With a lack of knowledge and supporting data on the regulation of BMAA production and the role of this compound in cyanobacteria, the association between BMAA and cyanobacteria is still subject to debate. In this study we investigated the biosynthesis of BMAA in axenic non-diazotrophic cyanobacterial cultures using the stable isotope ¹5N. Nitrogen starvation of nutritionally replete cells resulted in an increase in free cellular ¹5N BMAA suggesting that BMAA may be the result of catabolism to provide nitrogen or that BMAA is synthesised to serve a functional role in the cell in response to nitrogen deprivation. The addition of NO3⁻ and NH4⁺ to the culture medium following starvation resulted in a decrease of free cellular BMAA without a corresponding increase in the protein-associated fraction. The use of ammonia as a nitrogen source resulted in a more rapid reduction of BMAA when compared to nitrate. This study provides the first data regarding the regulation of intracellular BMAA concentrations in cyanobacteria with results conclusively showing the production of ¹5N BMAA by an axenic cyanobacterial culture.

Evaluation of the XRCC1 gene as a phenotypic modifier in BRCA1/2 mutation carriers. Results from the consortium of investigators of modifiers of BRCA1/BRCA2.

BACKGROUND: Single-nucleotide polymorphisms (SNPs) in genes involved in DNA repair are good candidates to be tested as phenotypic modifiers for carriers of mutations in the high-risk susceptibility genes BRCA1 and BRCA2. The base excision repair (BER) pathway could be particularly interesting given the relation of synthetic lethality that exists between one of the components of the pathway, PARP1, and both BRCA1 and BRCA2. In this study, we have evaluated the XRCC1 gene that participates in the BER pathway, as phenotypic modifier of BRCA1 and BRCA2. METHODS: Three common SNPs in the gene, c.-77C>T (rs3213245) p.Arg280His (rs25489) and p.Gln399Arg (rs25487) were analysed in a series of 701 BRCA1 and 576 BRCA2 mutation carriers. RESULTS: An association was observed between p.Arg280His-rs25489 and breast cancer risk for BRCA2 mutation carriers, with rare homozygotes at increased risk relative to common homozygotes (hazard ratio: 22.3, 95% confidence interval: 14.3-34, P<0.001). This association was further tested in a second series of 4480 BRCA1 and 3016 BRCA2 mutation carriers from the Consortium of Investigators of Modifiers of BRCA1 and BRCA2. CONCLUSIONS AND INTERPRETATION: No evidence of association was found when the larger series was analysed which lead us to conclude that none of the three SNPs are significant modifiers of breast cancer risk for mutation carriers.

Distinguishing the cyanobacterial neurotoxin ß-N-methylamino-L-alanine (BMAA) from other diamino acids.

ß-N-methylamino-L-alanine (BMAA) is produced by diverse taxa of cyanobacteria, and has been detected by many investigators who have searched for it in cyanobacterial blooms, cultures and collections. Although BMAA is distinguishable from proteinogenic amino acids and its isomer 2,4-DAB using standard chromatographic and mass spectroscopy techniques routinely used for the analysis of amino acids, we studied whether BMAA could be reliably distinguished from other diamino acids, particularly 2,6-diaminopimelic acid which has been isolated from the cell walls of many bacterial species. We used HPLC-FD, UHPLC-UV, UHPLC-MS, and triple quadrupole tandem mass spectrometry (UHPLC-MS/MS) to differentiate BMAA from the diamino acids 2,6-diaminopimelic acid, N-2(amino)ethylglycine, lysine, ornithine, 2,4-diaminosuccinic acid, homocystine, cystine, tryptophan, as well as other amino acids including asparagine, glutamine, and methionine methylsulfonium.

Distinguishing the cyanobacterial neurotoxin beta-N-methylamino-L-alanine (BMAA) from its structural isomer 2,4-diaminobutyric acid (2,4-DAB).

The cyanobacterial neurotoxin beta-N-methylamino-L-alanine (BMAA) has been associated with certain forms of progressive neurodegenerative disease, including sporadic Amyotrophic Lateral Sclerosis and Alzheimer's disease. Reports of BMAA in cyanobacterial blooms from lakes, reservoirs, and other water resources continue to be made by investigators in a variety of laboratories. Recently it was suggested that during analysis BMAA may be confused with its structural isomer 2,4-diaminobutyric acid (2,4-DAB), or that current detection methods may mistake other compounds for BMAA. We here review the evidence that BMAA can be consistently and reliably separated from 2,4-DAB during reversed-phase HPLC, and that BMAA can be confidently distinguished from 2,4-DAB during triple quadrupole LC-MS/MS analysis by i) different retention times, ii) diagnostic product ions resulting from collision-induced dissociation, and iii) consistent ratios between selected reaction monitoring (SRM) transitions. Furthermore, underivatized BMAA can be separated from 2,4-DAB with an amino acid analyzer with post-column visualization using ninhydrin. Other compounds that may be theoretically confused with BMAA during chloroformate derivatization during GC analysis are distinguished due to their different retention times.

Barriers to the delivery of the hepatitis B birth dose: a study of five Papua New Guinean hospitals in 2007.

Hepatitis B is highly endemic in Papua New Guinea (PNG). Vaccination at birth is a key mother-to-child transmission prevention strategy. Despite recommendations for newborns to be vaccinated within 24 hours of delivery, a 2005 survey showed 23% coverage among children born in health facilities. Our study examined hepatitis B birth-dose coverage and knowledge, practices and barriers to vaccine delivery in five major PNG hospitals. Data on births and vaccines administered were sourced from the National Department of Health (NDoH) and directly from the five hospitals. A maternity unit audit and staff survey were undertaken. Across the five hospitals, the hospital-level data of hepatitis B birth-dose coverage was 79% (range: 40-96%) compared to 19% from national data (range: 0-106%). Despite hospitals having adequate vaccine supply, access to appropriately stored vaccine in maternity units was compromised with only one unit having a vaccine-specific temperature-monitored refrigerator. In interviews of 25 staff, incorrect reasons given for delaying vaccination were prematurity (60%), low birthweight (48%) and difficult birth (36%). This study found encouraging birth-dose coverage rates in five major hospitals but 20% of babies still missed receiving the recommended vaccine. The NDoH Immunization Unit will use the results of this study to inform strategies to improve hepatitis B birth-dose coverage in hospitals.

Effect of platelet-activating factor on the electrophysiology of the human Fallopian tube: early mediation of embryo-maternal dialogue?

Platelet-activating factor (PAF) is produced by preimplantation embryos and may be involved in the earliest stages of embryo-maternal dialogue. This study explored the potential effects of PAF acting as a signalling agent on human Fallopian tubal epithelial cells grown as a polarized layer in primary culture. The response of the tubal epithelium was assessed in terms of the transepithelial potential difference and short-circuit current (I(scc)), which were recorded using a modified Ussing chamber. Resistance was calculated from the measurements of potential difference and I(scc). PAF (1.9 nmol to 1.9 micromol l(-1)) administered to the apical surface of the cells produced a marked, transient increase in both potential difference and I(scc) in a dose-dependent manner. The mode of action of PAF on the electrophysiological responses of human tubal epithelial cells was investigated. Blockers of Na(+), K(+) and voltage-operated Ca(2+) channels had little effect on PAF action. However, incubation of the epithelial cells in Cl(-)free medium or with a blocker of the Na(+)-K(+)-2Cl(-) cotransporter (Furosemide) reduced the effect of PAF. Blockade of chloride-bicarbonate channels with 4-acetamido-4'-iso-thiocyanostilbene-2.2'-disulphonic acid (SITS) reduced the effect of low doses of PAF only. These results indicate that PAF influences the movement of chloride ions across the tubal epithelial cell and is a candidate molecule for initial embryo-maternal dialogue.

Experience with spiral computed tomography as the sole diagnostic method for traumatic aortic rupture.

BACKGROUND: Spiral computed tomographic (CT) scan is an excellent screen for aortic trauma. Traditionally, aortography is performed when injury is suspected to confirm the diagnosis. We hypothesized that it is safe and expeditious to forgo aortography when the spiral CT demonstrates aortic injury. METHODS: Retrospective review of 54 patients undergoing aortic repair from July 1994 to December 1999. Spiral CT was the initial diagnostic study in 52 patients. Pseudoaneurysm or aortic wall defect in the presence of mediastinal hematoma was considered diagnostic. Angiography, initially routine, was later performed only when requested by the surgeon, and for all "nonnegative" studies (periaortic hematoma without detectable aortic injury). RESULTS: Twenty-six patients underwent angiography before operation (group 1). Nineteen group 1 spiral CTs were unequivocally diagnostic; 7 were nonnegative and angiography was required. Twenty-eight other patients underwent repair based on spiral CT alone (group 2). There was one false-positive result in both groups. There were no unexpected operative findings. Mean time from admission to diagnosis was 5.7+/-3.4 hours for group 1 and 1.7+/-1.7 hours for group 2 (p < 0.01). CONCLUSIONS: Operating on the basis of a diagnostic spiral CT is safe and expeditious. Aortography may be reserved for those with equivocal studies.

Mutations within the tumour suppressor gene p53 are not confined to a late event in prostate cancer progression. a review of the evidence.

Mutations in the p53 tumour suppressor gene are generally believed to be a late event in the progression of prostate cancer, and are associated with androgen independence, metastasis, and a worse prognosis. In this review, we examine the current literature available on p53 mutations and focus on stages A (T1) and B (T2) of prostate cancer. We report here that p53 mutations can be found in approximately one third of prostate cancers that are clinically localized to the prostate. In addition, high levels of p53 mutation are found in normal prostate tissue of prostate cancer patients, prostatic intraepithelial neoplasia, and benign prostatic hyperplasia. The limitations of techniques used to determine p53 mutations are discussed, as well as other modes of p53 loss in early stage prostate cancer.

Asymmetric synthesis of 2-alkyl-perhydroazepines from [5,3,0]-bicyclic lactams.

The synthesis and utility of a novel class of [5,3,0]-bicyclic lactams are described. Produced by the cyclodehydration of (R)-phenylglycinol with omega-keto acids, lactams 4-6 were obtained as separable diastereomeric mixtures ( approximately 2:1) in low yields ( approximately 40%). Higher chemical yield (up to 61%) was realized via an alternate route involving ring closure metathesis of 2-allyl-N-acroyl oxazolidines, 8. Stereoselective reductions of the syn-bicyclic lactams, 4a and 5a, occurred with the use of alane or lithiumaluminum hydride, affording azepine alcohols, 11a and 15a, of the R configuration at the 2-position, in good to moderate yields (50-88%). High selectivity was also observed in the diisobutylaluminum hydride reduction of the epimeric anti lactams, 4b and 5b, affording azepine alcohols, 11b and 15b, of the S configuration at C-2. Hydrogenolytic cleavage of the N-benzyl moiety afforded chiral 2-substituted perhydroazepines, (R)- and (S)-12, in good yields and good enantiomeric excesses (84-94%).

Formation of Fallopian tubal fluid: role of a neglected epithelium.

Fluid produced and secreted by the Fallopian tube provides the environment in which gamete transport and maturation, fertilization and early embryo development occur. This review describes the composition of oviductal fluid in terms of ions and nutrients such as glucose, lactate, pyruvate and amino acids. The function of oestrogen-specific glycoprotein is discussed. The mechanisms of fluid secretion and agents known to influence fluid production and secretion are described. Clinical implications of abnormal oviductal fluid production and secretion in hydrosalpinx and pelvic inflammatory disease are also discussed.

Heparinless partial cardiopulmonary bypass for the repair of aortic trauma.

OBJECTIVE: We hypothesized that partial cardiopulmonary bypass with a heparin-bonded system would be a technically simple, effective adjunct for reducing paraplegia during repair of traumatic aortic rupture. It avoids the risk of heparin, but, unlike left atrial-arterial bypass, it can heat, cool, oxygenate, and rapidly infuse volume if needed. METHODS: A retrospective review was conducted of patients admitted for aortic trauma from July 1994 to December 1999. Bypass consisted of femoral venous (right atrial) cannulation, a centrifugal pump, and an oxygenator-heater/cooler. Arterial return was to the femoral artery or distal aorta. The entire system was heparin-bonded and no systemic heparin was given. RESULTS: Heparin-bonded partial bypass was established in 50 patients (mean age 43 +/- 17 years). Crossclamp time was 32 +/- 11 minutes (range 14-70 minutes), mean flow 3.0 +/- 0.8 L/min, and bypass time 64 +/- 43 minutes. During repair, 58% of patients received volume through the system (mean 1.1 +/- 1.9 L). Core temperature rose slightly (35.9 degrees C +/- 0.7 degrees C to 36.3 degrees C +/- 0.8 degrees C). Three of the 15 patients who underwent percutaneous femoral arterial and venous cannulation concomitant with their angiograms had vessel injury, with one limb loss, and this procedure was discontinued. Thirty-five patients underwent percutaneous femoral vein and direct distal aortic cannulation without event. The mortality rate for patients supported by bypass was 10%, and all deaths were due to other injuries. There were no new cases of paraplegia and no worsening of intracranial or pulmonary injuries. CONCLUSIONS: Heparin-bonded bypass is technically simple to use and avoids the risk of anticoagulation. Paraplegia was avoided. The ability to correct hypothermia, oxygenate, and rapidly infuse volume may simplify management and improve outcomes.

Identification of monozygotic twins that are concordant for preeclampsia.

Preeclampsia is thought to be discordant in monozygotic twins. While recruiting for a genetic study into preeclampsia, we identified 4 sets of twins and a triplet gestation; all were monozygous on deoxyribonucleic acid "fingerprinting." Two twins were concordant for preeclampsia, and 2 of the triplets had pregnancy-induced hypertension, although only 1 of them had proteinuria. Hence we confirm the existence of monozygotic twins that are concordant for preeclampsia.

Evidence for post-transcriptional down-regulation of the apoptosis-related gene bcl-2 in human colorectal cancer.

This retrospective study was undertaken to investigate the expression of bcl-2 protein and messenger RNA in colorectal cancer (CRC). Immunohistochemical analysis using a monoclonal mouse antibody to the bcl-2 protein and in situ hybridization using a digoxigenin-labelled bcl-2 cRNA probe were carried out on formalin-fixed and paraffin-embedded specimens from 53 colorectal adenocarcinomas, 27 liver secondaries, and 60 adenomas with various degrees of dysplasia. Normal human tonsil sections were used as positive controls. Expression of bcl-2 protein and of messenger RNA was evaluated semiquantitatively. The expression of bcl-2 protein was gradually and significantly lost during the progression from moderately dysplastic adenoma to primary CRC (moderate/severe dysplasia: Mann-Whitney U-test, p=0.0001; severe dysplasia/primary CRC: p=0.027), whereas the cellular expression of bcl-2 mRNA was gradually increased during the dysplasia/adenoma-carcinoma neoplastic sequence. These observations suggest that in a proportion of colorectal cancer cases, the bcl-2 proto-oncogene expression may be down-regulated at a post-transcriptional level.

The effect of angiotensin II on mitogen-activated protein kinase in human cardiomyocytes.

The role of angiotensin II (Ang II)-receptors on mitogen-activated protein kinase (MAPK) activation in cardiomyocytes remains controversial. Therefore, the current study was designed to investigate the actions of AT(1)- and AT(2)-receptors on Ang II-induced extracellular signal-regulated kinase (ERK), p38 and the c-Jun N-terminal kinase (JNK) MAPK activities in human cardiomyocytes. Human cardiac tissue was obtained from open-heart surgery (n=6). The cardiac tissue was minced and incubated in the special tissue culture system for 24 hours in the absence or presence of Ang II (10(-7) M). These studies were repeated with the AT(1)-receptor antagonist losartan (10(-6) M) or the AT(2)-receptor antagonist PD-123319 (10(-6) M). Immunohistochemical staining and Western blot analysis with phospho-antibodies were performed to determine ERK, JNK and p38 activities. Ang II increased ERK and p38 activities in human cardiomyocytes. The effects of Ang II were abolished by losartan and enhanced by PD-123319. Co-incubation with both losartan and PD-123319 resulted in a decrease of ERK and p38 activities in cardiomyocytes. The immunohistochemical staining of JNK showed no significant differences between Ang II alone, Ang II plus losartan and Ang II plus PD-123319 groups. In conclusion, Ang II has a potent effect on ERK and p38 MAPK activities in cardiomyocytes, by acting through AT(1)-receptors. This effect of Ang II is modified by AT(2)-receptors. Therefore, Ang II, via AT(1)- and AT(2)-receptor stimulation, has a distinct effect on MAPK activity in cardiomyocytes.