Cell compartmentalisation in planctomycetes: novel types of structural organisation for the bacterial cell

The organisation of cells of the planctomycete species Pirellula marina, Isosphaera pallida, Gemmata obscuriglobus, Planctomyces mat-is and Candidatus Brocadia anammoxidans was investigated based on ultrastructure derived from thin-sections of cryosubstituted cells, freeze-fracture replicas, and in the case of Gemmata obscuriglobus and Pirellllla marina, computer-aided 3-D reconstructions from serial sections of cryosubstituted cells. All planctomycete cells display a peripheral ribosome-free region, termed here the paryphoplasm, surrounding the perimeter of the cell, and an interior region including any nucleoid regions as well as ribosome-like particles, bounded by a single intracytoplasmic membrane (ICM), and termed the pirellulosome in Pirellula species. Immunogold labelling and RNase-gold cytochemistry indicates that in planctomycetes all the cell DNA is contained wholly within the interior region bounded by the ICM, and the paryphoplasm contains no DNA but at least some of the cell's RNA. The ICM in Isosphaera pallida and Planctomyces mat-is is invaginated such that the paryphoplasm forms a major portion of the cell interior in sections, but in other planctomycetes it remains as a peripheral zone. In the anaerobic ammonium-oxidising (anammox process) chemoautotroph Candidatus Brocadia anammoxidans the interior region bounded by ICM contains a further internal single-membrane-bounded region, the anam-moxosome. In Gemmata obscuriglobus. the interior ICM-bounded region contains the nuclear body, a double-membrane-bounded region containing the cell's nucleoid and all genomic DNA in addition to some RNA. Shared features of cell compartmentalisation in different planctomycetes are consistent with the monophyletic nature of the planctomycetes as a distinct division of the Bacteria. The shared organisational plan for the planctomycete cell constitutes a new type not known in cells of other bacteria.

Melanocortin-1 receptor genotype is a risk factor for basal and squamous cell carcinoma

MC1R gene variants have previously been associated with red hair and fair skin color, moreover skin ultraviolet sensitivity and a strong association with melanoma has been demonstrated for three variant alleles that are active in influencing pigmentation: Arg151Cys, Arg160Trp, and Asp294His. This study has confirmed these pigmentary associations with MC1R genotype in a collection of 220 individuals drawn from the Nambour community in Queensland, Australia, 111 of whom were at high risk and 109 at low risk of basal cell squamous cell carcinoma. Comparative allele frequencies for nine MC1R variants that have been reported in the Caucasian population were determined for these two groups, and an association between prevalence of basal cell carcinoma, squamous cell carcinoma, solar keratosis and the same three active MC1R variant alleles was demonstrated [odds ratio=3.15 95% CI (1.7, 5.82)]. Three other commonly occurring variant alleles: Val60Leu, Val92Met, and Arg163Gln were identified as having a minimal impact on pigmentation phenotype as well as basal cell carcinoma and squamous cell carcinoma risk. A significant heterozygote effect was demonstrated where individuals carrying a single MC1R variant allele were more likely to have fair and sun sensitive skin as well as carriage of a solar lesion when compared with those individuals with a consensus MC1R genotype. After adjusting for the effects of pigmentation on the association between MC1R variant alleles and basal cell carcinoma and squamous cell carcinoma risk, the association persisted, confirming that presence of at least one variant allele remains informative in terms of predicting risk for developing a solar-induced skin lesion beyond that information gained through observation of pigmentation phenotype.

The spironolactone renaissance

Until recently, spironolactone was considered only as an antagonist at the aldosterone receptors of the epithelial cells of the kidney and was used clinically in the treatment of hyperaldosteronism and, occasionally, as a K+-sparing diuretic. The spironolactone renaissance started with the experimental finding that spironolactone reversed aldosterone-induced cardiac fibrosis by a cardiac action. Experimentally, spironolactone also has direct effects on blood vessels. Spironolactone reduces vascular fibrosis and injury, inhibits angiogenesis, reduces vascular tone and reduces portal hypertension. The rationale for the Randomized Aldactone Evaluation Study (RALES) of spironolactone in heart failure was that ‘aldosterone escape’ occurred through non-angiotensin II mechanisms. The RALES clinical trial was stopped early when it was shown that there was a 30% reduction in risk of death among the spironolactone patients. In RALES, spironolactone also reduced hospitalisation for worsening heart failure and improved the symptoms of heart failure. Other recent clinical trials have shown that spironolactone reduces cardiac and vascular collagen turnover, improves heart variability, reduces ventricular arrhythmias, improves endothelial dysfunction and dilates blood vessels in human heart failure and these effects probably all contribute to the increased survival in heart failure. Spironolactone may also be useful in the treatment of left ventricular hypertrophy, portal hypertension and cirrhosis. There have also been some recent small clinical trials of spironolactone as an anti-androgen showing potential in acne, hirsutism and precocious puberty.

Reversal of cardiovascular remodelling with candesart

Cardiovascular remodelling, defined as ventricular and vascular hypertrophy together with fibrosis, characterises hypertension following inhibition of the production of the endogenous vasodilator, nitric oxide (NO). This study has determined whether the cardiovascular remodelling following chronic NO synthase inhibition can e reversed by administration of the selective angiotensin II AT(1)-receptor antagonist, candesartan. Male Wistar rats were treated with L-nitroarginine methyl ester (L-NAME, 400 mg/l in drinking water) for eight weeks and with candesartan cilexetil (2 mg/kg/day by oral gavage) for the last four weeks. L-NAME-treated rats became hypertensive with systolic blood pressure increasing from 110 +/- 4 mmHg (control) to 170 +/- 10 mmHg. Rats developed left ventricular hypertrophy (control 1.70 +/- 0.06; L-NAME 2.10 +/- 0.04 mg/kg body wt) with markedly increased deposition of perivascular and interstitial collagen. Candesartan returned blood pressure, left ventricular weights and collagen deposition to control values. Echo cardiographic assessment showed concentric hypertrophy with an increased fractional shortening; this was reversed by candesartan treatment. Heart failure was not evident. In the isolated Langendorff heart, diastolic stiffness increased in L-NAME-treated rats while the rate of increase in pressure (+dP/dt) increased after eight weeks only; candesartan reduced collagen deposition and normalised +dP/dt. In isolated left ventricular papillary muscles, the potency (negative log EC50) of noradrenaline as a positive inotropic compound was unchanged, (control 6.56 +/- 0.14); maximal increase in force before ectopic beats was reduced from 5.0 +/- 0.4 mN to 2.0 +/- 0.2 mN. Noradrenaline potency as a vasoconstrictor in thoracic aortic rings was unchanged, but maximal contraction was markedly reduced from 25.2 +/- 2.0 mN to 3.0 +/- 0.3 mN; this was partially reversed by candesartan treatment. Thus, chronic inhibition of NO production with L-NAME induces hypertension, hypertrophy and fibrosis with increased toxicity and significant decreases in vascular responses to noradrenaline. These changes were at least partially reversible by treatment with candesartan, implying a significant role of AT(1)-receptors in L-NAME-induced cardiovascular changes.

A caveolin dominant negative mutant associates with lipid bodies and induces intracellular cholesterol imbalance

Recent studies have indicated a role for caveolin in regulating cholesterol-dependent signaling events. In the present study we have analyzed the role of caveolins in intracellular cholesterol cycling using a dominant negative caveolin mutant. The mutant caveolin protein, cav-3(DGV) specifically associates with the membrane surrounding large lipid droplets. These structures contain neutral lipids, and are accessed by caveolin 1-3 upon overexpression. Fluorescence, electron, and video microscopy observations are consistent with formation of the membrane-enclosed lipid rich structures by maturation of subdomains of the ER. The caveolin mutant causes the intracellular accumulation of free cholesterol (FC) in late endosomes, a decrease in surface cholesterol and a decrease in cholesterol efflux and synthesis. The amphiphile U18666A acts synergistically with cav(DGV) to increase intracellular accumulation of FC. Incubation of cells with oleic acid induces a significant accumulation of full-length caveolins in the enlarged lipid droplets. We conclude that caveolin can associate with the membrane surrounding lipid droplets and is a key component involved in intracellular cholesterol balance and lipid transport in fibroblasts.

Relationship between endosomes and lysosomes

Delivery of endocytosed macromolecules to lysosomes occurs by means of direct fusion of late endosomes with lysosomes. This has been formally demonstrated in a cell-free content mixing assay using late endosomes and lysosomes from rat liver. There is evidence from electron microscopy Studies that the same process occurs in intact cells. The fusion process results in the formation of hybrid organelles from which lysosomes are reformed. The discovery of the hybrid organelle has opened up three areas of investigation: (i) the mechanism of direct fusion of late endosomes and lysosomes, (ii) the mechanism of re-formation of lysosomes from the hybrid organelle, and (iii) the function of the hybrid organelle. Fusion has analogies with homotypic vacuole fusion in yeast. It requires syntaxin 7 as part of the functional trans-SNARE [SNAP receptor, where SNAP is soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein] complex and the release of lumenal calcium to achieve membrane fusion. Reformation of lysosomes from the hybrid organelle occurs by a maturation process involving condensation of lumenal content and probably removal of some membrane proteins by vesicular traffic. Lysosomes may thus be regarded as a type of secretory granule, storing acid hydrolases in between fusion events with late endosomes. The hybrid organelle is predicted to function as a 'cell stomach', acting as a major site of hydrolysis of endocytosed macromolecules.

Epizootic activity of Murray Valley encephalitis virus in an aboriginal community in the southeast Kimberley region of western Australia: Results of cross-sectional and longitudinal serologic studies

Murray Valley encephalitis (MVE) virus is a mosquito-borne flavivirus causing severe encephalitis with a resultant high morbidity and mortality. In the period 1989-1993. we undertook a cross-sectional and longitudinal studs by annually screening members of a small remote Aboriginal community in northwestern Australia for MVE virus antibodies. Of the estimated 250-300 people in the community. 249 were tested, and 52.6% had positive serology to MVE. The proportion testing positive increased with increasing age group. and males were slightly more likely to be positive than females. During the study period. a high proportion of the population seroconverted to MVE: the clinical/subclinical ratio seems to be lower than previously reported. Although MVE is mostly asymptomatic, the devastating consequences of clinical illness indicate that advice should be provided regarding the avoidance of mosquito bites. Our longitudinal study showed that the risk of seroconversion was similar for each age group. not just the young.

Expression of a truncated Brca1 protein delays lactational mammary development in transgenic mice

To address the hypothesis that certain disease-associated mutants of the breast-ovarian cancer susceptibility gene BRCA1 have biological activity in vivo, we have expressed a truncated Brca1 protein (trBrca1) in cell-lines and in the mammary gland of transgenic mice. Immunofluorescent analysis of transfected cell-lines indicates that trBRCA1 is a stable protein and that it is localized in the cell cytoplasm. Functional analysis of these cell-lines indicates that expression of trBRCA1 confers an increased radiosensitivity phenotype on mammary epithelial cells, consistent with abrogation of the BRCA1 pathway. MMTV-trBrca1 transgenic mice from two independent lines displayed a delay in lactational mammary gland development, as demonstrated by altered histological profiles of lobuloalveolar structures. Cellular and molecular analyses indicate that this phenotype results from a defect in differentiation, rather than altered rates of proliferation or apoptosis. The results presented in this paper are consistent with trBrca1 possessing dominant-negative activity and playing an important role in regulating normal mammary development. They may also have implications for germline carriers of BRCA1 mutations.