A microscopic survey is presented of the most commonly observed and morphologically conspicuous microorganisms found attached to natural surfaces or to artificial materials deposited in the immediate vicinity of thermal submarine vents at the Galapagos Rift ocean spreading zone at a depth of 2,550 meters. Of special interest were the following findings: (i) all surfaces intermittently exposed to H(2)S-containing hydrothermal fluid were covered by layers, ca. 5 to 10 mum thick, of procaryotic, gram-negative cells interspaced with amorphous metal (Mn-Fe) deposits; (ii) although some of the cells were encased by dense metal deposits, there was little apparent correlation between metal deposition and the occurrence of microbial mats, (iii) highly differentiated forms appeared to be analogues of certain cyanobacteria, (iv) isolates from massive mats of a prosthecate bacterium could be identified as Hyphomicrobium spp., (v) intracellular membrane systems similar to those found in methylotrophic and nitrifying bacteria were observed in approximately 20% of the cells composing the mats, (vi) thiosulfate enrichments made from mat material resulted in isolations of different types of sulfur-oxidizing bacteria including the obligately chemolithotrophic genus Thiomicrospira.

The behavioral response of single Beggiatoa sp. filaments moving on a gas-permeable membrane was studied by the combined use of microscopy and oxygen microelectrodes during controlled oscillations of oxygen tension. The bacteria reacted to increasing oxygen by reversing the direction of movement. The same step-up phobic response to oxygen was observed when a filament tip or loop glided into a stable microgradient of increasing oxygen. The response was sensitive to a change in oxygen tension of <5% of air saturation min. The response time was 20 to 50 s. Frequently, only part of the filament responded, which led to the formation of sharp bends, loops, and coils. This partial response facilitated the positioning of the long filaments within the narrow O(2)-H(2)S interface. The structure of whole Beggiatoa mats on sediment surfaces varied from loose to dense in relation to shallow or steep oxygen gradients in the 0.3- to 2-mm-thick, unstirred boundary layer. In an illuminated sediment Beggiatoa spp. lived together with photosynthetic organisms and migrated vertically in accordance with light/dark variations. The combined effect of phobic responses to light and oxygen can explain this migration.

The mating-type locus of Neurospora crassa regulates mating identity and entry into the sexual cycle. The mat A idiomorph encodes three genes, mat A-1, mat A-2, and mat A-3. Mutations in mat A-1 result in strains that have lost mating identity and vegetative incompatibility with mat a strains. A strain containing mutations in both mat A-2 and mat A-3 is able to mate, but forms few ascospores. In this study, we describe the isolation and characterization of a mutant deleted for mat (deltamatA), as well as mutants in either mat A-2 or mat A-3. The deltamatA strain is morphologically wild type during vegetative growth, but it is sterile and heterokaryon compatible with both mat A and mat a strains. The mat A-2 and mat A-3 mutants are also normal during vegetative growth, mate as a mat A strain, and produce abundant biparental asci in crosses with mat a, and are thus indistinguishable from a wild-type mat A strain. These data and the fact that the mat A-2 mat A-3 double mutant makes few asci with ascospores indicate that MAT A-2 and MAT A-3 are redundant and may function in the same pathway. Analysis of the expression of two genes (sdv-1 and sdv-4) in the various mat mutants suggests that the mat A polypeptides function in concert to regulate the expression of some sexual development genes.

The aim of this study was to determine the extent to which clinical tests of structural characteristics of the foot and ankle could account for variation in the magnitude of regional forces and pressures under the foot during walking in older people. Plantar forces and pressures were obtained from 172 older people (53 men, 119 women) aged 62-96 years (mean 80.0, S.D. 6.4) using a floor-mounted resistive sensor mat system. Subjects also completed tests of foot posture, range of motion, strength, sensation and toe deformity. Multiple regression analysis was then used to determine which clinical variables were most strongly correlated with plantar forces and pressures. Maximum forces and peak pressures under most regions of the foot were largely explained by differences in bodyweight, with some important exceptions. Loading under the midfoot was associated with the arch index, loading under the first metatarsophalangeal joint (1st MPJ) was associated with 1st MPJ range of motion, and loading under the hallux was associated with hallux plantarflexor strength, 1st MPJ range of motion and the degree of hallux valgus deformity. Clinical measurements accounted for 13-53% of the variance in maximum force and 4-40% of the variance in peak pressures. These findings indicate that structural foot and ankle characteristics identified from clinical measurements can explain some key aspects of plantar loading patterns of the foot. This information provides further insights into the dynamic function of the foot, which might assist in the development of interventions for pressure-related foot complaints in older people.

In order to analyze differential gene expression of putative prostate tumor markers we compared the expression levels of >400 cancer-related genes using the cDNA array technique in a set of prostate tumors and matched normal prostate tissues. Up-regulated expression of mammary tumor 8 kDa protein (MAT-8), complement component C1S (C1S), ferritin heavy chain (FTH1), peptidyl-prolyl cis-trans isomerase A (PPIA), RNA-binding protein regulatory subunit DJ-1 protein (DJ-1) and vacuolar ATP synthase subunit F (ATP6V1F) was determined in prostate carcinoma and confirmed by using quantitative real-time RT-PCR analyses. Furthermore, quantitative real time RT-PCR on intact RNAs from 11 paired laser microdissected epithelial tissue samples confirmed up-regulated MAT-8 expression in 6 out of 11 prostate tumors. To determine the function of MAT-8 in vitro, human PC-3 and LNCaP prostate carcinoma cells were transfected with small interfering double-stranded RNA (siRNA) oligonucleotides against the MAT-8 gene leading to a specific down-regulation of MAT-8 expression. In addition, suppression of MAT-8 expression caused a significant decrease in cellular proliferation of both prostate cancer cell lines, whereas invasive capacity and cellular apoptosis remained unaffected. Taken together, our results indicate that the human MAT-8 gene contains the potential to serve as a prostate cancer expression marker and that MAT-8 plays an important role in cellular growth of prostate carcinomas.

The Line Islands are calcium carbonate coral reef platforms located in iron-poor regions of the central Pacific. Natural terrestrial run-off of iron is non-existent and aerial deposition is extremely low. However, a number of ship groundings have occurred on these atolls. The reefs surrounding the shipwreck debris are characterized by high benthic cover of turf algae, macroalgae, cyanobacterial mats and corallimorphs, as well as particulate-laden, cloudy water. These sites also have very low coral and crustose coralline algal cover and are call black reefs because of the dark-colored benthic community and reduced clarity of the overlying water column. Here we use a combination of benthic surveys, chemistry, metagenomics and microcosms to investigate if and how shipwrecks initiate and maintain black reefs. Comparative surveys show that the live coral cover was reduced from 40 to 60% to <10% on black reefs on Millennium, Tabuaeran and Kingman. These three sites are relatively large (>0.75 km(2)). The phase shift occurs rapidly; the Kingman black reef formed within 3 years of the ship grounding. Iron concentrations in algae tissue from the Millennium black reef site were six times higher than in algae collected from reference sites. Metagenomic sequencing of the Millennium Atoll black reef-associated microbial community was enriched in iron-associated virulence genes and known pathogens. Microcosm experiments showed that corals were killed by black reef rubble through microbial activity. Together these results demonstrate that shipwrecks and their associated iron pose significant threats to coral reefs in iron-limited regions.

BACKGROUND

This multicenter study is aimed at estimating changes in the effect of high temperatures on elderly mortality before and after the 2003 heat waves and following the introduction of heat prevention activities.

METHODS

A total of sixteen cities were included in the study. City-specific relationships between maximum apparent temperature (MAT) and elderly daily mortality before (1998-2002) and after (2006-2010) intervention were modelled through non-linear distributed lag models and estimates were combined using a random effect meta-analysis. We estimated the percentage change in daily mortality for 3°C variations in MAT above the 25th percentile of the June city-specific 1998-2002 distribution. A time-varying analysis was carried out to describe intra-seasonal variations in the two periods.

RESULTS

We observed a reduction in high temperatures' effect post intervention; the greatest reduction was for increases in temperature from 9°C to 12°C above the 25th percentile, with a decrease from +36.7% to +13.3%. A weak effect was observed for temperatures up to 3°C above the 25th percentile only after. Changes were month-specific with a reduction in August and an increase in May, June and September in 2006-2010.

CONCLUSIONS

A change in the temperature-mortality relationship was observed, attributable to variations in temperature distributions during summer and to the introduction of adaptation measures. The reduction in the effect of high temperature suggests that prevention programs can mitigate the impact. An effect of lower temperature remains, indicating a relevant impact of temperature at the beginning of summer when the population has not yet adapted and intervention activities are not fully operational.

Polymer nanofibers, with diameters in the nanometer range, possess larger surface areas per unit mass and permit easier addition of surface functionalities compared with polymer microfibers. Hence, polymer nanofiber mats are being considered for use as filters, scaffolds for tissue engineering, protective clothing, reinforcement in composite materials and sensors. Although some of these applications are in the development stage, a few have been commercially exploited. Research on polymer nanofibers, nanofiber mats, and their applications has seen a remarkable growth over the last few years. However, a review of the various issues related to these nanofibers has not been published. This article presents a review of the recent trends in the processing methods and characterization techniques for polymer nanofibers. Research challenges and future trends in the processing and characterization of polymer nanofibers are discussed in the article. Five processing methods have been examined in this review, namely drawing, template synthesis, phase separation, self-assembly, and electrospinning. Among these methods, electrospinning has been used to convert a large variety of polymers into nanofibers and may be the only process that has the potential for mass production. The structure, morphology, and geometry of nanofibers and the porosity and tensile properties of nanofiber mats can be investigated through conventional techniques and instruments. But new techniques are needed for the mechanical testing of single nanofibers. Although measurement of mechanical properties such as tensile modulus, strength, and elongation is difficult because of the small diameters of the fibers, these properties are crucial for the proper use of nanofiber mats.

Prostate-specific membrane antigen (PSMA), a glutamyl preferring carboxypeptidase, is found in prostate and other carcinomas present on both tumor cells and associated microvascular lining cells. We find that the channel structures delineated by PSMA-expressing cells in human and rat prostate tumors are in functional continuity with the vasculature and thus form part of tumor microvasculature. The PSMA-positive cell-outlined channels are CD31 negative and mutually exclusive of CD31-positive cell-lined channels elsewhere in the tumor consistent with tumor cells adapted to a pseudoendothelial phenotype in vasculogenic mimicry. To assess the functional potential of such PSMA-lined microvasculature to selectively direct infarctive tumor therapy, we coupled the soluble extracellular domain of tissue factor to a PSMA catalytic site inhibitor to create a PSMA-directed selective tumor vascular thrombogen (STVT). This protein induced selective local in vivo infarctive necrosis of the rat Mat Lu prostate tumor when administered i.v. The combined administration of this STVT with low-dose doxorubicin produced a profound tumoricidal effect, resulting in complete eradication of some tumors. This is consistent with the therapeutic potential for a PSMA-directed STVT and expands the potential for selective infarctive ablation of tumors.

The Hippo signal transduction cascade controls cell growth, proliferation and death, all of which are frequently deregulated in tumour cells. Since initial studies in Drosophila melanogaster were instrumental in defining Hippo signalling, the machinery was named after the central Ste20-like kinase Hippo. Moreover, given that loss of Hippo signalling components Hippo, Warts, and Mats resulted in uncontrolled tissue overgrowth, Hippo signalling was defined as a tumour-suppressor cascade. Significantly, all of the core factors of Hippo signalling have mammalian orthologues that functionally compensate for loss of their counterparts in Drosophila. Furthermore, studies in Drosophila and mammalian cell systems showed that Hippo signalling represents a kinase cascade that is tightly regulated by PPIs (protein-protein interactions). Several Hippo signalling molecules contain SARAH (Salvador/RASSF1A/Hippo) domains that mediate specific PPIs, thereby influencing the activities of MST1/2 (mammalian Ste20-like serine/threonine kinase 1/2) kinases, the human Hippo orthologues. Moreover, WW domains are present in several Hippo factors, and these domains also serve as interaction surfaces for regulatory PPIs in Hippo signalling. Finally, the kinase activities of LATS1/2 (large tumour-suppressor kinase 1/2), the human counterparts of Warts, are controlled by binding to hMOB1 (human Mps one binder protein 1), the human Mats. Therefore Hippo signalling is regulated by PPIs on several levels. In the present paper, I review the current understanding of how these regulatory PPIs are regulated and contribute to the functionality of Hippo signalling.

LKB1, originally considered a tumor suppressor, plays an important role in hepatocyte proliferation and liver regeneration. Mice lacking the methionine adenosyltransferase (<em>MAT</em>) gene <em>MAT</em>1A exhibit a chronic reduction in hepatic S-adenosylmethionine (SAMe) levels, basal activation of LKB1, and spontaneous development of nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC). These results are relevant for human health because patients with liver cirrhosis, who are at risk to develop HCC, have a marked reduction in hepatic <em>MAT</em>1A expression and SAMe synthesis. In this study, we isolated a cell line (SAMe-deficient [SAMe-D]) from <em>MAT</em>1A knockout (<em>MAT</em>1A-KO) mouse HCC to examine the role of LKB1 in the development of liver tumors derived from metabolic disorders. We found that LKB1 is required for cell survival in SAMe-D cells. LKB1 regulates Akt-mediated survival independent of phosphoinositide 3-kinase, adenosine monophosphate protein-activated kinase (AMPK), and mammalian target of rapamycin complex (mTORC2). In addition, LKB1 controls the apoptotic response through phosphorylation and retention of p53 in the cytoplasm and the regulation of herpesvirus-associated ubiquitin-specific protease (HAUSP) and Hu antigen R (HuR) nucleocytoplasmic shuttling. We identified HAUSP as a target of HuR. Finally, we observed cytoplasmic staining of p53 and p-LKB1(Ser428) in a NASH-HCC animal model (from <em>MAT</em>1A-KO mice) and in liver biopsies obtained from human HCC derived from both alcoholic steatohepatitis and NASH.

CONCLUSIONS

The SAMe-D cell line is a relevant model of HCC derived from NASH disease in which LKB1 is the principal conductor of a new regulatory mechanism and could be a practical tool for uncovering new therapeutic strategies.

Nonalcoholic fatty liver disease (NAFLD) is highly prevalent in the Western population. By mechanisms that are not completely understood, this disease may progress to nonalcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). db/db mice spontaneously develop hepatic steatosis, which progresses to NASH when these mice are fed a methionine choline-deficient (MCD) diet. The goal of our studies was to identify lipid and methionine metabolism pathways affected by MCD feeding to determine potential causal events leading to the development of NASH from benign steatosis. db/db mice fed the MCD diet for 2 weeks exhibited signs of incipient NASH development such as upregulated cytokines and chemokines. At this time point, MCD diet feeding caused S-adenosylmethionine (SAMe) depletion in db/db mice, while wild-type mice on the same diet retained hepatic SAMe levels. SAMe depletion exerts pleiotropic effects upon liver homeostasis and is commonly associated with a variety of liver insults such as thioacetamide, CCL4, and alcohol treatment; thus, SAMe depletion may serve as the second hit in NASH development. It is possible that differences in hepatic lipid and/or methionine metabolism between wild-type and db/db mice underlay the differential maintenance of SAMe levels during methionine and choline restriction. Indeed, db/db mice exhibited inhibited lipid oxidation pathways, which may be a priming factor for NASH development, and db/db mice fed the MCD diet had differential methionine adenosyltransferase (MAT) expression. The occurrence of SAMe depletion at this early, benign stage of NASH development in db/db mice with fatty liver suggests that SAMe supplementation may be (A) targeted to individuals susceptible to NASH (i.e., NAFLD patients) and (B) preventative of NASH before substantial liver injury has occurred.

Liver-specific and non-liver-specific methionine adenosyltransferase (<em>MAT</em>) are products of two genes, <em>MAT</em>1A and <em>MAT</em>2A, respectively, that catalyze the formation of S-adenosylmethionine. We showed a switch from <em>MAT</em>1A to <em>MAT</em>2A expression at the transcriptional level in human hepatocellular carcinoma (HCC) that facilitates cancer cell growth. The purpose of the present study was to better understand the molecular mechanism of increased <em>MAT</em>2A expression in HCC. In vitro DNase I footprinting analysis revealed two protected sites (-354 to -312 and -73 to -28) using nuclear proteins from HCC and HepG2 cells, but not normal liver. These sites are also protected in HepG2 cells on in vivo DNase I footprinting analysis. These protected sites contain consensus binding sites for c-Myb and Sp1. In HCC, the mRNA levels of c-myb and Sp1 and binding to their respective sites increased. Mutation of the c-Myb or Sp1 site reduced <em>MAT</em>2A promoter activity by 67% and 50%, respectively. The importance of these cis-acting elements and trans-activating factors was confirmed using heterologous promoter and expression vectors. Increased expression of c-Myb and Sp1 and binding to the <em>MAT</em>2A promoter contribute to transcriptional up-regulation of <em>MAT</em>2A in HCC.-Yang, H., Huang, Z.-Z., Wang, J., Lu, S. C. The role of c-Myb and Sp1 in the up-regulation of methionine adenosyltransferase 2A gene expression in human hepatocellular carcinoma.

Mounting evidence underlines the role of genomic hypomethylation in the generation of genomic instability (GI) and tumorigenesis, but whether DNA hypomethylation is required for hepatocellular carcinoma (HCC) development and progression remains unclear. We investigated the correlation between GI and DNA methylation, and influence of methionine metabolism deregulation on these parameters and hepatocarcinogenesis in c-Myc and c-Myc/Tgf-alpha transgenic mice and human HCCs. S-adenosyl-L-methionine/S-adenosylhomocysteine ratio and liver-specific methionine adenosyltransferase (MatI/III) progressively decreased in dysplastic and neoplastic liver lesions developed in c-Myc transgenic mice and in human HCC with better (HCCB) and poorer (HCCP) prognosis (based on patient's survival length). Deregulation of these parameters resulted in a rise of global DNA hypomethylation both in c-Myc and human liver lesions, positively correlated with GI levels in mice and humans, and inversely correlated with the length of survival of HCC patients. No changes in MATI/III and DNA methylation occurred in c-Myc/Tgf-alpha lesions and in a small human HCC subgroup with intermediate prognosis, where a proliferative activity similar to that of c-Myc HCC and HCCB was associated with low apoptosis. Upregulation of genes involved in polyamine synthesis, methionine salvage and downregulation of polyamine negative regulator OAZ1, was highest in c-Myc/Tgf-alpha HCCs and HCCP. Our results indicate that alterations in the activity of MAT/I/III, and extent of DNA hypomethylation and GI are prognostic markers for human HCC. However, a small human HCC subgroup, as c-Myc/Tgf-alpha tumors, may develop in the absence of alterations in DNA methylation.

The Guerrero Negro (GN) hypersaline microbial mats have become one focus for biogeochemical studies of stratified ecosystems. The GN mats are found beneath several of a series of ponds of increasing salinity that make up a solar saltern fed from Pacific Ocean water pumped from the Laguna Ojo de Liebre near GN, Baja California Sur, Mexico. Molecular surveys of the laminated photosynthetic microbial mat below the fourth pond in the series identified an enormous diversity of bacteria in the mat, but archaea have received little attention. To determine the bulk contribution of archaeal phylotypes to the pond 4 study site, we determined the phylogenetic distribution of archaeal rRNA gene sequences in PCR libraries based on nominally universal primers. The ratios of bacterial/archaeal/eukaryotic rRNA genes, 90%/9%/1%, suggest that the archaeal contribution to the metabolic activities of the mat may be significant. To explore the distribution of archaea in the mat, sequences derived using archaeon-specific PCR primers were surveyed in 10 strata of the 6-cm-thick mat. The diversity of archaea overall was substantial albeit less than the diversity observed previously for bacteria. Archaeal diversity, mainly euryarchaeotes, was highest in the uppermost 2 to 3 mm of the mat and decreased rapidly with depth, where crenarchaeotes dominated. Only 3% of the sequences were specifically related to known organisms including methanogens. While some mat archaeal clades corresponded with known chemical gradients, others did not, which is likely explained by heretofore-unrecognized gradients. Some clades did not segregate by depth in the mat, indicating broad metabolic repertoires, undersampling, or both.

We investigated the genotypic diversity of oxygenic and anoxygenic phototrophic microorganisms in microbial mat samples collected from three hot spring localities on the east coast of Greenland. These hot springs harbour unique Arctic microbial ecosystems that have never been studied in detail before. Specific oligonucleotide primers for cyanobacteria, purple sulfur bacteria, green sulfur bacteria and Choroflexus/Roseiflexus-like green non-sulfur bacteria were used for the selective amplification of 16S rRNA gene fragments. Amplification products were separated by denaturing gradient gel electrophoresis (DGGE) and sequenced. In addition, several cyanobacteria were isolated from the mat samples, and classified morphologically and by 16S rRNA-based methods. The cyanobacterial 16S rRNA sequences obtained from DGGE represented a diverse, polyphyletic collection of cyanobacteria. The microbial mat communities were dominated by heterocystous and non-heterocystous filamentous cyanobacteria. Our results indicate that the cyanobacterial community composition in the samples were different for each sampling site. Different layers of the same heterogeneous mat often contained distinct and different communities of cyanobacteria. We observed a relationship between the cyanobacterial community composition and the in situ temperatures of different mat parts. The Greenland mats exhibited a low diversity of anoxygenic phototrophs as compared with other hot spring mats which is possibly related to the photochemical conditions within the mats resulting from the Arctic light regime.

The wettability of electrospun poly(epsilon-caprolactone) (PCL) mats was improved by co-electrospinning with poly(vinyl alcohol) (PVA), by double-spinneret electrospinning method. The improved hydrophilicity of the hybrid PCL/PVA mats was confirmed by water contact angle measurement. The in vitro cell attachment on the hydrophobic PCL and hydrophilically modified PCL/PVA mats was compared by culture studies using human prostate epithelial cells (HPECs). The stability of water-soluble PVA component in the electrospun PCL/PVA mats was checked by thermogravimetric analysis and intensity of fluorescence material after immersion in water for 7 days. The images from scanning electron microscopy, field emission scanning electron microscopy, and optical microscopy showed that the attachment and proliferation rate of HPECs were improved by introducing PVA into the electrospun PCL mats.

The vertical and temporal changes in microbial communities were investigated throughout the water column and sediment of the saline meromictic Lake Kaiike by PCR-denaturing gradient gel electrophoresis (DGGE) of 16S rDNA. Marked depth-related changes in microbial communities were observed at the chemocline and the sediment-water interface. However, no major temporal changes in the microbial community below the chemocline were observed during the sampling period, suggesting that the ecosystem in the anoxic zone of Lake Kaiike was nearly stable. Although the sequence of the most conspicuous DGGE band throughout the anoxic water and in the top of the microbial mat was most similar to that of an anoxic, photosynthetic, green sulphur bacterium, Pelodyction luteolum DSM273 (97% similarity), it represented a new phylotype. A comparison of DGGE banding patterns of the water column and sediment samples demonstrated that specific bacteria accumulated on the bottom from the anoxic water layers, and that indigenous microbial populations were present in the sediment. The measurements of bicarbonate assimilation rates showed significant phototrophic assimilation in the chemocline and lithoautotrophic assimilation throughout the anoxic water, but were not clearly linked with net sulphide turnover rates, indicating that sulphur and carbon metabolisms were not directly correlated.

To understand the molecular properties of matrilin-3, a newly discovered member of the novel extracellular matrix protein family, we cloned a MAT-3 cDNA from developing chicken sterna. Real time quantitative reverse-transcription polymerase chain reaction indicates that MAT-3 mRNA is mainly expressed in the proliferation zone of a growth plate. It is also expressed in the maturation zone, overlapping with that of the mature chondrocyte-abundant matrilin-1 mRNA. This suggests that matrilin-3 may self-assemble in the proliferation zone, in addition to its co-assembly with matrilin-1 during endochondral ossification. Transfection of a MAT-3 cDNA into COS-7 cells shows that MAT-3 predominantly forms a homotetramer but also a trimer and a dimer. Co-transfection of both MAT-3 and MAT-1 cDNAs results in three major matrilins as follows: (MAT-1)(3), (MAT-3)(4), and (MAT-1)(2)(MAT-3)(2). Thus matrilin-3 may assemble into both homotypic and heterotypic oligomers. Our analysis shows that the assembly of MAT-3 does not depend on the number of epidermal growth factor repeats within the molecule, but the presence of Cys(412) and Cys(414) within the coiled-coil domain, which form covalent disulfide linkage responsible for both homo-oligomerization of MAT-3 and hetero-oligomerization of MAT-3 and MAT-1. Our data suggest that the varying synthetic levels of matrilins in different zones of a growth plate may result in a change of matrilin oligomeric forms during endochondral ossification.

Lamivudine is a novel cytosine nucleoside analog, reverse transcriptase inhibitor that has shown activity against human immunodeficiency virus (HIV) types 1 and 2 and hepatitis B virus in vitro. This study was conducted to compare the absolute bioavailability, pharmacokinetics, and absorption characteristics of oral solution, 100-mg capsule, and 100-mg tablet formulations of lamivudine with those of intravenous lamivudine. Twelve patients with HIV were enrolled in a single-center, randomized, open-label, four-way cross-over study. Treatment arms consisted of 100 mg intravenous lamivudine (administered over 1 hour), 100 mg oral lamivudine (1 mg/mL), a 100-mg capsule, and a 100-mg tablet, each followed by a 3- to 14-day washout period. Serial blood samples over 24 hours were obtained after each dose administration. Serum concentration data were analyzed to determine pharmacokinetic parameter estimates including area under the curve (AUC), terminal half-life (t1/2), mean residence time (MRT) for each formulation, systemic clearance, oral clearance, and apparent volume of distribution (Vd). Absolute bioavailability and in vivo mean absorption time (MAT) and mean dissolution time (MDT) were calculated for the oral formulations. Deconvolution techniques were used to calculate the input rate for the oral solution, capsule, and tablet. The two one-sided t test was used to determine bioequivalency among oral formulations with respect to logarithmic transformed estimates of AUC and maximum peak concentration (Cmax). Mean (CV) systemic clearance and Vdss after intravenous administration of lamivudine were 22.6 L/h (15%) and 99 L (28%), respectively; mean t1/2 ranged from 8.41 to 9.11 hours for all formulations; and MRT ranged from 4.42 to 5.77 hours for all formulations. Mean absolute bioavailability ranged from 86% to 88% for the oral solution, capsule, and tablet. All oral formulations were considered bioequivalent for AUC and Cmax. The MAT was 1.32 hour for the oral solution, and MDT was 0.03 and -0.11 hours for the capsule and the oral solution, respectively. The oral formulations of lamivudine examined in this study demonstrated acceptable bioavailability for oral administration. The solid oral formulations (capsule and tablet) show rapid dissolution properties with an absorption rate similar to or exceeding those observed with the oral solution. This suggests that dissolution is not an important factor for the rate of absorption of lamivudine. The use of deconvolution techniques using PCDCON provides valuable insight into the absorption characteristics of lamivudine.

Mating-type (MAT) loci were cloned from two asexual (mitosporic) phytopathogenic ascomycetes, Fusarium oxysporum (a pyrenomycete) and Alternaria alternata (a loculoascomycete), by a polymerase chain reaction (PCR)-based strategy. The conserved high mobility group (HMG) box domain found in the MATMATMATMATMATMATMATMAT-specific primers were used to assess the mating type of F. oxysporum and A. alternata field isolates by PCR. MAT genes from A. alternata were expressed. The A. alternata genes were confirmed to be functional in a close sexual relative, Cochliobolus heterostrophus, by heterologous expression.

Growth inhibition mediated by Hippo (Hpo) signaling is essential for tissue growth and organ size control in Drosophila. However, the cellular mechanism by which the core components like Mob as tumor suppressor (Mats) and Warts (Wts) protein kinase are activated is poorly understood. In this work, we found that the endogenous Mats is located at the plasma membrane in developing tissues. Membrane targeting constitutively activates Mats to promote apoptosis and reduce cell proliferation, which leads to reduced tissue growth and organ size. Moreover, the ability of membrane-targeted Mats to inhibit tissue growth required the wts gene activity and Wts kinase activity was increased by the activated Mats in developing tissues. Consistent with the idea that Mats is a key component of the Hpo pathway, Mats is required and sufficient to regulate Yki nuclear localization. These results support a model in which the plasma membrane is an important site of action for Mats tumor suppressor to control tissue growth and organ size.

Sporulation in Saccharomyces cerevisiae normally occurs only in MATa/MAT alpha diploids. We show that mutations in RME1 bypassed the requirements for both a and alpha mating type information in sporulation and therefore allowed MATa/MATa and MAT alpha/MAT alpha diploids to sporulate. RME1 was located on chromosome VII, between LEU1 and ADE6.

The switching of yeast mating-type alleles involves a transposition of a copy of a sequence from HML or HMR to replace the sequences at MAT. Using diploid strains of yeast we have discovered that about 1% of the homothalic conversions of MAT alleles are accompanied by large intrachromosomal rearrangements. These rearrangements are highly specific fusions of part of MAT either with HMR (to produce a deficiency ring chromosome). We conclude that the mechanism of MAT conversions involves a highly specific pairing between the homologous sequences at MAT and the donor genes HML or HMR followed by a specialized gene conversion event, in which the original allele is replaced by a sequence copied from HMR or HML. At about a 1% frequency conversion of the MAT locus is accompanied by a reciprocal recombination event that results in an intrachromosomal deletion. This same preferential pairing is reflected in a high frequency >> 10(-3)) of site-specific mitotic recombination between MAT alleles on differenat chromosomes. A gene conversion model also allows us to explain the "illegal" transpositions of MAT alleles to HMR or HML that occur when normal excision of MAT is prevented.