Candidatus Chloracidobacterium thermophilum, which naturally inhabits microbial mats of alkaline siliceous hot springs in Yellowstone National Park, is the only known chlorophototroph in the phylum Acidobacteria. The Ca. C. thermophilum genome was composed of two chromosomes (2,683,362 bp and 1,012,010 bp), and both encoded essential genes. The genome included genes to produce chlorosomes, the Fenna-Matthews-Olson protein, bacteriochlorophylls a and c as principal pigments, and type-1, homodimeric reaction centres. Ca. C. thermophilum is an aerobic photoheterotroph that lacks the ability to synthesize several essential nutrients. Key genes of all known carbon fixation pathways were absent, as were genes for assimilatory nitrate and sulfate reduction and vitamin B(12) synthesis. Genes for the synthesis of branched-chain amino acids (valine, isoleucine and leucine) were also absent, but genes for catabolism of these compounds were present. This observation suggested that Ca. C. thermophilum may synthesize branched-chain amino acids from an intermediate(s) of the catabolic pathway by reversing these reactions. The genome encoded an aerobic respiratory electron transport chain that included NADH dehydrogenase, alternative complex III and cytochrome oxidase. The chromosomes of the laboratory isolate were compared with assembled, metagenomic scaffolds from the major Ca. C. thermophilum population in hot-spring mats. The larger chromosomes of the two populations were highly syntenous but significantly divergent (~13%) in sequence. In contrast, the smaller chromosomes have undergone numerous rearrangements, contained many transposases, and might be less constrained by purifying selection than the large chromosomes. Some transposases were homologous to those of mat community members from other phyla.

In eukaryotic cells, premature termination of translation at nonsense codons has been implicated as the cause of a variety of posttranscriptional events, including rapid mRNA decay in the cytoplasm or the nucleus, altered splice site selection, and exon skipping. In the yeast Saccharomyces cerevisiae, nonsense codons promote accelerated mRNA decay, and we sought to determine the cellular location in which this degradation occurs. In this report, we demonstrate that six different mRNAs, including nonsense-containing transcripts of the LEU2, HIS4, PGK1, and CYH2 genes, and two wild-type mRNAs (the MAT(alpha)1 and CYH2 mRNAs), were stabilized when the translation elongation inhibitor cycloheximide was added to cellular growth media. Subsequent removal of cycloheximide resulted in resumption of translation and degradation of wild-type and nonsense-containing mRNAs. A significant fraction of the CYH2 pre-mRNA that accumulated in the presence of cycloheximide was associated with polysomes, but disappeared from that fraction when decay resumed in the absence of the drug. Moreover, the abundance of the spliced and unspliced forms of the untranslated U3 snRNA was shown to be unaffected in strains harboring mutations that stabilize nonsense-containing mRNAs. Taken together, these observations indicate that nonsense-containing mRNAs in yeast are degraded within the polysome compartment of the cell.

Although chemotherapy has advanced into the era of targeted drugs, the antitumor efficacies of current therapies are limited, most likely because of the high degree of cancer clonal heterogeneity, intratumor genetic heterogeneity and cell signal complexity. As shutdown of a single target does not necessarily eradicate the cancer, the use of combinations of molecular-targeted agents (MATs) has been proposed, and some pioneering research has been conducted to examine the efficacy of this strategy. In this article, the clinical and preclinical studies that are underway in an attempt to improve the anticancer efficacy of chemotherapies through combination strategies are summarized. Studies of combining cytotoxic agents with MATs, coinhibiting two or more targets in a single pathway or coinhibiting parallel or compensatory pathways as well as specific combinations will be introduced, and the antitumor potentials of each combination strategy will be evaluated.

OBJECTIVE

Methionine adenosyltransferase (<em>MAT</em>) catalyzes S-adenosylmethionine biosynthesis. Two genes (<em>MAT</em>1A and <em>MAT</em>2A) encode for the catalytic subunit of <em>MAT</em>, while a third gene (<em>MAT</em>2beta) encodes for a regulatory subunit that modulates the activity of <em>MAT</em>2A-encoded isoenzyme. We uncovered multiple splicing variants while characterizing its 5'-flanking region. The aims of our current study are to examine the expression pattern, regulation, and functions of the 2 major variants: V1 and V2.

METHODS

Studies were conducted using RNA from normal human tissues, resected hepatocellular carcinoma specimens, and cell lines. Gene expression, promoter and nuclear binding activities, growth, and apoptosis were measured by routine assays.

RESULTS

<em>MAT</em>2beta is expressed in most but not all tissues, and the 2 variants are differentially expressed. The messenger RNA levels of both variants are markedly increased in hepatocellular carcinoma. Tumor necrosis factor (TNF)-alpha, which induces <em>MAT</em>2A in HepG2 cells, also induced V1 (but not V2) expression. TNF-alpha induced the promoter activity of <em>MAT</em>2beta V1, likely via nuclear factor kappaB and activator protein 1. Both variants regulate growth, but only V1 regulates apoptosis. Reduced expression of V1 led to c-Jun-N-terminal kinase (JNK) activation, apoptosis, and sensitized HepG2 cells to TNF-alpha-induced apoptosis, while overexpression of V1 was protective. However, blocking JNK1 or JNK2 activation did not prevent apoptosis induced by V1 knockdown. V1 (but not V2) knockdown also leads to apoptosis in a colon cancer cell line, suggesting these variants play similar roles in many cell types.

CONCLUSIONS

Different variants of <em>MAT</em>2beta regulate growth and death, which broadens their importance in biology.

Unlike antibiotics, biocides are multi-targeted antimicrobial agents. Several of the damaging effects reported to occur in the most widely studied organisms, bacteria, may also take place to varying degrees in other organisms. Nevertheless, there is considerable variation in the response of different microorganisms to biocides. Bacteria themselves (Gram-positive and Gram-negative vegetative organisms, mycobacteria and spores) respond differently to biocides and this disparity is widened when yeasts, moulds, protozoa and algae are considered. The underlying reasons for these varied responses are poorly understood at present, but the chemical composition of outer cellular layers is likely to be a factor of prime importance. Other possible contributory factors may be differences in stress responses, the presence of efflux pumps and cells occurring within biofilms or algal mats.

To obtain a broad perspective of the events leading to spontaneous loss of heterozygosity (LOH), we have characterized the genetic alterations that functionally inactivated the URA3 marker hemizygously or heterozygously situated either on chromosome III or chromosome V in diploid Saccharomyces cerevisiae cells. Analysis of chromosome structure in a large number of LOH clones by pulsed-field gel electrophoresis and PCR showed that chromosome loss, allelic recombination, and chromosome aberration were the major classes of genetic alterations leading to LOH. The frequencies of chromosome loss and chromosome aberration were significantly affected when the marker was located in different chromosomes, suggesting that chromosome-specific elements may affect the processes that led to these alterations. Aberrant-sized chromosomes were detected readily in approximately 8% of LOH events when the URA3 marker was placed in chromosome III. Molecular mechanisms underlying the chromosome aberrations were further investigated by studying the fate of two other genetic markers on chromosome III. Chromosome aberration caused by intrachromosomal rearrangements was predominantly due to a deletion between the MAT and HMR loci that occurred at a frequency of 3.1 x 10(-6). Another type of chromosome aberration, which occurred at a frequency slightly higher than that of the intrachromosomal deletion, appeared to be caused by interchromosomal rearrangement, including unequal crossing over between homologous chromatids and translocation with another chromosome.

The oxidation of hydrogen sulfide is essential to sulfur cycling in marine habitats. However, the role of microbial sulfur oxidation in marine sediments and the microorganisms involved are largely unknown, except for the filamentous, mat-forming bacteria. In this study we explored the diversity, abundance and activity of sulfur-oxidizing prokaryotes (SOP) in sulfidic intertidal sediments using 16S rRNA and functional gene sequence analyses, fluorescence in situ hybridization (FISH) and microautoradiography. The 16S rRNA gene analysis revealed that distinct clades of uncultured Gammaproteobacteria are important SOP in the tidal sediments. This was supported by the dominance of gammaproteobacterial sequences in clone libraries of genes encoding the reverse dissimilatory sulfite reductase (rDSR) and the adenosine phosphosulfate reductase (APR). Numerous sequences of all three genes grouped with uncultured autotrophic SOP. Accordingly, Gammaproteobacteria accounted for 40-70% of all ¹⁴CO₂ -incorporating cells in surface sediments as shown by microautoradiography. Furthermore, phylogenetic analysis of all three genes consistently suggested a discrete population of SOP that was most closely related to the sulfur-oxidizing endosymbionts of the tubeworm Oligobrachia spp. FISH showed that members of this population (WS-Gam209 group) were abundant, reaching up to 1.3 × 10⁸ cells ml⁻¹ (4.6% of all cells). Approximately 25% of this population incorporated CO₂, consistent with a chemolithoautotrophic metabolism most likely based on sulfur oxidation. Thus, we hypothesize that novel, gammaproteobacterial SOP attached to sediment particles may play a more important role for sulfide removal and primary production in marine sediments than previously assumed.

Theoretical models predict that selfish DNA elements require host sex to persist in a population. Therefore, a transposon that induces sex would strongly favor its own spread. We demonstrate that a protein homologous to transposases, called alpha3, was essential for mating type switch in Kluyveromyces lactis. Mutational analysis showed that amino acids conserved among transposases were essential for its function. During switching, sequences in the 5' and 3' flanking regions of the alpha3 gene were joined, forming a DNA circle, showing that alpha3 mobilized from the genome. The sequences encompassing the alpha3 gene circle junctions in the mating type alpha (MATalpha) locus were essential for switching from MATalpha to MATa, suggesting that alpha3 mobilization was a coupled event. Switching also required a DNA-binding protein, Mating type switch 1 (Mts1), whose binding sites in MATalpha were important. Expression of Mts1 was repressed in MATa/MATalpha diploids and by nutrients, limiting switching to haploids in low-nutrient conditions. A hairpin-capped DNA double-strand break (DSB) was observed in the MATa locus in mre11 mutant strains, indicating that mating type switch was induced by MAT-specific DSBs. This study provides empirical evidence for selfish DNA promoting host sexual reproduction by mediating mating type switch.

The results of our study of the origin of the additional X chromosome in 39 males with a 47,XXY chromosome constitution are reported. We used a total of 20 X-linked RFLPs and successfully determined the origin of all 32 patients in whom DNA from both parents was available, and a further 3 in whom DNA was available from the patient and mother only. Males whose additional X chromosome was maternal in origin were further investigated using an X-linked centromere specific probe to determine the cell division at which the error occurred. Our results showed 53% of the non-disjunction to be attributable to pat mei I errors, 34% to mat mei I errors, 9% to mat mei II errors and 3% to a post-zygotic mitotic error. In the great majority of patients resulting from an error of maternal meiosis there was clear evidence of recombination involving the non-disjoined chromosomes, suggesting that absence of recombination is not an important aetiological factor in non-disjunction of the X chromosome in female meiosis. There was no alteration of parental age associated with the paternally derived 47,XXY males but a marked increase in maternal age among the maternally derived 47,XXY males, the increase being associated with mat mei I but not mat mei II errors. The proportion of paternally and maternally derived cases was similar among different ascertainment classes, suggesting that there is no dramatic effect of parental origin of the additional X chromosome on the phenotype of 47,XXY males.

The diversity associated with a microbial mat sample collected from a deep-sea hydrothermal vent on the Southern East Pacific Rise was determined using a molecular phylogenetic approach based on the comparison of sequences from the small subunit ribosomal RNA gene (16S rDNA). The DNA was extracted from the sample and the 16S rDNA was amplified by PCR. Sixteen different phylotypes were identified by restriction fragment length polymorphism analysis; four phylotypes were later identified as putative chimeras. Analysis of the 16S rDNA sequences placed all the phylotypes within the Proteobacteria. The majority of the sequences (98%) were most closely related to a new clade of epsilon-Proteobacteria that were initially identified from an in situ growth chamber deployed on a deep-sea hydrothermal vent on the Mid-Atlantic Ridge in 1995. The similarity between phylotypes identified from Atlantic and Pacific deep-sea hydrothermal vent sites indicates that this new clade of Proteobacteria may be endemic to and widely distributed among deep-sea hydrothermal vents.

We studied the diel migrations of several species of microorganisms in a hypersaline, layered microbial mat. The migrations were quantified by repeated coring of the mat with glass capillary tubes. The resulting minicores were microscopically analyzed by using bright-field and epifluorescence (visible and infrared) microscopy to determine depths of coherent layers and were later dissected to determine direct microscopic counts of microorganisms. Microelectrode measurements of oxygen concentration, fiber optic microprobe measurements of light penetration within the mat, and incident irradiance measurements accompanied the minicore sampling. In addition, pigment content, photosynthesis and irradiance responses, the capacity for anoxygenic photosynthesis, and gliding speeds were determined for the migrating cyanobacteria. Heavily pigmented Oscillatoria sp. and Spirulina cf. subsalsa migrated downward into the mat during the early morning and remained deep until dusk, when upward migration occurred. The mean depth of the migration (not more than 0.4 to 0.5 mm) was directly correlated with the incident irradiance over the mat surface. We estimated that light intensity at the upper boundary of the migrating cyanobacteria was attenuated to such an extent that photoinhibition was effectively avoided but that intensities which saturated photosynthesis were maintained through most of the daylight hours. Light was a cue of paramount importance in triggering and modulating the migration of the cyanobacteria, even though the migrating phenomenon could not be explained solely in terms of a light response. We failed to detect diel migration patterns for other cyanobacterial species and filamentous anoxyphotobacteria. The sulfide-oxidizing bacterium Beggiatoa sp. migrated as a band that followed low oxygen concentrations within the mat during daylight hours. During the nighttime, part of this population migrated toward the mat surface, but a significant proportion remained deep.

To distinguish among possible mechanisms of repair of a double-strand break (DSB) by gene conversion in budding yeast, Saccharomyces cerevisiae, we employed isotope density transfer to analyze budding yeast mating type (MAT) gene switching in G2/M-arrested cells. Both of the newly synthesized DNA strands created during gene conversion are found at the repaired locus, leaving the donor unchanged. These results support suggestions that mitotic DSBs are primarily repaired by a synthesis-dependent strand-annealing mechanism. We also show that the proportion of crossing-over associated with DSB-induced ectopic recombination is not affected by the presence of nonhomologous sequences at one or both ends of the DSB or the presence of additional sequences that must be copied from the donor.

The genetic diversity of Desulfovibrio species in environmental samples was determined by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified [NiFe] hydrogenase gene fragments. Five different PCR primers were designed after comparative analysis of [NiFe] hydrogenase gene sequences from three Desulfovibrio species. These primers were tested in different combinations on the genomic DNAs of a variety of hydrogenase-containing and hydrogenase-lacking bacteria. One primer pair was found to be specific for Desulfovibrio species only, while the others gave positive results with other bacteria also. By using this specific primer pair, we were able to amplify the [NiFe] hydrogenase genes of DNAs isolated from environmental samples and to detect the presence of Desulfovibrio species in these samples. However, only after DGGE analysis of these PCR products could the number of different Desulfovibrio species within the samples be determined. DGGE analysis of PCR products from different bioreactors demonstrated up to two bands, while at least five distinguishable bands were detected in a microbial mat sample. Because these bands most likely represent as many Desulfovibrio species present in these samples, we conclude that the genetic diversity of Desulfovibrio species in the natural microbial mat is far greater than that in the experimental bioreactors.

Since the pioneering model for homologous recombination proposed by Robin Holliday in 1964, there has been great progress in understanding how recombination occurs at a molecular level. In the budding yeast Saccharomyces cerevisiae, one can follow recombination by physically monitoring DNA after the synchronous induction of a double-strand break (DSB) in both wild-type and mutant cells. A particularly well-studied system has been the switching of yeast mating-type (MAT) genes, where a DSB can be induced synchronously by expression of the site-specific HO endonuclease. Similar studies can be performed in meiotic cells, where DSBs are created by the Spo11 nuclease. There appear to be at least two competing mechanisms of homologous recombination: a synthesis-dependent strand annealing pathway leading to noncrossovers and a two-end strand invasion mechanism leading to formation and resolution of Holliday junctions (HJs), leading to crossovers. The establishment of a modified replication fork during DSB repair links gene conversion to another important repair process, break-induced replication. Despite recent revelations, almost 40 years after Holliday's model was published, the essential ideas he proposed of strand invasion and heteroduplex DNA formation, the formation and resolution of HJs, and mismatch repair, remain the basis of our thinking.

Yeast cells normally display either an axial (for MATa or MAT alpha cells) or bipolar (for MATa/alpha cells) pattern of bud-site selection. The RSR1 gene, which was previously identified as a multicopy suppressor of Ts- mutations in the bud-emergence gene CDC24, encodes a GTPase of the Ras family that is required for both budding patterns. Mutations in Rsr1p that presumably block its ability to bind or hydrolyze GTP cause a randomized budding phenotype, suggesting that regulators of Rsr1p will prove to be required for proper bud positioning. The BUD5 gene product is required for proper bud-site selection and contains similarity to GDP-dissociation stimulators (GDS) for Ras-type proteins, suggesting that Bud5p may be a GDS for Rsr1p. Here I report that BUD5 is required for wild-type RSR1, but not for mutationally activated rsr1val12, to serve as a multicopy suppressor of cdc24, indicating that Bud5p functions as a GDS for Rsr1p in vivo. To identify the GAP (GTPase-activating protein) for Rsr1p, a genetic selection was designed based on the observation that mutationally activated rsr1val12, but not wild-type RSR1, can serve as a multicopy suppressor of yeast RAS2(Ts) mutants. Mutants were selected that allowed wild-type RSR1 to act as a multicopy suppressor of RAS2(Ts). Two such mutations proved to be in the BUD2 gene, suggesting that Bud2p functions as a GAP for Rsr1p in vivo.

We enrolled consecutive febrile admissions to two hospitals in Moshi, Tanzania. Confirmed leptospirosis was defined as a ≥ 4-fold increase in microscopic agglutination test (MAT) titer; probable leptospirosis as reciprocal MAT titer ≥ 800; and exposure to pathogenic leptospires as titer ≥ 100. Among 870 patients enrolled in the study, 453 (52.1%) had paired sera available, and 40 (8.8%) of these met the definition for confirmed leptospirosis. Of 832 patients with ≥ 1 serum sample available, 30 (3.6%) had probable leptospirosis and an additional 277 (33.3%) had evidence of exposure to pathogenic leptospires. Among those with leptospirosis the most common clinical diagnoses were malaria in 31 (44.3%) and pneumonia in 18 (25.7%). Leptospirosis was associated with living in a rural area (odds ratio [OR] 3.4, P < 0.001). Among those with confirmed leptospirosis, the predominant reactive serogroups were Mini and Australis. Leptospirosis is a major yet underdiagnosed cause of febrile illness in northern Tanzania, where it appears to be endemic.

There is a lack of clinical tools to facilitate communication between clinicians and patients about chemotherapy-induced nausea and vomiting (CINV). The Multinational Association of Supportive Care in Cancer (MASCC) has developed such a tool, which is an eight-item scale for the assessment of acute and delayed nausea and vomiting, and is completed once per cycle of chemotherapy. The aim of the current study was to assess its psychometric properties, specifically reliability and validity, cultural transferability and equivalence, and congruence with proxy assessments, as well as to determine if accuracy of recall of CINV events using the MASCC Antiemesis Tool (MAT) differed over time from chemotherapy. A prospective study was carried out with adult cancer patients and their informal carers from two hospitals, one each in the United Kingdom (UK) and United States of America (U.S.). Patients completed the Rhodes Index for nausea, vomiting and retching (INVR) daily for the first five days after chemotherapy and were then asked to complete the MAT at one week, two weeks, or three weeks after chemotherapy. Carers completed an adapted MAT concurrently with patients. The sample consisted of 87 patients and 22 informal carers. The internal consistency reliability of the scale was high, with Cronbach alphas of 0.77 (patient sample) and 0.82 (carer sample). Responses were similar between the UK and U.S. samples in terms of nausea and vomiting, and both samples found the scale easy to use. Contrasted-groups validity (using age as a grouping variable) and concurrent validity (MAT compared with INVR) suggested that the scale is sensitive to detect the different dimensions of CINV and performed well against a daily assessment of nausea/vomiting (total score correlation r=0.86, P<0.001). Recall of events was high even three weeks after chemotherapy (correlations with INVR of 0.44-0.99, all P<0.01). Factor analysis clearly identified three factors, namely vomiting, acute nausea, and delayed nausea. Proxy assessments by carers were congruent with the patients' responses, especially in relation to vomiting. The MAT is a reliable, valid, clear, and easy-to-use clinical tool that could facilitate discussion between clinicians and patients about their nausea and vomiting experience, thereby potentially aiding treatment decisions. Regular assessment of nausea and vomiting after chemotherapy has the potential to significantly improve CINV management.

Bacterial aromatic polyketides are important therapeutic compounds including front line antibiotics and anticancer drugs. It is one of the last remaining major classes of natural products of which the biosynthesis has not been reconstituted in the genetically superior host Escherichia coli. Here, we demonstrate the engineered biosynthesis of bacterial aromatic polyketides in E. coli by using a dissected and reassembled fungal polyketide synthase (PKS). The minimal PKS of the megasynthase PKS4 from Gibberella fujikuroi was extracted by using two approaches. The first approach yielded a stand-alone Ketosynthase (KS)_malonyl-CoA:ACP transferase (MAT) didomain and an acyl-carrier protein (ACP) domain, whereas the second approach yielded a compact PKS (PKS_WJ) that consists of KS, MAT, and ACP on a single polypeptide. Both minimal PKSs produced nonfungal polyketides cyclized via different regioselectivity, whereas the fungal-specific C2-C7 cyclization mode was not observed. The kinetic properties of the two minimal PKSs were characterized to confirm both PKSs can synthesize polyketides with similar efficiency as the parent PKS4 megasynthase. Both minimal PKSs interacted effectively with exogenous polyketide cyclases as demonstrated by the synthesis of predominantly PK8 3 or NonaSEK4 6 in the presence of a C9-C14 or a C7-C12 cyclase, respectively. When PKS_WJ and downstream tailoring enzymes were expressed in E. coli, the expected nonaketide anthraquinone SEK26 was recovered in good titer. High-cell density fermentation was performed to demonstrate the scale-up potential of the in vivo platform for the biosynthesis of bacterial polyketides. Using engineered fungal PKSs can therefore be a general approach toward the heterologous biosynthesis of bacterial aromatic polyketides in E. coli.

Spinal manipulative therapy has been widely recognized in the medical fields as a conservative treatment modality for spinal dysfunction and pain. Spinal manipulative therapy consists of an application of a thrusting force on a specific part of the spine in a well-defined direction. The magnitude of this force has been associated with positive treatment effects, such as realigning vertebral bodies, mobilizing spinal joints, relaxing back musculature through reflex pathways, and producing a respiratory burst. However, direct force measurements during spinal manipulative therapy in a clinically relevant situation have not been performed to date. The purpose of this study was to measure the forces exerted onto patients during spinal manipulative therapy on various locations of the spinal column. Force measurements were obtained using a thin, flexible pressure mat. The results indicate that peak and preload forces are considerably smaller for spinal manipulative therapy performed on the cervical spine compared to corresponding values obtained on the thoracic spine and sacroiliac joint. Furthermore, for treatments on the thoracic spine and sacroiliac joint, a significant relation was found to exist between preload and peak forces.

Sodium alginate (SA)/poly (vinyl alcohol) (PVA) fibrous mats were prepared by electrospinning technique. ZnO nanoparticles of size ∼160nm was synthesized and characterized by UV spectroscopy, dynamic light scattering (DLS), XRD and infrared spectroscopy (IR). SA/PVA electrospinning was further carried out with ZnO with different concentrations (0.5, 1, 2 and 5%) to get SA/PVA/ZnO composite nanofibers. The prepared composite nanofibers were characterized using FT-IR, XRD, TGA and SEM studies. Cytotoxicity studies performed to examine the cytocompatibility of bare and composite SA/PVA fibers indicate that those with 0.5 and 1% ZnO concentrations are less toxic where as those with higher concentrations of ZnO is toxic in nature. Cell adhesion potential of this mats were further proved by studying with L929 cells for different time intervals. Antibacterial activity of SA/PVA/ZnO mats were examined with two different bacteria strains; Staphylococcus aureus and Escherichia coli, and found that SA/PVA/ZnO mats shows antibacterial activity due to the presence of ZnO. Our results suggest that this could be an ideal biomaterial for wound dressing applications once the optimal concentration of ZnO which will give least toxicity while providing maximum antibacterial activity is identified.f.

Abstract Nitrogen (N) availability is a key nutritional factor controlling microbial production in Antarctic freshwater and soil habitats. Since there are no significant sources of biologically available N entering these ecosystems, nitrogen fixation may be a major source of "new" N supporting primary and secondary production. The role of N2 fixation was examined in cyanobacteria-dominated microbial aggregates embedded in the permanent ice cover of Lake Bonney, McMurdo Dry Valley (Victoria Land) lakes area, and in cyanobacterial mats found in soils adjacent to the ice edge. Nitrogenase activity was extremely low compared to temperate and tropical systems, but N2 fixation was found at all study sites. N2 fixation occurred under both dark and light conditions, indicating the potential involvement of both phototrophic and heterotrophic diazotrophs. Nitrogenase activity measurements (acetylene reduction assay) and molecular characterization (PCR amplification of nifH fragments) demonstrated a diverse and periodically active (when liquid water is present) diazotrophic community in this arid, nutrient-limited environment. As a result of the close proximity to other microorganisms and the nutritional constraints of this environment, these diazotrophs may be involved in mutually beneficial consortial relationships that enhance their growth when water is available.

Solutions of poly(ethylene-co-vinyl alcohol) or EVOH, ranging in composition from 56 to 71 wt% vinyl alcohol, can be readily electrospun at room temperature from solutions in 70% 2-propanol/water (rubbing alcohol). The solutions are prepared at 80 degrees C and allowed to cool to room temperature. Interestingly, the solutions are not stable at room temperature and eventually the polymer precipitates after several hours. However, prior to precipitation, electrospinning is extensive and rapid, allowing coverage of fibers on various substrates, including a grounded metal plate, dielectrics interposed between the charged jet and the metal ground, and on the human body. Fiber diameters of ca. 0.2-8.0 microm were obtained depending upon the solution concentration, an attractive range for tissue engineering, wound healing, and related applications. Electrospun EVOH mats have been shown to support the culturing of smooth muscle cells and fibroblasts.

In the context of an imitation game, 12- and 18-month-old infants saw an adult do such things as make a toy mouse hop across a mat (with sound effects). In one condition (House), the adult ended by placing the mouse in a toy house, whereas in another condition (No House) there was no house present at the final location. Infants at both ages usually simply put the mouse in the house (ignoring the hopping motion and sound effects) in the House condition, presumably because they interpreted the adult's action in terms of this final goal and so ignored the behavioral means. In contrast, infants copied the adult's action (both the hopping motion and the sound effects) when no house was present, presumably because here infants saw the action itself as the adult's only goal. From very early, infants' social learning is flexible: infants focus on and copy either the end or the means of an adult action as required by the context.

Methionine metabolism starts with the formation of S-adenosylmethionine (AdoMet), the most important biological methyl donor. This reaction is catalyzed by methionine adenosyltransferase (<em>MAT</em>). <em>MAT</em> is the product of two different genes: <em>MAT</em>1A, which is expressed only in the adult liver, and <em>MAT</em>2A, which is widely distributed, expressed in the fetal liver, and replaces <em>MAT</em>1A in hepatocarcinoma. In the liver, preservation of high expression of <em>MAT</em>1A and low expression of <em>MAT</em>2A is critical for the maintenance of a functional and differentiated organ. Here we describe that in cultured rat hepatocytes <em>MAT</em>1A expression progressively decreased, as described for other liver-specific genes, and <em>MAT</em>2A expression was induced. We find that this switch in gene expression was prevented by adding AdoMet to the culture medium. We also show that in cultured hepatocytes with decreased <em>MAT</em>1A expression AdoMet addition markedly increased <em>MAT</em>1A transcription in a dose-dependent fashion. This effect of AdoMet was mimicked by methionine, and blocked by 3-deazaadenosine and L-ethionine, but not D-ethionine, indicating that the effect was specific and mediated probably by a methylation reaction. These findings identify AdoMet as a key molecule that differentially regulates <em>MAT</em>1A and <em>MAT</em>2A expression and helps to maintain the differentiated status of the hepatocyte.