A prospective study in Thailand identified 106 patients with culture-proven leptospirosis. The accuracy of the microscopic agglutination test (MAT) in predicting the infecting serovar was evaluated in 78/106 (74%) patients with a diagnostic titer. MAT correctly determined the infecting serovar in 26 cases (33%), indicating that this assay is a poor predictor of infecting serovar in our setting.

The mating-type loci located at the ends of chromosome III in Saccharomyces cerevisiae are transcriptionally repressed by a region-specific but sequence-nonspecific silencing apparatus, mediated by cis-acting silencer sequences. Previous deletion analyses have defined the locations and organizations of the silencers in their normal context and have shown that they are composed of various combinations of replication origins and binding sites for specific DNA-binding proteins. We have evaluated what organization of silencer sequences is sufficient for establishing silencing at a novel location, by inserting individual silencers next to the MAT locus and then assessing expression of MAT. The results of this analysis indicate that efficient silencing can be achieved by inserting either a single copy of the E silencer from HMR or multiple, tandem copies of either the E or I silencer from HML. These results indicate that while all silencers are functionally equivalent, they have different efficiencies; HMR E is more active than HML E, which itself is more active than HML I. Both HMR E and HML E exhibit orientation-dependent silencing, and the particular organization of binding elements within the silencer domain is critical for function. In some situations, silencing of MAT is conditional: complete silencing is obtained when cells are grown on glucose, and complete derepression occurs when cells are shifted to a nonfermentable carbon source, a process mediated in part by the RAS/cyclic AMP signaling pathway. Finally, the E silencer from HMR is able to reestablish repression immediately upon a shift back to glucose, while the silencers from HML exhibit a long lag in reestablishing repression, thus indicating distinctions between the two silencers in their reestablishment capacities. These results demonstrate that silencers can serve as nonspecific gene inactivation centers and that the attendant silencing can be rendered responsive to potential developmental cues.

Exposure to moderate doses of UV B (0.35 to 0.79 W m(sup-2) s(sup-1) or 0.98 to 2.2 (mu)mol of photons m(sup-2) s(sup-1) at 310 nm) caused the surface layers of microbial mats from Solar Lake, Sinai, Egypt, to become visibly lighter green. Concurrent with the color change were rapid and dramatic reductions in gross photosynthesis and in the resultant high porewater oxygen concentrations in the surface layers of the mats. The depths at which both maximum gross photosynthesis and maximum oxygen concentrations occurred were displaced downward. In contrast, gross photosynthesis in the deeper layers of the mats increased in response to UV B incident upon the surface. The cessation of exposure to UV B partially reversed all of these changes. Taken together, these responses suggest that photoautotrophic members of the mat community, most likely the dominant cyanobacterium Microcoleus chthonoplastes, were migrating in response to the added UV B. The migration phenomenon was also observed in response to increases in visible radiation and UV A, but UV B was ca. 100-fold more effective than visible radiation and ca. 20-fold more effective than UV A in provoking the response. Migrating microorganisms within this mat are apparently able to sense UV B directly and respond behaviorally to limit their exposure to UV. Because of strong vertical gradients of light and dissolved substances in microbial mats, the migration and the resultant vertical redistribution of photosynthetic activity have important consequences for both the photobiology of the cyanobacteria and the net primary productivity of the mat ecosystem.

A putative pheromone precursor gene of Neurospora crassa, mfa-1 (which encodes mating factor a-1), was identified as the most abundant clone in starved mycelial and perithecial cDNA libraries. Northern analysis demonstrated high mfa-1 expression in all mating type a tissues and suggested low expression levels in mat A tissues. The mfa-1 gene was expressed as an approximately 1.2-kb transcript predicted to encode a 24-residue peptide, followed by a long 3' untranslated region (3' UTR). The predicted MFA1 sequence showed 100% sequence identity to PPG2 of Sordaria macrospora and structural similarity (a carboxy-terminal CAAX motif) to many hydrophobic fungal pheromone precursors. Mutants with a disrupted open reading frame (ORF) in which the critical cysteine residue had been changed to a nonprenylatable residue, tyrosine (YAAX mutants), were isolated, as were mfa-1 mutants with intact ORFs but multiple mutations in the 3' noncoding region (CAAX mutants). The 3' UTR is required for the full range of mfa-1 gene activity. Both classes of mutants showed delayed and reduced vegetative growth (which was suppressed by supplementation with a minute amount [30 micro M] of ornithine, citrulline, or arginine), as well as aberrant sexual development. When crossed as female parents to wild-type males, the CAAX and YAAX mutants showed greatly reduced ascospore production. No ascospores were produced in homozygous mfa-1 crosses. As males, YAAX mat a mutants were unable to attract wild-type mat A trichogynes (female-specific hyphae) or to initiate sexual development, while CAAX mat a mutants were able to mate and produce sexual progeny despite their inability to attract mat A trichogynes. In the mat A background, both CAAX and YAAX mutants showed normal male fertility but defective vegetative growth and aberrant female sexual development. Thus, the mfa-1 gene appears to have multiple roles in N. crassa development: (i) it encodes a hydrophobic pheromone with a putative farnesylated and carboxymethylated C-terminal cysteine residue, required by mat a to attract trichogynes of mat A; (ii) it is involved in female sexual development and ascospore production in both mating types; and (iii) it functions in vegetative growth of both mating types.

Lung metastases resulting from the intravenous (i.v.) injection of cells from the rat mammary adenocarcinoma 13762 MAT were significantly reduced by a variety of sulphated polysaccharides, the most effective being heparin, fucoidan and Carrageenan lambda. Although all the inhibitory polysaccharides were anticoagulants, it is unlikely that anticoagulation is the total explanation of their antimetastatic effect because: (i) heparin preparations from 2 different suppliers, although exhibiting comparable anticoagulant activities, differed 10-fold in their antimetastatic capability; (ii) certain sulphated polysaccharides consistently gave a 30% difference in the number of metastatic lesions, yet exhibited identical anticoagulant activity; and (iii) the entrapment of 13762 MAT cells in the lung was not impaired by heparin or fucoidan. It was more probable that the sulphated polysaccharides were interfering with the passaging of tumour cells across the capillary wall as heparin significantly inhibited metastasis when injected up to 3 hr after lodgement, and heparin and fucoidan caused a gradual loss of tumour cells from the lung which only became apparent greater than 1 hr following cell lodgement. The data did not eliminate the possibility that tumour cell adhesion to the endothelium occurred via sulphated polysaccharide recognition. A negative correlation existed between the sulphated polysaccharides that bound to the surface of the tumour cells and those that inhibited metastasis.

Although the notion of an early origin and diversification of life on Earth during the Archaean eon has received increasing support in geochemical, sedimentological and palaeontological evidence, ambiguities and controversies persist regarding the biogenicity and syngeneity of the record older than Late Archaean. Non-biological processes are known to produce morphologies similar to some microfossils, and hydrothermal fluids have the potential to produce abiotic organic compounds with depleted carbon isotope values, making it difficult to establish unambiguous traces of life. Here we report the discovery of a population of large (up to about 300 mum in diameter) carbonaceous spheroidal microstructures in Mesoarchaean shales and siltstones of the Moodies Group, South Africa, the Earth's oldest siliciclastic alluvial to tidal-estuarine deposits. These microstructures are interpreted as organic-walled microfossils on the basis of petrographic and geochemical evidence for their endogenicity and syngeneity, their carbonaceous composition, cellular morphology and ultrastructure, occurrence in populations, taphonomic features of soft wall deformation, and the geological context plausible for life, as well as a lack of abiotic explanation falsifying a biological origin. These are the oldest and largest Archaean organic-walled spheroidal microfossils reported so far. Our observations suggest that relatively large microorganisms cohabited with earlier reported benthic microbial mats in the photic zone of marginal marine siliciclastic environments 3.2 billion years ago.

Leptospirosis is a common and underdiagnosed zoonosis. Two rapid assays for serological diagnosis of acute leptospirosis in diagnostic laboratories, the immunoglobulin M (IgM)-dipstick assay and the indirect hemagglutination assay (IHA), were evaluated and compared with standard assays. Sera were examined from 104 patients admitted to a hospital for investigation in a leptospirosis diagnostic protocol. Specimens for serology were taken on days 1 and 4 of the patients' hospital stay. Antibodies were detected using an IgM-enzyme-linked immunosorbent assay (ELISA), microscopic agglutination test (MAT), an IgM-dipstick assay, and an IHA. Fifty-one patients were found to have leptospirosis. The sensitivity of the IgM-dipstick assay was 98%, its specificity was 90.6%, its positive predictive value was 90.9%, and its negative predictive value was 98%. The sensitivity of the IHA was 92.2%, its specificity was 94.4%, its positive predictive value was 95.9%, and its negative predictive value was 92.7%. The standard IgM-ELISA and MAT, were positive in the first samples tested from 67 and 55% of the cases, respectively, and the rapid IgM-dipstick assay and IHA were positive in 71 and 49%, respectively, in the first sample tested. Both rapid assays are highly sensitive and specific. Neither requires specialized equipment, and both are suitable for use in diagnostic laboratories.

Previous studies investigating microbial diversity in the Octopus Spring cyanobacterial mat community (Yellowstone National Park) have shown a discrepancy between bacterial populations observed by molecular retrieval and cultivation techniques. To investigate how selective enrichment culture techniques affect species composition, we used denaturing gradient gel electrophoresis (DGGE) separation of PCR-amplified 16S rRNA gene fragments to monitor the populations contained within enrichment cultures of aerobic chemoorganotrophic bacteria from the ca. 50 degrees C region of the mat community. By varying the degree of dilution of the inoculum, medium composition, and enrichment conditions and duration and by analyzing the cultures by DGGE, we detected 14 unique 16S rRNA sequence types. These corresponded to alpha-, beta-, gamma-, and delta-proteobacteria, Thermus relatives, and gram-positive bacteria with high G + C ratio and, at the highest inoculum dilutions, Chloroflexus aurantiacus relatives, which were estimated to still be approximately 300 times less abundant than cells of the mat primary producer, Synechococcus spp. Only three of these populations were previously cultivated on solidified medium after similar enrichment. Only two of these population have 16S rRNA sequences which were previously cloned directly from the mat. These results reveal a diversity of bacterial populations in enrichment culture which were not detected by either molecular retrieval or strain purification techniques.

Perception of external stimuli and generation of an appropriate response are crucial for host colonization by pathogens. In pathogenic fungi, mitogen activated protein kinase (MAPK) pathways regulate dimorphism, biofilm/mat formation, and virulence. Signaling mucins, characterized by a heavily glycosylated extracellular domain, a transmembrane domain, and a small cytoplasmic domain, are known to regulate various signaling pathways. In Candida albicans, the mucin Msb2 regulates the Cek1 MAPK pathway. We show here that Msb2 is localized to the yeast cell wall and is further enriched on hyphal surfaces. A msb2Δ/Δ strain formed normal hyphae but had biofilm defects. Cek1 (but not Mkc1) phosphorylation was absent in the msb2Δ/Δ mutant. The extracellular domain of Msb2 was shed in cells exposed to elevated temperature and carbon source limitation, concomitant with germination and Cek1 phosphorylation. Msb2 shedding occurred differentially in cells grown planktonically or on solid surfaces in the presence of cell wall and osmotic stressors. We further show that Msb2 shedding and Cek1 phosphorylation were inhibited by addition of Pepstatin A (PA), a selective inhibitor of aspartic proteases (Saps). Analysis of combinations of Sap protease mutants identified a sap8Δ/Δ mutant with reduced MAPK signaling along with defects in biofilm formation, thereby suggesting that Sap8 potentially serves as a major regulator of Msb2 processing. We further show that loss of either Msb2 (msb2Δ/Δ) or Sap8 (sap8Δ/Δ) resulted in higher C. albicans surface β-glucan exposure and msb2Δ/Δ showed attenuated virulence in a murine model of oral candidiasis. Thus, Sap-mediated proteolytic cleavage of Msb2 is required for activation of the Cek1 MAPK pathway in response to environmental cues including those that induce germination. Inhibition of Msb2 processing at the level of Saps may provide a means of attenuating MAPK signaling and reducing C. albicans virulence.

Black band disease (BBD) consists of a mat-forming microbial consortium that migrates across coral colonies causing rapid tissue loss. Although BBD-associated microbial communities have been well characterized, little is known regarding how these complex bacterial consortia develop. This study analyzed successional changes in microbial communities leading to the development of BBD. Long-term monitoring of tagged corals throughout outbreaks of BBD in the central Great Barrier Reef documented cyanobacterium-infected lesions, herein termed cyanobacterial patch(es) (CP), which were macroscopically distinct from BBD and preceded the onset of BBD in 19% of the cases. Dominant cyanobacteria within CP lesions were morphologically distinct from ones dominating BBD lesions. Clone libraries and terminal restriction fragment length polymorphism analysis confirmed shifts within cyanobacterial assemblages, from Blennothrix sp.-affiliated sequences dominating CP lesions, to Oscillatoria sp.-affiliated sequences, similar to those retrieved from other BBD samples worldwide, dominating BBD lesions. Bacterial 16S ribosomal RNA clone libraries also showed shifts in bacterial ribotypes during transitions from CP to BBD, with Alphaproteobacteria-affiliated sequences dominant in CP libraries, whereas gammaproteobacterial and cyanobacterial ribotypes were more abundant in BBD clone libraries. Sequences affiliated with organisms identified in sulfur cycling were commonly retrieved from lesions showing characteristic field signs of BBD. As high sulfide concentrations have been implicated in BBD-mediated coral tissue degradation, proliferation of a microbial community actively involved in sulfur cycling potentially contributes to the higher progression rates found for BBD compared with CP lesions. Results show how microbial colonization of indistinct lesions may facilitate a common coral disease with proven ecological effects on coral populations.

Mating in basidiomycetous fungi is often controlled by two unlinked, multiallelic loci encoding homeodomain transcription factors or pheromones/pheromone receptors. In contrast to this tetrapolar organization, Cryptococcus neoformans/Cryptococcus gattii have a bipolar mating system, and a single biallelic locus governs sexual reproduction. The C. neoformans MAT locus is unusually large (>100 kb), contains >20 genes, and enhances virulence. Previous comparative genomic studies provided insights into how this unusual MAT locus might have evolved involving gene acquisitions into two unlinked loci and fusion into one contiguous locus, converting an ancestral tetrapolar system to a bipolar one. Here we tested this model by studying Cryptococcus heveanensis, a sister species to the pathogenic Cryptococcus species complex. An extant sexual cycle was discovered; co-incubating fertile isolates results in the teleomorph (Kwoniella heveanensis) with dikaryotic hyphae, clamp connections, septate basidia, and basidiospores. To characterize the C. heveanensis MAT locus, a fosmid library was screened with C. neoformans/C. gattii MAT genes. Positive fosmids were sequenced and assembled to generate two large probably unlinked MAT gene clusters: one corresponding to the homeodomain locus and the other to the pheromone/receptor locus. Strikingly, two divergent homeodomain genes (SXI1, SXI2) are present, similar to the bE/bW Ustilago maydis paradigm, suggesting one or the other homeodomain gene was recently lost in C. neoformans/C. gattii. Sequencing MAT genes from other C. heveanensis isolates revealed a multiallelic homeodomain locus and at least a biallelic pheromone/receptor locus, similar to known tetrapolar species. Taken together, these studies reveal an extant C. heveanensis sexual cycle, define the structure of its MAT locus consistent with tetrapolar mating, and support the proposed evolutionary model for the bipolar Cryptococcus MAT locus revealing transitions in sexuality concomitant with emergence of a pathogenic clade. These studies provide insight into convergent processes that independently punctuated evolution of sex-determining loci and sex chromosomes in fungi, plants, and animals.

BACKGROUND

The strongly metastatic MAT-LyLu and the weakly metastatic AT-2 rat prostatic cancer cell lines have been shown to express voltage-gated ion channels differentially. In the present study, the possible contribution of voltage-gated ion channel activity to the proliferation of these cell lines was investigated, in a comparative approach.

METHODS

Several voltage-gated ion channel modulators were tested for their effects on proliferation over 54 hr, using an in vitro assay. The modes of action of the chemicals were monitored by electrophysiological (patch-clamp) recording.

RESULTS

The voltage-gated K(+) channel blockers 4-aminopyridine (4-AP; 2 mM), margatoxin (5 nM), charybdotoxin (4.5 nM), and verapamil (50 microM) inhibited the K(+) channels of both cell lines by between 38-65% and reduced the proliferation of the AT-2 cell line, in a dose-dependent manner, by 8-51%. However, only 4-AP reduced proliferation of the MAT-LyLu cell line. Tetrodotoxin (6 microM) blocked completely the voltage-gated Na(+) channel expressed selectively in the MAT-LyLu cell line, but had no effect on the proliferation of either cell line. On the other hand, the presumed Na(+) channel "opener" veratridine (10-50 microM) reduced significantly, in a dose-dependent manner, the proliferation of both cell lines by up to approximately 30%.

CONCLUSIONS

We conclude that the mechanism(s) controlling the proliferation of the weakly metastatic AT-2 cells involves voltage-gated K(+) channels. In contrast, the proliferation of strongly metastatic MAT-LyLu cells is much less dependent upon voltage-gated K(+) channel activity.

Fifty strains representing 38 species of the genus Legionella were examined for biofilm formation on glass, polystyrene, and polypropylene surfaces in static cultures at 25 degrees C, 37 degrees C, and 42 degrees C. Strains of Legionella pneumophila, the most common causative agent of Legionnaires' disease, were found to have the highest ability to form biofilms among the test strains. The quantity, rate of formation, and adherence stability of L. pneumophila biofilms showed considerable dependence on both temperature and surface material. Glass and polystyrene surfaces gave between two- to sevenfold-higher yields of biofilms at 37 degrees C or 42 degrees C than at 25 degrees C; conversely, polypropylene surface had between 2 to 16 times higher yields at 25 degrees C than at 37 degrees C or 42 degrees C. On glass surfaces, the biofilms were formed faster but attached less stably at 37 degrees C or 42 degrees C than at 25 degrees C. Both scanning electron microscopy and confocal laser scanning microscopy revealed that biofilms formed at 37 degrees C or 42 degrees C were mycelial mat like and were composed of filamentous cells, while at 25 degrees C, cells were rod shaped. Planktonic cells outside of biofilms or in shaken liquid cultures were rod shaped. Notably, the filamentous cells were found to be multinucleate and lacking septa, but a recA null mutant of L. pneumophila was unaffected in its temperature-regulated filamentation within biofilms. Our data also showed that filamentous cells were able to rapidly give rise to a large number of short rods in a fresh liquid culture at 37 degrees C. The possibility of this biofilm to represent a novel strategy by L. pneumophila to compete for proliferation among the environmental microbiota is discussed.

We have isolated a hybrid plasmid, pDB(mei2)2, containing a 7.4-kilobases (kb) DNA fragment from a Schizosaccharomyces pombe genomic library which is able to complement the mei2 mutation of S. pombe. Integration of the cloned DNA sequence at the mei2 site on chromosome I demonstrated that it contained the mei2 gene. This gene was localized on a 4.7-kb HindIII-PvuII fragment in the subclone pFMV402. Transcriptional regulation was studied by Northern blot analysis in which polyadenylated RNA was prepared from a heterozygous (h+N/h-S) diploid strain cultured either in nitrogen-rich growth medium or in nitrogen-free sporulation medium. The size of the major mei2 mRNA, which always gave a broad band, was estimated to be 4.2 +/- 0.2 kb, and a few minor bands (e.g., 3.2 and 1.8 kb) appeared as well. These transcripts appeared more abundantly in sporulating cells than in growing cells. Neither the mating type genes (mat) nor the mei3 gene was essential for transcription of the mei2 gene, since ample mei2 mRNA was detected in sporulation-deficient cells transferred to sporulation medium, such as h+N/h+N and h-S/h-S homozygotes, as well as mei1 and mei3 mutants.

We have excised a 28-base-pair DNA fragment from the MAT alpha intergenic region and tested its ability to direct diploid-specific transcriptional repression. This fragment (1643-1671, 5'-GCTTCCCAATGTAGAAAAGTACA-TCATA-3') lies within a region required for the normal diploid-specific repression of the MAT alpha transcripts. First, the fragment was inserted into a 53-base-pair MAT alpha deletion that expresses alpha 1 and alpha 2 constitutively. Insertion of the fragment restores proper diploid regulation to the MAT alpha transcripts: alpha 1 mRNA is strongly repressed and alpha 2 mRNA is reduced by a factor of approximately equal to 10 from its haploid level. The fragment works equally well in either orientation, and two copies of the fragment do not lead to stronger repression than a single copy. We also inserted the fragment at three sites upstream of the CYC1-lacZ fusion gene. Insertions placing the regulatory fragment between the CYC1 upstream activator sequence (UAS) and the coding region make beta-galactosidase efficiently in alpha haploids but produce 1/40th the enzyme in a/alpha diploids. This diploid-specific repression requires functional MATa-1 gene product. Insertion of the MAT fragment on the opposite side of the UAS (37 base pairs upstream of the UAS) also caused diploid repression of the fusion gene, but only by a factor of 7. When the regulatory fragment is inserted at a large distance on the far side of the UAS (375 base pairs), it has little if any effect on beta-galactosidase expression. We postulate that this sequence is the operator recognized by the diploid-specific repressor.

NSP1 is related to a group of nuclear pore proteins termed 'nucleoporins'. We observe that in thermosensitive nsp1 mutants lacZ fusion proteins which contain the nuclear targeting sequence of Mat alpha 2 or Pho2 are located inside the nucleus at the permissive temperature (23 degrees C), but are mislocalized in the cytoplasm at the restrictive temperature (37 degrees C). No evidence was obtained that the large lacZ reporter protein leaks out from the nucleus. Another nuclear passenger protein consisting of the NLS of ribosomal protein L25 and cytosolic dihydrofolate reductase (DHFR) is also accumulating in the cytoplasm after shifting ts nsp1 cells to 37 degrees C. In the latter case, this could be attributed to an increased leakage of the reporter protein from the nucleus into the cytoplasm. These data suggest that NSP1 mutation affects nuclear transport and nuclear retention, but the effects depend on the used NLS and the reporter protein.

The repression of transcription of the silent mating-type locus HMRa in the yeast Saccharomyces cerevisiae requires the four SIR proteins, histone H4 and a flanking site designated HMR-E. The SUM1-1 mutation alleviated the need for many of these components in transcriptional repression. In the absence of each of the SIR proteins, SUM1-1 restored repression in MAT alpha strains; thus, SUM1-1 appeared to bypass the need for the SIR genes in repression of HMRa. Repression was not specific to the genes normally present at HMR, since the TRP1 gene placed at HMR was repressed by SUM1-1 in a sir3 strain. Therefore, like the mechanisms of silencing normally used at HMR, silencing by SUM1-1 was gene-nonspecific. SUM1-1 suppressed point mutations in histone H4, but failed to suppress strongly a deletion mutation in histone H4. Similarly, SUM1-1 suppressed mutations in the three known elements of HMR-E, but was unable to suppress a deletion of HMR-E. These epistasis analyses implied that the functions required for repression at HMR can be ordered, with the SIR genes and silencer elements acting upstream of SUM1-1. SUM1-1 itself may function at the level of chromatin in the assembly of inactive DNA at the silent mating-type loci.

Mutants requiring S-adenosyl methionine (SAM) for growth have been selected in Saccharomyces cerevisiae. Two classes of mutants have been found. One class corresponds to the simultaneous occurrence of mutations at two unlinked loci SAM1 and SAM2 and presents a strict SAM requirement for growth on any medium. The second class corresponds to special single mutations in the gene SAM2 which lead to a residual growth on minimal medium but to normal growth on SAM supplemented medium or on a complex medium like YPGA not containing any SAM. These genetic data can be taken as an indication that Saccharomyces cerevisiae possesses two isoenzymatic methionine adenosyl transferases (MAT). In addition, SAM1 and SAM2 loci have been identified respectively with the ETH-10 and ETH2 loci previously described. Biochemical evidences corroborate the genetic results. Two MAT activities can be dissociated in a wild type extract (MATI and MATII) by DEAE cellulose chromatography. Mutations at the SAM1 locus lead to the absence or to the modification of MATII whereas mutations at the SAM2 locus lead to the absence or to the modification of MATI. Moreover, some of our results seem to show that MATI and MATII are associated in vivo.

"Haemophilus somnus" has been identified in the etiology of bovine abortion on the basis of the isolation of the organism from aborted fetal and placental tissues. To investigate the role of hematogenous dissemination of "H. somnus" in the pathogenesis of abortion and to monitor the humoral immune response to infection, 19 pregnant cows (gestation ages, 1.4 to 7 months) were challenged intravenously (11 cows) or intrabronchially (8 cows). Five cows challenged intravenously aborted, and one cow challenged intrabronchially resorbed her fetus. "H. somnus" was isolated in large numbers from aborted tissues, and placental lesions were similar to those reported in a field case of "H. somnus" abortion. Antibody titers in serum were measured by the microagglutination test (MAT) and by enzyme-linked immunosorbent assay (ELISA). A response to challenge was measured by MAT; it was also measured by ELISA within the immunoglobulin G1 (IgG1), IgG2, and IgM isotypes. On comparison of pre- and postchallenge antibody titers, the greatest and most persistent response was detected within the IgG2 isotype. Prechallenge antibody titers (measured by MAT and by IgG2 ELISA) were lower in animals that aborted than in normal calving animals, indicating that IgG2 antibody may have a role in limiting hematogenous dissemination of "H. somnus."

We evaluated the larvicidal activity of the granular formulation of Bacillus thuringiensis israelensis (Bti) serotype H-14 (Vectobac G, 200 ITU/mg) and Bacillus sphaericus (Bsph) serotype H5a5b (Vectolex CG, 670 Bs ITU/mg) against Anopheles arabiensis and other mosquitoes in breeding habitats in 3 sites, Gash-Barka, Anseba, and Debub zones, in Eritrea. The primary objective was to determine the optimal application rate and duration of effect for Bti and Bsph in representative larval habitats as compared with the organophosphate temephos. The biolarvicides were tested at 100% (high) and 50% (low) of the maximum recommended application rate. Temephos was applied at a rate of 100 ml/ha. At least 4 replicate experiments with Vectobac G (5.6 and 11.2 kg/ha), Vectolex CG (11.2 and 22.4 kg/ha) were conducted in each study site. All 3 larvicides caused significant mortality of the main malaria vector species, An. arabiensis, and other mosquito species (Anopheles cinereus, Anopheles pretoriensis, Culex quinquefasciatus). The larvicidal activity for Bti and Bsph was variable depending upon breeding habitat, mosquito species, and general ecology of the area. Both biopesticides had a similar duration of activity (2-3 wk) and were generally as effective as temephos for these time periods. In some cases, the high and low application rates for Bti and Bsph produced equivalent control over 2-3 wk. The 2 Bacillus biopesticides were less effective in habitats with high algal content and in fast flowing streams primarily because of the inability to penetrate algal mats and dilution effect, respectively. The results show that application of the 2 biolarvicides bimonthly to streambed pools, rain pools, and similar habitats would maintain control of the anopheline mosquito population.

The ipt-type MAT vector uses the ipt gene for regeneration of marker-free transgenic plants. However, it was pointed out that this system was not suitable for most economically important crops that regenerated through auxin-dependent embryogenesis. We report a single-step transformation system of rice using MAT vector. When we transformed scutellum tissues of 5 days pre-cultured rice seeds, marker-free transgenic rice plants directly regenerated from 25.5% infected scutellum tissues without forming ipt-intermediates within 4 weeks after an infection. Excision of the ipt gene caused the regeneration of marker-free transgenic rice plants through embryogenic tissues. Therefore, this system needs no selective agent and no sexual crossing for identification of transgenic plants not containing a selectable marker gene. This system is highly effective for generation of marker-free transgenic plants in economically important crops.

Matriptase/MT-SP1, a type II membrane serine protease widely expressed in normal epithelial cells and human carcinoma cells, is thought to be involved in cancer progression. To clarify this possibility, we overexpressed exogenous matriptase in the human stomach cancer cell line AZ521. In vitro, the matriptase transfectant (Mat-AZ521) and the control transfectant (Mock-AZ521) showed a similar growth rate, although the saturation cell density was significantly higher with the Mat-AZ521. When implanted into nude mice subcutaneously or intraperitoneally, Mat-AZ521 cells grew faster and produced much larger solid tumors than Mock-AZ521 cells. The overexpression of matriptase in AZ521 cells shortened the survival time of tumor-bearing mice. Histological analysis showed that both the number and the size of blood vessels in tumor tissues were significantly higher in the Mat-AZ521 tumors than the Mock-AZ521 ones. Moreover, it was found that purified matriptase activated one of the important matrix metalloproteinases, stromelysin (MMP-3). These results suggest the possibility that the matriptase-dependent activation of MMP-3, as well as the direct activity of matriptase, promotes tumor growth and angiogenesis by enhancing extracellular matrix degradation in tumor cell microenvironments.

OBJECTIVE

The purposes of this study were to evaluate the effect on tibiofemoral contact mechanics of repair of the posterior root of the medial meniscus and the effect of meniscal allograft transplantation (MAT) with medial collateral ligament (MCL) release at different flexion angles.

METHODS

Ten fresh-frozen human cadaveric knees (five pairs) were used. A digital pressure sensor was inserted by capsulotomy, and experiments were performed serially under the following six conditions, that is, with an intact medial meniscus (normal controls), with a root tear, after root repair, after total meniscectomy, after MAT, and after MAT plus MCL release. During each experiment, knees were positioned at 0°, 30°, 60°, and 90° of flexion, and peak pressure (kPa) and contact area (cm2) were measured.

RESULTS

At 0° of flexion, contact pressure did not differ among the six experimental settings. However, at 30° and 60° of flexion, contact pressure differed significantly between root tear and root repair specimens (p = 0.04 and 0.03, respectively), and between total meniscectomy and MAT specimens (p = 0.02 and 0.03, respectively). On the other hand, mean contact pressures were different between normal (476.7 ± 473.1 and 573.3 ± 479.1 kPa) and root repair (575.7 ± 357.8 and 598.6 ± 415.8), and between normal and MAT (635.7 ± 437.4 and 674.3 ± 533.2). At 0°, 30°, 60°, and 90° of flexion, contact areas differed significantly between normal and total meniscectomy specimens (p = 0.02, 0.01, 0.02, and 0.02, respectively), and between MAT and total meniscectomy specimens (p = 0.03, 0.02, 0.02, and 0.03, respectively). Contact areas differed significantly between root tear and root repair specimens at 60° of flexion (p = 0.04), and between normal control and root repair specimens at 60° and 90° of flexion (p = 0.03 and 0.04, respectively). The effects of MAT plus MCL release on contact mechanics were not different from the effects of MAT alone (n.s.).

CONCLUSIONS

Both meniscal root repair and transplantation of meniscus improved contact mechanics, but it did not appear that repair of the meniscal root or transplantation of meniscus restores the biomechanical function back to normal level. The MAT plus MCL release was similar to those after MAT alone. Therefore, it is better to preserve meniscus and MCL release could be done during the MAT.

A novel moderately thermophilic, facultatively anaerobic chemoorganotrophic bacterium strain P3M-2(T) was isolated from a microbial mat developing on the wooden surface of a chute under the flow of hot water (46°C) coming out of a 2775-m-deep oil exploration well (Tomsk region, Russia). Strain P3M-2(T) is a moderate thermophile and facultative anaerobe growing on mono-, di- or polysaccharides by aerobic respiration, fermentation or by reducing diverse electron acceptors [nitrite, Fe(III), As(V)]. Its closest cultivated relative (90.8% rRNA gene sequence identity) is Ignavibacterium album, the only chemoorganotrophic member of the phylum Chlorobi. New genus and species Melioribacter roseus are proposed for isolate P3M-2(T) . Together with I. album, the new organism represents the class Ignavibacteria assigned to the phylum Chlorobi. The revealed group includes a variety of uncultured environmental clones, the 16S rRNA gene sequences of some of which have been previously attributed to the candidate division ZB1. Phylogenetic analysis of M. roseus and I. album based on their 23S rRNA and RecA sequences confirmed that these two organisms could represent an even deeper, phylum-level lineage. Hence, we propose a new phylum Ignavibacteriae within the Bacteroidetes-Chlorobi group with a sole class Ignavibacteria, two families Ignavibacteriaceae and Melioribacteraceae and two species I. album and M. roseus. This proposal correlates with chemotaxonomic data and phenotypic differences of both organisms from other cultured representatives of Chlorobi. The most essential differences, supported by the analyses of complete genomes of both organisms, are motility, facultatively anaerobic and obligately organotrophic mode of life, the absence of chlorosomes and the apparent inability to grow phototrophically.