OBJECTIVE

To assess the effect of a neuromuscular training program on the incidence of anterior cruciate ligament injuries in female team handball players.

METHODS

Prospective intervention study.

METHODS

Female team handball: Division I-III in Norway.

METHODS

Players from the three top divisions: control season (1998-1999), 60 teams (942 players); first intervention season (1999-2000), 58 teams (855 players); second intervention season (2000-2001), 52 teams (850 players).

METHODS

A five-phase program (duration, 15 min) with three different balance exercises focusing on neuromuscular control and planting/landing skills was developed and introduced to the players in the autumn of 1999 and revised before the start of the season in 2000. The teams were instructed in the program and supplied with an instructional video, poster, six balance mats, and six wobble boards. Additionally, a physical therapist was attached to each team to follow up with the intervention program during the second intervention period.

METHODS

The number of anterior cruciate ligament injuries during the three seasons and compliance with the program.

RESULTS

There were 29 anterior cruciate ligament injuries during the control season, 23 injuries during the first intervention season (OR, 0.87; CI, 0.50-1.52; p = 0.62), and 17 injuries during the second intervention season (OR, 0.64; CI, 0.35-1.18; p = 0.15). In the elite division, there were 13 injuries during the control season, six injuries during the first intervention season (OR, 0.51; CI, 0.19-1.35; p = 0.17), and five injuries in the second intervention season (OR, 0.37; CI, 0.13-1.05; p = 0.06). For the entire cohort, there was no difference in injury rates during the second intervention season between those who complied and those who did not comply (OR, 0.52; CI, 0.15-1.82; p = 0.31). In the elite division, the risk of injury was reduced among those who completed the anterior cruciate ligament injury prevention program (OR, 0.06; CI, 0.01-0.54; p = 0.01) compared with those who did not.

CONCLUSIONS

This study shows that it is possible to prevent anterior cruciate ligament injuries with specific neuromuscular training.

Biofilms are formed by the aggregation of microorganisms into multicellular structures that adhere to surfaces. Here we show that bakers' yeast Saccharomyces cerevisiae can initiate biofilm formation. When grown in low-glucose medium, the yeast cells adhered avidly to a number of plastic surfaces. On semi-solid (0.3% agar) medium they formed "mats": complex multicellular structures composed of yeast-form cells. Both attachment to plastic and mat formation require Flo11p, a member of a large family of fungal cell surface glycoproteins involved in adherence. The ability to study biofilm formation in a tractable genetic system may facilitate the identification of new targets for antifungal therapy.

In wild-type diploid cells of Saccharomyces cerevisiae, an HO endonuclease-induced double-strand break (DSB) at the MAT locus can be efficiently repaired by gene conversion using the homologous chromosome sequences. Repair of the broken chromosome was nearly eliminated in rad52delta diploids; 99% lost the broken chromosome. However, in rad51delta diploids, the broken chromosomes were repaired approximately 35% of the time. None of these repair events were simple gene conversions or gene conversions with an associated crossover, instead, they created diploids homozygous for the MAT locus and all markers in the 100-kb region distal to the site of the DSB. In rad51delta diploids, the broken chromosome can apparently be inherited for several generations, as many of these repair events are found as sectored colonies, with one part being repaired and the other part being lost the broken chromosome. Similar events occur in about 2% of wild-type cells. We propose that a broken chromosome end can invade a homologous template in the absence of RAD51 and initiate DNA replication that may extend to the telomere, 100 or more kb away. Such break-induced replication appears to be similar to recombination-initiated replication in bacteria.

Carbon nanotubes (CNT) are an important new class of technological materials that have numerous novel and useful properties. The forecast increase in manufacture makes it likely that increasing human exposure will occur, and as a result, CNT are beginning to come under toxicological scrutiny. This review seeks to set out the toxicological paradigms applicable to the toxicity of inhaled CNT, building on the toxicological database on nanoparticles (NP) and fibers. Relevant workplace regulation regarding exposure is also considered in the light of our knowledge of CNT. CNT could have features of both NP and conventional fibers, and so the current paradigm for fiber toxicology, which is based on mineral fibers and synthetic vitreous fibers, is discussed. The NP toxicology paradigm is also discussed in relation to CNT. The available peer-reviewed literature suggests that CNT may have unusual toxicity properties. In particular, CNT seem to have a special ability to stimulate mesenchymal cell growth and to cause granuloma formation and fibrogenesis. In several studies, CNT have more adverse effects than the same mass of NP carbon and quartz, the latter a commonly used benchmark of particle toxicity. There is, however, no definitive inhalation study available that would avoid the potential for artifactual effects due to large mats and aggregates forming during instillation exposure procedures. Studies also show that CNT may exhibit some of their effects through oxidative stress and inflammation. CNT represent a group of particles that are growing in production and use, and therefore, research into their toxicology and safe use is warranted.

A double-stranded DNA cut has been observed in the mating type (MAT) locus of the yeast Saccharomyces cerevisiae in cultures undergoing homothallic cassette switching. Cutting is observed in exponentially growing cells of genotype HO HML alpha MAT alpha HMR alpha or HO HMLa MATa HMRa, which switch continuously, but not in a/alpha HO/HO diploid strains, in which homothallic switching is known to be shut off. Stationary phase cultures do not exhibit the cut. Although this site-specific cut occurs in a sequence (Z1) common to the silent HML and HMR cassettes and to MAT, only the Z1 sequence at the MAT locus is cut. The cut at MAT occurs in the absence of the HML and HMR donor cassettes, suggesting that cutting initiates the switching process. An assay for switching on hybrid plasmids containing mata- cassettes has been devised, and deletion mapping has shown that the cut site is required for efficient switching. Thus a double-stranded cut at the MAT locus appears to initiate cassette transposition-substitution and defines MAT as the recipient in this process.

The Hippo pathway defined originally in Drosophila melanogaster is conserved in mammals. The fly core components Hippo, Sav, Wts, and Mats are conserved in mammals as Mst1/2, WW45, LATS1/2, and Mob1. The pathway impinges on transcriptional coactivator Yorkie in fly and YAP in mammals to coordinate cell proliferation and apoptosis. Several recent publications establish that the pathway is one major conserved mechanism governing cell contact inhibition, organ size control, and cancer development. This advance opens new vistas in exploring fundamental mechanisms in cell and developmental biology and offers potential targets to interfere with cancer development.

Attachment of ubiquitin to proteins is catalyzed by a family of ubiquitin-conjugating (UBC) enzymes. Although these enzymes are essential for many cellular processes; their molecular functions remain unclear because no physiological target has been identified for any of them. Here we show that four UBC proteins (UBC4, UBC5, UBC6, and UBC7) target the yeast MAT alpha 2 transcriptional regulator for intracellular degradation by two distinct ubiquitination pathways. UBC6 and UBC7 define one of the pathways and can physically associate. The UBC6/UBC7-containing complex targets the Deg1 degradation signal of alpha 2, a conclusion underscored by the finding that UBC6 is encoded by DOA2, a gene previously implicated in Deg1-mediated degradation. These data reveal an unexpected overlap in substrate specificity among diverse UBC enzymes and suggest a combinatorial mechanism of substrate selection in which UBC enzymes partition into multiple ubiquitination complexes.

We developed a procedure to measure mRNA decay rates in the yeast Saccharomyces cerevisiae and applied it to the determination of half-lives for 20 mRNAs encoded by well-characterized genes. The procedure utilizes Northern (RNA) or dot blotting to quantitate the levels of individual mRNAs after thermal inactivation of RNA polymerase II in an rpb1-1 temperature-sensitive mutant. We compared the results of this procedure with results obtained by two other procedures (approach to steady-state labeling and inhibition of transcription with Thiolutin) and also evaluated whether heat shock alter mRNA decay rates. We found that there are no significant differences in the mRNA decay rates measured in heat-shocked and non-heat-shocked cells and that, for most mRNAs, different procedures yield comparable relative decay rates. Of the 20 mRNAs studied, 11, including those encoded by HIS3, STE2, STE3, and MAT alpha 1, were unstable (t1/2 less than 7 min) and 4, including those encoded by ACT1 and PGK1, were stable (t1/2 greater than 25 min). We have begun to assess the basis and significance of such differences in the decay rates of these two classes of mRNA. Our results indicate that (i) stable and unstable mRNAs do not differ significantly in their poly(A) metabolism; (ii) deadenylation does not destabilize stable mRNAs; (iii) there is no correlation between mRNA decay rate and mRNA size; (iv) the degradation of both stable and unstable mRNAs depends on concomitant translational elongation; and (v) the percentage of rare codons present in most unstable mRNAs is significantly higher than in stable mRNAs.

The MAT alpha 2 homeodomain regulates the expression of cell type-specific genes in yeast. We have determined the 2.7 A resolution crystal structure of the alpha 2 homeodomain bound to a biologically relevant DNA sequence. The DNA in this complex is contacted primarily by the third of three alpha-helices, with additional contacts coming from an N-terminal arm. Comparison of the yeast alpha 2 and the Drosophila engrailed homeodomain-DNA complexes shows that the protein fold is highly conserved, despite a 3-residue insertion in alpha 2 and only 27% sequence identity between the two homeodomains. Moreover, the orientation of the recognition helix on the DNA is also conserved. This docking arrangement is maintained by side chain contacts with the DNA--primarily the sugar-phosphate backbone--that are identical in alpha 2 and engrailed. Since these residues are conserved among all homeodomains, we propose that the contacts with the DNA are also conserved and suggest a general model for homeodomain-DNA interactions.

Saccharomyces cerevisiae can change its mating type as often as every generation by a highly choreographed, site-specific recombination event that replaces one MAT allele with different DNA sequences encoding the opposite allele. The study of this process has yielded important insights into the control of cell lineage, the silencing of gene expression, and the formation of heterochromatin, as well as the molecular events of double-strand break-induced recombination. In addition, MAT switching provides a remarkable example of a small locus control region--the Recombination Enhancer--that controls recombination along an entire chromosome arm.

Entry into meiosis is a key developmental decision. We show here that meiotic entry in Saccharomyces cerevisiae is controlled by antisense-mediated regulation of IME4, a gene required for initiating meiosis. In MAT a/alpha diploids the antisense IME4 transcript is repressed by binding of the a1/alpha2 heterodimer at a conserved site located downstream of the IME4 coding sequence. MAT a/alpha diploids that produce IME4 antisense transcript have diminished sense transcription and fail to initiate meiosis. Haploids that produce the sense transcript have diminished antisense transcription and manifest several diploid phenotypes. Our data are consistent with transcription interference as a regulatory mechanism at the IME4 locus that determines cell fate.

In order to identify determinants governing nuclear protein localization, we constructed a set of hybrid genes by fusing the S. cerevisiae gene, MAT alpha 2, coding for a presumptive nuclear protein, and the E. coli gene, lacZ, coding for beta-galactosidase. The resultant hybrid proteins contain 3, 13, 25, 67, or all 210 amino acids of wild-type alpha 2 protein at the amino terminus and a constant, enzymatically active portion of beta-galactosidase at the carboxy terminus. Indirect immunofluorescence and subcellular fractionation studies with yeast cells containing the alpha 2-LacZ hybrid proteins indicate that the alpha 2 segment can direct localization of beta-galactosidase to the nucleus. A segment as small as 13 amino acids from alpha 2 is sufficient for this localization. Comparison of amino acid sequences of other nuclear proteins with this region of alpha 2 reveals a sequence that may be necessary for nuclear targeting. Production of some alpha 2-LacZ hybrid proteins causes cell death, perhaps as a result of improper or incomplete localization. These studies also indicate that the alpha 2 protein, argued on genetic grounds to be a negative regulator, acts in the yeast nucleus.

The mating-type region of fission yeast consists of three components, matmatmatmatmatmatmation that is transposed to matmating type. We have determined the nucleotide sequence of each component of mat. The P and M specific regions are 1104 and 1128 bp, respectively, and bounded by sequences common to each mating-type cassette (H1; 59 bp and H2; 135 bp). A third sequence is present at matmatmatmatmatmating and confer an h+ or h- mating type respectively. All four genes are required for meiotic competence in an h+/h- diploid. The transcription of each mat gene is strongly influenced by nutritional conditions and full induction was observed only in nitrogen-free medium. The predicted product of the Pi gene contains a region of homology with the homeobox sequence, suggesting that this gene encodes a DNA binding protein that directly regulates the expression of other genes.

The coding region of the mat K gene and two intergenic spacers, psb A-trn H and trn L(UAA)-trn F(GAA), of cpDNA were sequenced to study phylogenetic relationships of 32 Paeonia species. In the psb A-trn H intergenic spacer, short sequences bordered by long inverted repeats have undergone inversions that are often homoplasious mutations. Insertions/deletions found in the two intergenic spacers, mostly resulting from slipped-strand mispairing, provided relatively reliable phylogenetic information. The mat K coding region, evolving more rapidly than the trnL-trn F spacer and more slowly than the psb A-trn H spacer, produced the best resolved phylogenetic tree. The mat K phylogeny was compared with the phylogeny obtained from sequences of internal transcribed spacers (ITS) of nuclear ribosomal DNA. A refined hypothesis of species phylogeny of section Paeonia was proposed by considering the discordance between the nuclear and cpDNA phylogenies to be results of hybrid speciation followed by inheritance of cpDNA of one parent and fixation of ITS sequences of another parent. The Eurasian and western North American disjunct distribution of the genus may have resulted from interrruption of the continuous distribution of ancestral populations of extant peony species across the Bering land bridge during the Miocene. Pleistocene glaciation may have played an important role in triggering extensive reticulate evolution within section Paeonia and shifting distributional ranges of both parental and hybrid species.

We have cloned and sequenced a gene (MF alpha) coding for alpha-factor, a tridecapeptide mating factor secreted by yeast alpha cells. A plasmid carrying the MF alpha gene was identified by screening for production of alpha-factor by mat alpha 2 mutants, which fail to secrete alpha-factor because of simultaneous synthesis and degradation of the factor. The cloned segment codes for four mature alpha-factor within a putative precursor of 165 amino acids. The putative precursor begins as a signal sequence for secretion. The next segment, of approximately 60 amino acids, contains three potential glycosylation sites. The carboxy-terminal half of the precursor contains four tandem copies of mature alpha-factor, each preceded by spacer peptides of six or eight amino acids (variations of Lys-Arg-Glu-Ala-Asp-Ala-Glu-Ala), which are hypothesized to contain proteolytic processing signals.

Moments analysis has been applied to the calculation of mean (in vivo) dissolution time (MDT) and mean absorption time (MAT) from plasma level of drug versus time data. Methods for accurately estimating the MDT under varying conditions, limitations of the methods, and interpretation of the data are presented. The importance of accurate estimates of the terminal rate constant (lambda 2) and the drug concentration at the time of withdrawing the final plasma sample (Cz) is emphasized in connection with extrapolation to t = affinity. The appropriate use of a logarithmic trapezoidal equation for calculating the area under the moments curve (AUMC) is shown to increase the accuracy of estimating MDT.

Mammalian tumours displaying multidrug resistance overexpress a plasma membrane protein (P-glycoprotein), which is encoded by the MDR1 gene and apparently functions as an energy-dependent drug efflux pump. Tissue-specific expression of MDR1 and other members of the MDR gene family has been observed in normal cells, suggesting a role for P-glycoproteins in secretion. We have isolated a gene from the yeast Saccharomyces cerevisiae that encodes a protein very similar to mammalian P-glycoproteins. Deletion of this gene resulted in sterility of MATa, but not of MAT alpha cells. Subsequent analysis revealed that the yeast P-glycoprotein is the product of the STE6 gene, a locus previously shown to be required in MATa cells for production of a-factor pheromone. Our findings suggest that the STE6 protein functions to export the hydrophobic a-factor lipopeptide in a manner analogous to the efflux of hydrophobic cytotoxic drugs catalysed by the related mammalian P-glycoprotein. Thus, the evolutionarily conserved family of MDR-like genes, including the hlyB gene of Escherichia coli and the STE6 gene of S. cerevisiae, encodes components of secretory pathways distinct from the classical, signal sequence-dependent protein translocation system.

Interactions between polymorphisms at different quantitative trait loci (QTLs) are thought to contribute to the genetics of many traits, and can markedly affect the power of genetic studies to detect QTLs. Interacting loci have been identified in many organisms. However, the prevalence of interactions, and the nucleotide changes underlying them, are largely unknown. Here we search for naturally occurring genetic interactions in a large set of quantitative phenotypes--the levels of all transcripts in a cross between two strains of Saccharomyces cerevisiae. For each transcript, we searched for secondary loci interacting with primary QTLs detected by their individual effects. Such locus pairs were estimated to be involved in the inheritance of 57% of transcripts; statistically significant pairs were identified for 225 transcripts. Among these, 67% of secondary loci had individual effects too small to be significant in a genome-wide scan. Engineered polymorphisms in isogenic strains confirmed an interaction between the mating-type locus MAT and the pheromone response gene GPA1. Our results indicate that genetic interactions are widespread in the genetics of transcript levels, and that many QTLs will be missed by single-locus tests but can be detected by two-stage tests that allow for interactions.

For cultivation-independent detection of sulfate-reducing prokaryotes (SRPs) an oligonucleotide microarray consisting of 132 16S rRNA gene-targeted oligonucleotide probes (18-mers) having hierarchical and parallel (identical) specificity for the detection of all known lineages of sulfate-reducing prokaryotes (SRP-PhyloChip) was designed and subsequently evaluated with 41 suitable pure cultures of SRPs. The applicability of SRP-PhyloChip for diversity screening of SRPs in environmental and clinical samples was tested by using samples from periodontal tooth pockets and from the chemocline of a hypersaline cyanobacterial mat from Solar Lake (Sinai, Egypt). Consistent with previous studies, SRP-PhyloChip indicated the occurrence of Desulfomicrobium spp. in the tooth pockets and the presence of Desulfonema- and Desulfomonile-like SRPs (together with other SRPs) in the chemocline of the mat. The SRP-PhyloChip results were confirmed by several DNA microarray-independent techniques, including specific PCR amplification, cloning, and sequencing of SRP 16S rRNA genes and the genes encoding the dissimilatory (bi)sulfite reductase (dsrAB).

The pilus of the bacterium Neisseria gonorrhoeae is a fimbriate surface structure which promotes attachment of the bacterium to host epithelial cells. Gonococcal pilus phase variation is characterized by a rapid on/off switch in which piliated (P+) cells throw off non-piliated (P-) variants and vice versa. Two regions of the gonococcal chromosome (pilE1 and pilE2) act as pilin expression loci, reminiscent of the MAT locus in the yeast Saccharomyces cerevisiae, while several other chromosomal regions contain silent (non-expressing) pilin sequences. Biochemical and antigenic diversity is seen in pili from a wide variety of clinical isolates. Pilins (pilus subunits) are composed of conserved N-terminal and variable C-terminal regions; the conserved region of gonococcal pilin is also found in pilins produced by widely disparate bacteria. We show here that the gonococcal pilin undergoes antigenic variation in vitro and in vivo. The protein consists of constant, semi-variable and hypervariable regions. This antigenic variation probably involves gene conversion of mini-cassettes of pilin information.

The product of the yeast MAT alpha 2 gene, alpha 2 protein, negatively regulates expression of a class of yeast cell type specific genes, the a-specific genes. In this paper we show that alpha 2 is a sequence-specific DNA binding protein. It recognizes a 32 base pair DNA sequence (operator) located upstream of an a-specific gene, STE6. A strongly homologous sequence is present upstream of each of the other four known a-specific genes. A synthetic STE6 operator, when placed in a test promoter (CYC1), brings the promoter under negative control by alpha 2 in vivo. This operator brings about repression when it is placed between the CYC1 upstream activation sequence (UAS) and the transcription start and when it is placed upstream of the UAS, outside the promoter. Thus, the operator need not overlap with essential promoter sequences to permit repression by alpha 2.

The Roseobacter lineage is a phylogenetically coherent, physiologically heterogeneous group of alpha-Proteobacteria comprising up to 25% of marine microbial communities, especially in coastal and polar oceans, and it is the only lineage in which cultivated bacteria are closely related to environmental clones. Currently 41 subclusters are described, covering all major marine ecological niches (seawater, algal blooms, microbial mats, sediments, sea ice, marine invertebrates). Members of the Roseobacter lineage play an important role for the global carbon and sulfur cycle and the climate, since they have the trait of aerobic anoxygenic photosynthesis, oxidize the greenhouse gas carbon monoxide, and produce the climate-relevant gas dimethylsulfide through the degradation of algal osmolytes. Production of bioactive metabolites and quorum-sensing-regulated control of gene expression mediate their success in complex communities. Studies of representative isolates in culture, whole-genome sequencing, e.g., of Silicibacter pomeroyi, and the analysis of marine metagenome libraries have started to reveal the environmental biology of this important marine group.

Candida albicans, the most prevalent fungal pathogen in humans, is thought to lack a sexual cycle. A set of C. albicans genes has been identified that corresponds to the master sexual cycle regulators a1, alpha1, and alpha2 of the Saccharomyces cerevisiae mating-type (MAT) locus. The C. albicans genes are arranged in a way that suggests that these genes are part of a mating type-like locus that is similar to the mating-type loci of other fungi. In addition to the transcriptional regulators a1, alpha1, and alpha2, the C. albicans mating type-like locus contains several genes not seen in other fungal MAT loci, including those encoding proteins similar to poly(A) polymerases, oxysterol binding proteins, and phosphatidylinositol kinases.

Mating type in the yeast Saccharomyces cerevisiae is determined by the MAT (a or alpha) locus. HML and HMR, which usually contain copies of alpha and a mating type information, respectively, serve as donors in mating type interconversion and are under negative transcriptional control. Four trans-acting SIR (silent information regulator) loci are required for repression of transcription. A defect in any SIR gene results in expression of both HML and HMR. The four SIR genes were isolated from a genomic library by complementation of sir mutations in vivo. DNA blot analysis suggests that the four SIR genes share no sequence homology. RNA blots indicate that SIR2, SIR3, and SIR4 each encode one transcript and that SIR1 encodes two transcripts. Null mutations, made by replacement of the normal genomic allele with deletion-insertion mutations created in the cloned SIR genes, have a Sir- phenotype and are viable. Using the cloned genes, we showed that SIR3 at a high copy number is able to suppress mutations of SIR4. RNA blot analysis suggests that this suppression is not due to transcriptional regulation of SIR3 by SIR4; nor does any SIR4 gene transcriptionally regulate another SIR gene. Interestingly, a truncated SIR4 gene disrupts regulation of the silent mating type loci. We propose that interaction of at least the SIR3 and SIR4 gene products is involved in regulation of the silent mating type genes.