Facilitation of bone resorption activities in synovial lavage fluid patients with mandibular condyle fractures.

The aim of this study was to investigate the bone resorption effect of the mediators delivered in joint cavity of patients with mandibular condyle fractures by detecting osteoclast markers using cellular biochemistry methods, and by analysing bone resorption activities via inducing osteoclast differentiation of the infiltrated cells from arthrocentesis. Sixteen joints in 10 patients with mandibular condyle fractures were evaluated. The control group consisted of synovial fluid (SF) samples from seven joints of four volunteers who had no clinical signs or symptoms involving the temporomandibular joint (TMJ) or disc displacement. We collected SF cells from all patients during therapeutic arthrocentesis. The infiltrating cells from TMJ SF were cultured, differentiated into tartrate-resistant acid phosphatase (TRAP)-positive osteoclast-like cells and examined bone resorption activities. We also investigated factors related to osteoclast induction of SF, using ELISA procedures. Osteoclast-like cells were induced from the SF cells obtained from all patients with condylar fractures. These multinucleated giant cells were positive for TRAP and actin, and had the ability to absorb dentin slices. The levels of macrophage colony-stimulating factor (M-CSF), prostaglandin E2 (PGE2), soluble form of receptor activator of nuclear factor kappa-B ligand (sRANKL) and osteoprotegerin (OPG), in SF samples from the patients, were significantly higher than in the controls. These findings indicate that bone resorption activities in SF from patients with mandibular condyle fractures were upregulated and may participate in the pathogenesis and wound healing.

EFFECT OF SEASONAL CHANGES ON TESTICULAR MORPHOLOGY AND THE EXPRESSION OF CIRCADIAN CLOCK GENES IN JAPANESE WOOD MICE (APODEMUS SPECIOSUS).

This study aimed to determine the seasonality of reproduction throughout the year in Japanese wood mice (Apodemus speciosus). The effect of seasonal changes on testicular morphology and the periodic expression of circadian clock genes in the hypothalamus and testes of male individuals was evaluated. We also examined the morphology of the testes and caudae epididymides of male mice. In addition, RT-PCR analysis was carried out with mRNA extracted from the hypothalamus and testes to evaluate the expression of the circadian clock genes Clock, Bmal1, Per1, and Cry1. The complete induction of testicular activity was detected from February to April and from August to October, with testes weight increasing with the completion of spermatogenesis (reproductive season). From May to early June and from November to early January, testicular weight declined, the seminiferous tubules reduced in size, spermatogenesis was arrested, and sperm were not produced (non-reproductive season). From mid- June to July and mid-January, the re-induction of testicular activity for spermatogenesis was observed in the seminiferous tubules (transitional season). Out of the four examined genes, Cry1 had the highest expression level in both the hypothalamus and testes throughout the year, followed by Bmal1, Per1, and Clock. The expression of Bmal1 was significantly lower in the hypothalamus and testes during the transitional season compared to the reproductive and non-reproductive seasons. Cry1 transcript levels were also significantly lower in the hypothalamus and testes during the transitional season compared to the reproductive season. In conclusion, the results indicating changes in testicular morphology revealed annual reproductive, non-reproductive, and transmission periods in Japanese wood mice. When an increase in testicular activity was observed indicating the onset of the reproductive season, the mean day length was approximately 11–13 h. The expression of the circadian clock genes Bmal1 and Cry1 in the hypothalamus and testes during the reproductive season was significantly higher than that of the same genes during the transitional season. Consequently, completion of spermatogenesis occurred in the seminiferous tubules of Japanese wood mice testes during the reproductive period.

How do employment types and job stressors relate to occupational injury? A cross-sectional investigation of employees in Japan.

OBJECTIVE: This study investigated whether 1) the risk of occupational injury differs among permanent employees and specific types of temporary workers, 2) the risk of occupational injury differs across different employment types depending on the degree of job stressors. STUDY DESIGN: A cross-sectional study design based on self-report survey data. METHODS: A total of 36,688 full-time workers (28,868 men and 7820 women; average age = 35.4) were surveyed by means of a self-administered questionnaire. Employment types consisted of permanent employment and two forms of temporary employment: direct-hire and temporary work agent (TWA). Job characteristics including job demands, job control, and social support at work were measured. Occupational injury was measured by asking whether the participant had an injury on the job in the past 12 months that required a medical treatment. To investigate the relationships between employment types, job stressors, and occupational injury, hierarchical moderated logistic regression tests were conducted. RESULTS: High job demands (OR = 1.44) and low job control (OR = 1.21) were significantly associated with an increased risk of occupational injury, while controlling for demographic, life style, health, and occupational factors. In addition, direct-hires (OR = 1.85) and temporary agent workers (OR = 3.26) had a higher risk of occupational injury compared with permanent employees. However, the relationship between employment types and the risk of occupational injury depended on the levels of job demands and job control. Specifically, the magnitude of the relationship between job demands and the risk of occupational injury was substantially greater for temporary work agents than for permanent employees when they reported low levels of job control. Such an interaction effect between job demands and job control on the risk of occupational injury was not observed between permanent employees and direct-hire temporary workers. CONCLUSION: The current study indicated that temporary workers might be more vulnerable to occupational injury than permanent employees. High levels of job demands and low levels of job control might also add to temporary workers' risk of occupational injury, particularly for TWAs.

Source efficiency of alpha-emitters applied to the skin surface.

Skin surface contamination by alpha-emitters is in itself not hazardous, but it would cause significant internal exposure in the case of injured skin as well as misjudgment in direct in vivo measurements (e.g. lung counting). The present study determined the source efficiency of alpha-emitters (241Am) applied to swine skin samples by analysing the observed alpha-particle energy spectra using advanced alpha-spectrometric simulation. Based on our results, the source efficiency was determined to be 0.365 (alpha-particle s-1 per Bq) on average (c.f. 0.5 in the case of no self-absorption in the source). The decrease in source efficiency would be attributed primarily to the radionuclide entering hair follicles or deep wrinkles. The degradation of the measured spectra from the skin samples indicates the penetration of some radionuclides into the upper layers of the stratum corneum. Although this study was limited to results obtained from swine skin samples, it suggests that irregularities in the skin surface may affect direct alpha measurements.

Extended-spectrum ß-lactamase-producing Escherichia coli strains in the feces of carriers contribute substantially to urinary tract infections in these patients.

INTRODUCTION: The current increase in the incidence of urinary tract infections (UTIs) worldwide caused by extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli may be due to the high number of ESBL-producing Enterobacteriaceae carriers in the community. However, whether ESBL-producing bacteria can cause UTIs in carriers remains uncertain. MATERIALS AND METHODS: In this study, 21 fecal carriers of ESBL-producing Enterobacteriaceae were assessed for UTIs caused by ESBL-producing E. coli. Bacterial isolates obtained from patients' urine and stool specimens were phenotypically and genotypically examined. Clonal similarities of isolates were assessed by multilocus sequence typing (MLST) and random amplified polymorphic DNA (RAPD) fingerprinting. RESULTS: The study revealed that 9 of 21 carriers developed UTIs, and genetic analysis showed that 44% of the UTIs developed were caused by the same ESBL-producing E. coli as that found in the feces of the patients. CONCLUSIONS: The ESBL-producing E. coli in carriers can cause UTIs.

Active cigarette smoking, secondhand smoke exposure at work and home, and self-rated health.

OBJECTIVES: Although active smoking has been reported to be associated with poor self-rated health (SRH), its association with secondhand smoke (SHS) is not well understood. STUDY DESIGN: A cross-sectional study was conducted to examine the association of active smoking and SHS exposure with SRH. METHODS: A total of 2558 workers (1899 men and 689 women), aged 16-83 (mean 45) years, in 296 small and medium-sized enterprises were surveyed by means of a self-administered questionnaire. Smoking status and exposure levels to SHS (no, occasional or regular) among lifetime non-smokers were assessed separately at work and at home. SRH was assessed with the question: How would you describe your health during the past 1-year period (very poor, poor, good, very good)? SRH was dichotomized into suboptimal (poor, very poor) and optimal (good, very good). Odds ratios (ORs) with 95% confidence intervals (CIs) for reporting suboptimal vs optimal SRH according to smoking status and smoke exposure were calculated. RESULTS: Current heavy smokers (20+ cigarettes/day) had a significantly increased suboptimal SRH than lifetime non-smokers after adjusting for sociodemographic, lifestyle, physical and occupational factors (OR 1.34, 95% CI 1.06-1.69). Similarly, lifetime non-smokers occasionally exposed to SHS at work alone had worse SRH than their unexposed counterparts (OR 1.50, 95% CI 1.02-2.11). In contrast, lifetime non-smokers exposed at home alone had no significant increase in suboptimal SRH. CONCLUSIONS: The present study indicates an increase in suboptimal SRH among current heavy smokers, and suggests that SHS exposure at work is a possible risk factor for non-smokers. Whether or not the association is causal, control of smoking at work may protect workers from developing future health conditions.

Reduced uptake of oxidized low density lipoproteins in monocyte-derived macrophages from CD36-deficient subjects.

To clarify the physiological roles of CD36 as an oxidized low density lipoprotein (OxLDL) receptor, we analyzed the monocyte-derived macrophages from normal and two CD36-deficient subjects, since we identified the molecular abnormalities (Kashiwagi, H., Y. Tomiyama, Y. Kosugi, M. Shiraga, R. H. Lipsky, Y. Kanayama, Y. Kurata, and Y. Matsuzawa 1994. Blood. 83:3545-3552; and Kashiwagi, H., Y. Tomiyama, S. Honda, S. Kosugi, M. Shiraga, N. Nagao, S. Sekiguchi, Y. Kanayama, Y. Kurata, and Y. Matsuzawa. 1995. J. Clin. Invest. 95:1040-1046). Scatchard analysis of 125I-OxLDL binding showed a linear plot and the maximum binding was lower by approximately 40% in the macrophages from subjects with CD36 deficiency than those from normal controls. Competition studies showed that the uptake of 125I-OxLDL was suppressed by OKM5, an antibody against CD36, by 53% in normal control macrophages, but not in the CD36-deficient macrophages. After incubation with OxLDL for 24 h, cholesteryl ester mass accumulation was reduced by approximately 40% in the macrophages from CD36-deficient subjects than those from normal controls. These results suggest that CD36 is one of the physiological receptors for OxLDL. Since specific binding of OxLDL was only reduced by approximately 40% in spite of the complete deficiency of CD36, several other receptors also may have some role in OxLDL uptake. Further studies will be needed to assess the quantitative role of CD36 in foam cell formation in vivo.

Identification of the human beta-actin enhancer and its binding factor.

An enhancer of the human beta-actin gene and a factor that specifically interacts with it were detected. A mobility shift assay showed that the factor bound to the 25-base-pair sequence (between +759 and +783 downstream from the cap site) with high specificity. This finding correlated with those of DNase I protection and exonuclease III digestion assays. This binding region of the beta-actin enhancer contained a hyphenated dyad symmetry and an enhancer core-like sequence. In vitro competition experiments indicated that the factor did not bind to the simian virus 40 enhancer core region.

The characteristics of karyotype and telomeric satellite DNA sequences in the cricket, Gryllus bimaculatus (Orthoptera, Gryllidae).

The chromosomes derived from the Japanese population of Gryllus bimaculatus were characterized by C-banding and Ag-NOR staining. The chromosome number, 2n = 28 + XX (female)/XO (male), corresponded with that of other populations of G. bimaculatus, but the chromosome configuration in idiograms varied between the populations. NORs were carried on one pair of autosomes and appeared polymorphous. The positive C-bands located at the centromere of all chromosomes and the distal regions of many chromosome pairs, and the size and the distribution pattern of the distal C-heterochromatin showed differences among the chromosomes. In addition, this paper reports on the characteristics of HindIII satellite DNA isolated from the genome of G. bimaculatus. The HindIII repetitive fragments were about 0.54 kb long, and localized at the distal C-bands of the autosomes and the interstitial C-bands of the X chromosome. Molecular analysis showed two distinct satellite DNA sequences, named the GBH535 and GBH542 families, with high AT contents of about 67 and 66%, respectively. The two repetitive families seem to be derived from a common ancestral sequence, and both families possessed the same 13-bp palindrome sequence. The results of Southern blot hybridization suggest that the sequence of the GBH535 family is conserved in the genomic DNAs of Gryllus species, whereas the GBH542 family is a species-specific sequence.

Molecular cytogenetic characterization and chromosomal distribution of the satellite DNA in the genome of Oxya hyla intricata (Orthoptera: Catantopidae).

The genomic DNA of the grasshopper (Oxya hyla intricata) was subjected to electrophoresis after digestion with HaeIII, and the result showed two bands of highly repetitive DNA, approximately 200 and 400 bp in length. The 200-bp HaeIII-digested fragment was cloned and characterized by sequencing and fluorescence in situ hybridization (FISH). The results showed the presence of two distinct satellite DNA (stDNA) families: one consisting of a 169-bp repeated element having an A+T content of 60.9% and the other consisting of a 204-bp repeated element having an A+T content of 53.9%. No significant homology between the two stDNA families was observed. FISH showed that the chromosomal locations of these families are different from each other. The 169-bp element was located in the C-band-positive regions of the short arms of most of the chromosomes, whereas the 204-bp element was located in the centromeric regions of three chromosome pairs. These results imply that the origins of these two DNA families are different. The results of zoo-blot hybridization to the genomic DNA from four Oxya species, O. hyla intricata, O. japonica japonica, O. chinensis formosana, and O. yezoensis, suggest that the two stDNA families found in the present study are species-specific for O. hyla intricata.

Evaluation and characterization of catabolite-responsive elements (cre) of Bacillus subtilis.

A global mechanism of catabolite repression of the genus Bacillus comprises negative regulation exerted through the binding of the CcpA protein to the catabolite-responsive elements (cres) of the target genes. We searched for cre sequences in the Bacillus subtilis genome using a query sequence, WTGNAANCGNWNNCW (N and W stand for any base and A or T, respectively), picking out 126 putative and known cre sequences. To examine their cre function, we integrated spac promoter (P spac )-cre-lacZ fusions into the amyE locus. Examination of catabolite repression of beta-galactosidase synthesis in the integrants led us to the following conclusions: (i) lower mismatching of cre sequences to the query sequence is required for their function; (ii) although cre sequences are partially palindromic, low mismatching in the same direction as that of transcription of the target genes is more critical for their function than that in the inverse direction; and (iii) yet, a more palindromic nature of cre sequences is desirable for a better function. Furthermore, the alignment of 22 cre s that function in vivo implicated a consensus sequence, WWTGNAARCGNWWWCAWW (R stands for G or A). Interestingly, in the case where cre sequences are located in the protein-coding regions of the target genes, their conserved bases are preferentially the third bases of codons where base degeneracy is allowed.

Karyotypic evolution and organization of the highly repetitive DNA sequences in the Japanese shrew-moles, Dymecodon pilirostris and Urotrichus talpoides.

The karyological relationship and organization of highly repetitive DNA sequences in Japanese shrew-moles were studied by zoo-blot hybridization and fluorescence in situ hybridization (FISH). When the genomic DNA of the eastern race of Urotrichus talpoides was digested with PstI, three fragments of highly repetitive DNA sequences, approximately 0.7, 0.9, and 1.4 kb in length, were observed as distinct bands. The results of FISH in the eastern race of U. talpoides using these three fragments separately as probes showed that the 0.7-kb PstI fragment was distributed in the centromeric regions of most chromosomes, and that the 0.9- and 1.4-kb fragments were predominantly located in the C-heterochromatin region of chromosome 13p. Although the western race of U. talpoides also had three PstI fragments, 0.9- and 1.4-kb PstI fragments were more ambiguous than those of the eastern race. The PstI- digested genomic DNA in Dymecodonpilirostris produced only a faint 0.9-kb band, and its signal patterns obtained by zoo-blot hybridization were clearly different from those of U. talpoides. The 0.7-kb fragment of U. talpoides hybridized strongly with the 0.9-kb fragment of D. pilirostris. In a FISH analysis, the 0.9-kb fragment of D. pilirostris hybridized with highly repetitive DNA in the centromeric regions of most chromosomes from both D. pilirostris and U. talpoides. Zoo-blot hybridization and FISH analyses suggest that the 0.9- and 1.4-kb PstI fragments were generated specifically in the genome of U. talpoides after the common ancestor differentiated into two extant shrew-mole species. A difference in the length of the centromeric elements between U. talpoides and D. pilirostris might be observed due to certain modifications of the repeating unit.

Regulation of the phosphate regulon of Escherichia coli K-12: regulation and role of the regulatory gene phoR.

The phoR gene product functions as a negative regulator with excess of phosphate and as a positive regulator with limited phosphate for the phosphate-starvation-inducible pho regulon of Escherichia coli. We constructed recombinant plasmids that contain a phoR'-'lacZ fusion gene to study the regulation of phoR expression. The genetic and physiological regulation of phoR expression was found to be very similar to that of phoB, a positive regulatory gene for the pho regulon, and phoA, the structural gene for alkaline phosphatase, both of which are inducible by phosphate limitation. The synthesis of the PhoR protein became non-inducible when the phoB promoter upstream of phoR, was removed from the hybrid plasmid, or when a transcriptional terminator was inserted in the phoB structural gene, irrespective of phosphate concentration in the medium. The results suggest that phoB and phoR constitute a single operon whose promoter is located proximal to phoB. The same low level of the PhoR protein in the cell can function as a positive regulator with limited phosphate and as a negative regulator with excess phosphate for the phoB-phoR operon. These results suggest that the maximal level of the operon is induced as consequences of both the increase in the quantity of the PhoR protein and of functional change of the protein as a positive regulator, which are induced by phosphate limitation.

Regulation of the pho regulon in Escherichia coli K-12. Genetic and physiological regulation of the positive regulatory gene phoB.

phoB is a positive regulatory gene for phoA, which codes for alkaline phosphatase, as well as for other genes belonging to the phosphate (pho) regulon whose expression is inducible by phosphate limitation in Escherichia coli. A hybrid plasmid that contains a phoB-lacZ fused gene was constructed in vitro. This plasmid enabled us to study phoB gene expression by measuring the beta-galactosidase level in the cells. The plasmid was introduced into various regulatory mutants related to the phosphate regulon, and phoB gene expression in these strains was studied under limited and excess phosphate conditions. It was found that the regulation of phoB expression was very similar to that of phoA expression. Expression of both genes was induced by phosphate starvation. Both genes were constitutively expressed in phoR, phoS, phoT and phoU mutants and were not expressed in a phoR-phoM double mutant. The implications of these findings for the regulatory mechanism of the pho regulon are discussed.

Nucleotide sequence of the phoB gene, the positive regulatory gene for the phosphate regulon of Escherichia coli K-12.

phoB encodes a positive regulator for a number of the genes belonging to the phosphate regulon of Escherichia coli, including phoA, phoS, phoE and ugpAB. We have determined the nucleotide sequence of the chromosomal segment containing the promoter and the coding region of the phoB gene. The sequence data combined with the known amino-terminal amino acid sequence of a PhoB-LacZ hybrid protein suggest that the PhoB protein consists of 228 amino acid residues with a Mr of 26,302. In the regulatory region of the gene, a consensus nucleotide sequence shared by the regulatory regions of the phoA, phoS and phoE genes, which we name the "phosphate box", was found. Since these genes are positively regulated by the product of phoB, this suggests that transcription of the phoB gene is also regulated positively by its own product. Extensive homology was found in the amino acid sequences of the products of phoB, ompR and dye, all of which are positive regulatory genes for a number of genes coding for envelope proteins. This implies that these genes were originally duplications of a protogene that evolved to have divergent but related functions.

Nucleotide sequence of the phoR gene, a regulatory gene for the phosphate regulon of Escherichia coli.

Genes in the phosphate regulon of Escherichia coli are positively regulated by the products of the phoB and phoR genes with limited phosphate, and negatively regulated by the product of the phoR gene with excess phosphate. We present here the complete nucleotide sequence of the phoR gene. Together with the DNA sequence of the upstream phoB gene that we determined previously, this region shows the following features. The flanking regions of the operon are abundant in A-T base-pairs. A possible stem-and-loop structure of the transcript followed by several U residues characteristic of rho-independent transcriptional terminators was distal to the phoR coding region. The operon is probably composed of only two cistrons. The nucleotide sequence of phoR indicates that its protein consists of 431 amino acid residues and has a molecular weight of 49,666. The amino acid sequence of the PhoR protein has significant homology with that of the EnvZ protein, which is a regulator for the omp regulon. Therefore, the sequences of the PhoB and PhoR proteins have considerable homologies with those of the OmpR and EnvZ proteins, respectively, indicating that the two operons share a common ancestor. The PhoR protein contains an extensive hydrophobic region in the amino-terminal portion. Thus the protein may be a membrane protein and function as a component of a signal transducer.

Nucleotide sequence of the genes involved in phosphate transport and regulation of the phosphate regulon in Escherichia coli.

The pstA(=phoT), pstB and phoU genes are situated at 84 minutes on the Escherichia coli genetic map. All of them are involved in the negative regulation of the phosphate regulon, and all of them except for phoU are required for the binding-protein-mediated, highly specific phosphate transport. We have determined the DNA sequence of about 4 X 10(3) bases of chromosomal segment containing these genes. Four translational reading frames (TRFs) were detected in the region. We attempted to assign the TRFs to the mutant alleles. Plasmids were constructed so that each contained only one of the TRFs, downstream from the lac promoter, to be used for the complementation tests. By this test, TRF-2, TRF-3 and TRF-4 were identified with the pstA(=phoT), pstB and phoU genes, respectively. Alkaline phosphatase-constitutive mutations of the two strains in our collection were complemented by the plasmid with the TRF-1 region. Therefore, we propose to designate the allele phoW. The order of the genes in this region has been established to be phoS-phoW-pstA(=phoT)-pstB-phoU counterclockwise on the E. coli genetic map.

Regulation of the phosphate regulon of Escherichia coli. Activation of pstS transcription by PhoB protein in vitro.

Expression of the genes in the phosphate regulon, including the pstS (phoS) and phoB genes, is positively regulated by PhoB protein when phosphate is limited. We purified PhoB protein from overproducing cells and studied its interaction with the pstS gene. It binds specifically to the DNA fragment containing the promoter region of pstS. The transcription initiation site of the gene in vivo was identified by S1 nuclease mapping and primer-extension experiments. In-vitro transcription of pstS was activated by the PhoB protein, and the initiation site of transcription agreed with the in-vivo initiation site. Activation of in-vitro transcription by PhoB protein required both the normal sigma factor (sigma 70) and core RNA polymerase. PhoB protein binding sites on the promoter regions of pstS and phoB were determined by footprinting experiments with DNase I and a methylating agent. In both cases the protein binds to the pho box, the concensus sequence shared by regulatory regions of genes in the phosphate regulon. Our findings indicate that PhoB protein recognizes and binds to the pho box and activates transcription of the genes in the phosphate regulon.

Signal transduction in the phosphate regulon of Escherichia coli involves phosphotransfer between PhoR and PhoB proteins.

PhoB protein is the transcriptional activator for genes in the phosphate regulon of Escherichia coli, such as phoA and pstS, that are induced by phosphate deprivation. PhoR protein activates PhoB when phosphate is limiting and inactivates it when phosphate is in excess. We constructed a plasmid with a mutant phoR gene (phoR1084), which encoded a PhoR protein (PhoR1084) lacking the amino-terminal hydrophobic region of the intact protein. The cells carrying the plasmid overproduced PhoR1084, which was recovered in the soluble fraction of the cell lysate. We purified the Phor1084 protein and showed that it was autophosphorylated in the presence of ATP, and the phosphate group on the protein was rapidly transferred to PhoB. The phosphorylation of PhoB protein occurred concurrently with the acquisition of the ability to activate transcription from the pstS promoter. On the basis of these findings, we propose that phosphorylated PhoB protein activates transcription from the promoters of the phosphate regulon, and that the role of PhoR is to catalyze the formation and breakdown of phosphorylated PhoB in response to phosphate concentrations in the medium.