Properties of Spontaneous rpsL Mutant of Streptomyces albus KO-1297.

The Streptomyces albus J1074 strain remains one of the most popular platforms for the discovery of new natural compounds due to the expression of biosynthetic gene clusters (BGCs) from the microorganisms of the Actinobacteria class. Different methods were tested to provide a maximal expression of heterologous BGCs in this strain. However, there is still no description of the properties of spontaneous J1074 mutants in the rpsL gene encoding a ribosomal protein S12. The interest in such mutations in actinobacteria is due to the fact that they provide a considerable increase in the antibiotic activity. In this work, we describe the isolation and characterization of the S. albus KO-1297 strain, which contains a spontaneous missense mutation in the rpsL gene leading to a Lys88Glu substitution in the protein S12. As compared with the initial strain, this mutant exhibits an increased resistance to streptomycin and higher antibiotic productivity. The KO-1297 strain and genetically engineered rpsL K88E mutant K88E are not identical in their ability to produce antibiotics. KO-1297 also exhibits a certain level of instability of rpsL mutation. The genomes of KO-1297 and its rpsL WT revertant contain the mutations that can cause phenotypic differences between these strains (as well as between them and SAM2 and K88E strains).

Feeding frequency, but not dietary water content, affects voluntary physical activity in young lean adult female cats.

The objective of this study was to investigate whether increased dietary water content and feeding frequency increased voluntary physical activity of young, lean adult female cats. A replicated 4 × 4 Latin square design with a 2 × 2 factorial treatment arrangement (feeding frequency and water content) was used. The 4 treatments consisted of 1 meal daily dry pet food without added water (1D; 12% moisture as is), 1 meal daily dry pet food with added water (1W; 70% total water content), 4 meals daily dry pet food without added water (4D; 12% moisture as is), and 4 meals daily dry pet food with added water (4W; 70% total water content). Eight healthy adult, lean, intact, young, female domestic shorthair cats were used in this experiment. Voluntary physical activity was evaluated using Actical activity monitors placed on collars and worn around the cats' necks for the last 7 d of each experimental period of 14 d. Food anticipatory activity (FAA) was calculated based on 2 h prior to feeding periods and expressed as a percentage of total daily voluntary physical activity. Increased feeding frequency (4 vs. 1 meal daily) resulted in greater average daily activity (P = 0.0147), activity during the light period (P = 0.0023), and light:dark activity ratio (P = 0.0002). In contrast, physical activity during the dark period was not altered by feeding frequency (P > 0.05). Cats fed 4 meals daily had increased afternoon FAA (P= 0.0029) compared with cats fed once daily. Dietary water content did not affect any measure of voluntary physical activity. Increased feeding frequency is an effective strategy to increase the voluntary physical activity of cats. Thus, it may assist in the prevention and management of obesity.

Ten-year incidences of self-reported non-vertebral fractures in Japanese patients with rheumatoid arthritis: discrepancy between disease activity control and the incidence of non-vertebral fracture.

UNLABELLED: Despite improvements in rheumatoid arthritis disease activity of in the past 10 years, the incidence of self-reported non-vertebral fractures did not decrease in our cohort of 9,987 patients. This study may indicate that osteoporosis treatment and non-vertebral fracture prevention remain important regardless of the rheumatoid arthritis disease activity. INTRODUCTION: Although rheumatoid arthritis (RA) is a risk factor for osteoporosis and fractures, few studies have described the association between disease activity and the fracture incidence in patients with RA. This study aimed to investigate changes in the non-vertebral fracture incidence between 2001 and 2010 in our Institute of Rheumatology Rheumatoid Arthritis (IORRA) cohort. METHODS: The IORRA is a prospective observational cohort study of Japanese RA patients. A total of 9,987 patients with RA were enrolled in this cohort from 2000 to 2010. The clinical parameter and non-vertebral fracture occurrence data were collected biannually through self-reported questionnaires. Incidences of self-reported non-vertebral fractures were also analyzed via standardization according to gender, age, and disease activity during each 2-year period. RESULTS: From 2001 to 2010, the percentage of patients with 28-joint disease activity score remission increased from 7.8 to 39.7%, prednisolone intake decreased from 51.4 to 41.3%, and bisphosphonate intake increased from 5.0 to 23.4%. The non-vertebral fracture incidence rates were 24.6/1,000 person-years in 2001 and 35.5/1,000 person-years in 2010, with no apparent change even after standardization. The overall non-vertebral fracture incidence was significantly higher in the autumn/winter than in the spring/summer (p = 0.02). CONCLUSION: Despite improvements in disease activity and functional disability, the non-vertebral fracture incidence exhibited no apparent change between 2001 and 2010 in our patients with RA. Osteoporosis treatment and non-vertebral fracture prevention remain important regardless of the disease control in patients with RA.

Ulnar nerve strain at the elbow in patients with cubital tunnel syndrome: effect of simple decompression.

Simple decompression of the ulnar nerve at the elbow has not been shown to reduce nerve strain in cadavers. In this study, ulnar nerve strain at the elbow was measured intraoperatively in 11 patients with cubital tunnel syndrome, before and after simple decompression. Statistical analysis was performed using a paired Student's t-test. Mean ulnar nerve strain before and after simple decompression was 30.5% (range 9% to 69%) and 5.5% (range -2% to 11%), respectively; this difference was statistically significant (p < 0.01) with a statistical power of 96%. Simple decompression reduced ulnar nerve strain in all patients by an average of 24.5%. Our results suggest that the pathophysiology of cubital tunnel syndrome may be multifactorial, being neither a simple compression neuropathy nor a simple traction neuropathy, and simple decompression may be a favourable surgical procedure for cubital tunnel syndrome in terms of decompression and reduction of strain in the ulnar nerve.

Immunohistochemical and functional studies for M3 muscarinic receptors and cyclo-oxygenase-2 expressed in the mouse atrium.

In mouse atrium, M2 and M3 muscarinic receptors (M2R and M3R) are involved in biphasic (negative and positive) inotropic actions of muscarinic agonists, and the positive inotropic action is reduced by indomethacin. The aim of our study was to determine the localization of M2R, M3R and cyclo-oxygenase (COX) in mouse atrium and to characterize muscarinic receptor-mediated positive inotropy. M2R immunoreactivity was found only on atrial myocardium, but M3R immunoreactivity was localized on both the myocardium and endocardial endothelium. COX-1 and COX-2 immunoreactivities were identified in both myocardial and endocardial endothelium. In electrically stimulated left atria, carbachol caused M2R-mediated negative inotropy followed by M3R-mediated positive inotropy. Removal of atrial endothelium reduced the positive inotropy without affecting the negative inotropy, suggesting that stimulation of endothelial M3R mediates the positive inotropy. N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide (NS398, COX-2 inhibitor) decreased the carbachol-induced positive inotropy; however, 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethylpyrazole (SC560, COX-1 inhibitor), 1-[[4,5-bis(4-methoxyphenyl)-2-thiazolyl]carbonyl]-4-methylpiperazine (FR122047, COX-1 inhibitor) and L-nitroarginine methylester did not affect the inotropic response. M3R activation caused positive chronotropy in spontaneously beating right atria when M2R-mediated negative chronotropy was suppressed and rate of contraction was low, <350 beats min⁻¹. Our results indicate that although M3Rs are located on both myocardial cells and endocardial endothelial cells, only endothelial M3Rs mediate positive inotropy in response to muscarinic agonists via activation of COX-2 in the mouse atrium. M3R-mediated positive chronotropy counteracting M2R-mediated negative chronotropy was also demonstrated.

Surgical treatment of spontaneous posterior interosseous nerve palsy: a retrospective study of 50 cases.

We have reviewed 38 surgically treated cases of spontaneous posterior interosseous nerve palsy in 38 patients with a mean age of 43 years (13 to 68) in order to identify clinical factors associated with its prognosis. Interfascicular neurolysis was performed at a mean of 13 months (1 to 187) after the onset of symptoms. The mean follow-up was 21 months (5.5 to 221). Medical Research Council muscle power of more than grade 4 was considered to be a good result. A further 12 cases in ten patients were treated conservatively and assessed similarly. Of the 30 cases treated surgically with available outcome data, the result of interfascicular neurolysis was significantly better in patients < 50 years old (younger group (18 nerves); good: 13 nerves (72%), poor: five nerves (28%)) than in cases > 50 years old (older group (12 nerves); good: one nerve (8%), poor: 11 nerves (92%)) (p < 0.001). A pre-operative period of less than seven months was also associated with a good result in the younger group (p = 0.01). The older group had a poor result regardless of the pre-operative delay. Our recommended therapeutic approach therefore is to perform interfascicular neurolysis if the patient is < 50 years of age, and the pre-operative delay is < seven months. If the patient is > 50 years of age with no sign of recovery for seven months, or in the younger group with a pre-operative delay of more than a year, we advise interfascicular neurolysis together with tendon transfer as the primary surgical procedure.

A 100 nm thick InGaN/GaN multiple quantum-well column-crystallized thin film deposited on Si(111) substrate and its micromachining.

A 100 nm thick InGaN/GaN multiple quantum-well column-crystallized thin film was deposited on Si(111) substrate, with InN as the interlayer, by molecular beam epitaxy. The diameter of the column crystal is about 40 nm. Transmission electron microscopy images showed clear five-period well layers. Photoluminescence measurements demonstrated a wide emission wavelength from about 500 to 800 nm with the full width at half maximum of 107 nm at room temperature. An unusual photoluminescence peak position shift was observed from the optical measurement. The selected area electron diffraction image demonstrated the hexagonal wurtzite structure of the column crystal. A self-supported GaN-based active subwavelength grating was proposed, and the active subwavelength grating structure was fabricated from the InGaN/GaN quantum-well thin film by a Si micromachining process.

Listeria monocytogenes isolated from cold-smoked fish products in Osaka City, Japan.

Listeria monocytogenes contamination of ready-to-eat seafood products commercially available in Osaka was examined between 1999 and 2000. L. monocytogenes was isolated from 12 (13%) of the 95 products tested. All positive samples were from cold-smoked fish with 9 being obtained during the summer. Thirteen isolates of L. monocytogenes were typed by pulsed-field gel electrophoresis (PFGE) and polymerase chain reaction (PCR)-based typing methods. Isolates of the same serotype originating from the same manufacturer gave similar DNA profiles, irrespective of the type of sample or date of isolation. The finding suggest that persistent strains in each manufacturing facility proliferate during the summer and contaminate products during manufacturing processes.

The novel mutation K87E in ribosomal protein S12 enhances protein synthesis activity during the late growth phase in Escherichia coli.

Resistance to streptomycin in bacterial cells often results from a mutation in the rpsL gene that encodes the ribosomal protein S12. We found that a particular rpsL mutation (K87E), newly identified in Escherichia coli, causes aberrant protein synthesis activity late in the growth phase. While protein synthesis decreased with age in cells in the wild-type strain, it was sustained at a high level in the mutant, as determined using living cells. This was confirmed using an in vitro protein synthesis system with poly(U) and natural mRNAs (GFP mRNA and CAT mRNA). Other classical rpsL mutations (K42N and K42T) tested did not show such an effect, indicating that this novel characteristic is typical of ribosomes bearing the K87E mutant form of S12, although the K87E mutation conferred the streptomycin resistance and error-restrictive phenotypes also seen with the K42N and K42T mutations. The K87E (but not K42N or K42T) mutant ribosomes exhibited increased stability of the 70S complex in the presence of low concentrations of magnesium. We propose that the aberrant activation of protein synthesis at the late growth phase is caused by the increased stability of the ribosome.

A rifampicin resistance mutation in the rpoB gene confers ppGpp-independent antibiotic production in Streptomyces coelicolor A3(2).

In Streptomyces coelicolor A3(2), deletion of relA or a specific mutation in rplK ( relC) results in an inability to synthesize ppGpp (guanosine 5'-diphosphate 3'-diphosphate) and impairs production of actinorhodin. We have found that certain rifampicin-resistant ( rif) mutants isolated from either relA or relC strains regain the ability to produce actinorhodin at the same level as the wild-type strain, although their capacity to synthesize ppGpp is unchanged. These rif mutants were found to have a missense mutation in the rpoB gene that encodes the RNA polymerase beta-subunit. This rpoB mutation was shown to be responsible for the observed changes in phenotype, as demonstrated by gene replacement experiments. Gene expression analysis revealed that the restoration of actinorhodin production in both relA and relC strains is accompanied by increased expression of the pathway-specific regulator gene actII-ORF4, which is normally decreased in the rel mutants. In addition to the restoration of antibiotic production, the rif mutants also exhibited a lower rate of RNA synthesis compared to the parental strain when grown in a rich medium, suggesting that these mutant RNA polymerases behave like "stringent" RNA polymerases. These results indicate that rif mutations can alter gene expression patterns independently of ppGpp. We propose that RNA polymerases carrying particular rif mutations in the beta-subunit can functionally mimic the modification induced by binding of ppGpp.

The inhibitory effect of the autoantigen La on in vitro 3' processing of mammalian precursor tRNAs.

Mammalian tRNA 3' processing endoribonuclease (3' tRNase) can remove a 3' trailer from various precursor (pre)-tRNAs. We investigated what effect the autoantigen La has on 3' processing, since the La protein is known to bind to a 3'-terminal uridine tract of pre-tRNAs. We tested sixteen different pre-tRNA(Arg) substrates containing various 3' trailers with or without a 5' leader sequence for in vitro processing by pig 3' tRNase, and for gel-retardation in the presence or absence of human La protein. The R-TUUU series consists of four pre-tRNAs containing 6, 8, 11 and 15 nt 3' trailers ending with UUU and no 5' leader, while the R-TAGC series consists of the same four pre-tRNAs as R-TUUU except that the terminal sequence is AGC. The R-6LTUUU and R-6LTAGC series are derived from R-TUUU and R-TAGC, respectively, by adding a 6 nt 5' leader. La differentially inhibited their processing and bound to the pre-tRNAs; the 50 % inhibitory concentrations for the R-TUUU, R-TAGC, R-6LTUUU, and R-6LTAGC series were 82 to >850, >850, 2 to 292 and 573 to 785 nM, respectively, and the dissociation constants were 10 to 840, >850, 3 to 203 and 155 to 520 nM, respectively. These results indicate that both the terminal sequence UUU and the 5' leader contribute to more severe inhibition of 3' processing via tighter interaction with La. With respect to the R-TUUU and R-6LTUUU series, on the whole, the La inhibition was enhanced as the 3' trailer lengths decreased. Taken together, our results suggest that the La protein sterically hinders 3' tRNase from binding a pre-tRNA molecule probably near the cleavage site.

Condylar bony change and self-reported parafunctional habits in prospective orthognathic surgery patients with temporomandibular disorders.

OBJECTIVE: The purpose of this study was to investigate the relationship between self-reported parafunctional habits and condylar bony change and disk displacement in orthognathic surgery patients with signs and symptoms of temporomandibular joint disorders. STUDY DESIGN: This is a cross-sectional retrospective study of pretreatment helical computed tomography scans and questionnaires of 94 female orthognathic surgery patients. RESULTS: Condylar bony change, unilaterally or bilaterally, was found in 56.4% of the subjects, or 43.6% of the joints. Disk displacement, unilaterally or bilaterally, was seen in 59.6% of the subjects, or 45.7% of the joints. Bruxism and clenching was significantly associated with condylar bony change and disk displacement. Subjects with 3 or more parafunctional habits showed a significantly higher rate of bilateral condylar bony change. CONCLUSION: Our results suggest that bruxism and clenching might be related to deterioration of the temporomandibular joint and that the greater the number of parafunctional habits a subject has, the higher the risk of condylar bony change.

Molecular and functional analyses of the gene (eshA) encoding the 52-kilodalton protein of Streptomyces coelicolor A3(2) required for antibiotic production.

Analysis of proteins recovered in the S100 precipitate fraction of Streptomyces griseus after ultracentrifugation led to the identification of a 52-kDa protein which is produced during the late growth phase. The gene (eshA) which codes for this protein was cloned from S. griseus, and then its homologue was cloned from Streptomyces coelicolor A3(2). The protein was deduced to be 471 amino acids in length. The protein EshA is characterized by a central region that shows homology to the eukaryotic-type cyclic nucleotide-binding domains. Significant homology was also found to MMPI in Mycobacterium leprae, a major antigenic protein to humans. The eshA gene mapped near the chromosome end and was not essential for viability, as demonstrated by gene disruption experiments, but its disruption resulted in the abolishment of an antibiotic (actinorhodin but not undecylprodigiosin) production. Aerial mycelium was produced as abundantly as by the parent strain. Expression analysis of the EshA protein by Western blotting revealed that EshA is present only in late-growth-phase cells. The eshA gene was transcribed just preceding intracellular accumulation of the EshA protein, as determined by S1 nuclease protection, indicating that EshA expression is regulated at the transcription level. The expression of EshA was unaffected by introduction of the relA mutation, which blocks ppGpp synthesis.

Construction of an in vivo nonsense readthrough assay system and functional analysis of ribosomal proteins S12, S4, and S5 in Bacillus subtilis.

To investigate the function of ribosomal proteins and translational factors in Bacillus subtilis, we developed an in vivo assay system to measure the level of nonsense readthrough by utilizing the LacZ-LacI system. Using the in vivo nonsense readthrough assay system which we developed, together with an in vitro poly(U)-directed cell-free translation assay system, we compared the processibility and translational accuracy of mutant ribosomes with those of the wild-type ribosome. Like Escherichia coli mutants, most S12 mutants exhibited lower frequencies of both UGA readthrough and missense error; the only exception was a mutant (in which Lys-56 was changed to Arg) which exhibited a threefold-higher frequency of readthrough than the wild-type strain. We also isolated several ribosomal ambiguity (ram) mutants from an S12 mutant. These ram mutants and the S12 mutant mentioned above (in which Lys-56 was changed to Arg) exhibited higher UGA readthrough levels. Thus, the mutation which altered Lys-56 to Arg resulted in a ram phenotype in B. subtilis. The efficacy of our in vivo nonsense readthrough assay system was demonstrated in our investigation of the function of ribosomal proteins and translational factors.

Electrocochleogram after transection of vestibulo-cochlear nerve in a patient with a large acoustic neurinoma.

This study reports pre- and post-operative compound action potentials (CAPs) that were recorded from a 27-year-old woman with an acoustic neurinoma. During surgery it was necessary to totally sever her vestibulo-cochlear nerve to excise a large tumor. A pure tone audiogram changed to the scale-out pattern immediately after operation. However, CAP, the waveform of which was broadened, has been recorded 3 years post-operatively with a threshold elevation of 10 dB over the pre-operative threshold. This phenomenon suggests that CAP may originate from the extreme periphery of the auditory nerve within the cochlea. The broadening of the CAP was assumed to result from enhancement of the negative summating potential included in the CAP. We studied the effect of preceding stimulus on CAP using paired click stimuli pre- and post-operatively. A reduction of CAP amplitude in response to the second click of paired clicks was markedly suppressed in the inter-click interval between 3 and 80 ms post-operatively. We speculate that depletion of adaptation induced the abnormal CAP recovery described above and that the lateral efferent nerve system was involved in abnormal CAP adaptation with transection of the vestibulo-cochlear nerve in this case.

Novel approach for improving the productivity of antibiotic-producing strains by inducing combined resistant mutations.

We developed a novel approach for improving the production of antibiotic from Streptomyces coelicolor A3(2) by inducing combined drug-resistant mutations. Mutants with enhanced (1.6- to 3-fold-higher) actinorhodin production were detected at a high frequency (5 to 10%) among isolates resistant to streptomycin (Str(r)), gentamicin (Gen(r)), or rifampin (Rif(r)), which developed spontaneously on agar plates which contained one of the three drugs. Construction of double mutants (str gen and str rif) by introducing gentamicin or rifampin resistance into an str mutant resulted in further increased (1.7- to 2.5-fold-higher) actinorhodin productivity. Likewise, triple mutants (str gen rif) thus constructed were found to have an even greater ability for producing the antibiotic, eventually generating a mutant able to produce 48 times more actinorhodin than the wild-type strain. Analysis of str mutants revealed that a point mutation occurred within the rpsL gene, which encodes the ribosomal protein S12. rif mutants were found to have a point mutation in the rpoB gene, which encodes the beta-subunit of RNA polymerase. Mutation points in gen mutants still remain unknown. These single, double, and triple mutants displayed in hierarchical order a remarkable increase in the production of ActII-ORF4, a pathway-specific regulatory protein, as determined by Western blotting analysis. This reflects the same hierarchical order observed for the increase in actinorhodin production. The superior ability of the triple mutants was demonstrated by physiological analyses under various cultural conditions. We conclude that by inducing combined drug-resistant mutations we can continuously increase the production of antibiotic in a stepwise manner. This new breeding approach could be especially effective for initially improving the production of antibiotics from wild-type strains.