Anatomy and physiology of hamstring injury.

The hamstring muscles were analyzed anatomically and physiologically to clarify the specific reasons for the incidence of muscle strain of the hamstrings. For the anatomical study, hamstring muscles of 13 embalmed cadavers were dissected. For the physiological study, the knee flexor torque and surface electromyographic (EMG) signals were measured during isometric contraction of hamstring muscles in 10 healthy adults. The biceps femoris muscle long head (BF-L) and semimembranosus muscle (SM) had hemi-pennate architecture and their fiber length per total muscle length (FL/TML) was smaller than that of semtendinosus muscle (ST) and biceps femoris muscle short head (BF-S) with other architecture. The decrease of total muscle length per fiber length (ΔTML/FL) was larger in BF-L and SM than in ST and BF-S. The EMG activities at 0° of knee angle were at maximal compared with other knee angles and were of similar level in BF-L, in SM and in ST, whereas they were considerably smaller in BF-S. The EMG at 0° of knee angle activity per physiological cross-sectional area (PCSA) was about 1.6 times greater in BF-L than in SM. These results indicate the highest risk of muscle strain was in BF-L followed by SM.

Follistatin-related protein gene (FRP) is expressed in the synovial tissues of rheumatoid arthritis, but its polymorphisms are not associated with genetic susceptibility.

OBJECTIVE: To examine the expression level and function of follistatin-related protein gene (FRP, also referred to as FSTL1) in rheumatoid arthritis (RA), and possible association of its polymorphisms with genetic susceptibility to RA. METHODS: FRP mRNA expression levels in the synovial tissues from 10 patients with RA and 5 patients with OA were measured using real-time RT-PCR. Effects on the growth of synovial cells were evaluated by stably introducing FRP cDNA into a rheumatoid synovial cell line, E11. Screening of genomic DNA variations was done using DNA from 12 patients with RA and 12 healthy individuals by direct sequencing. Genotypes at the detected polymorphic sites were determined in 224 patients with RA and 220 healthy individuals using PCR-single strand conformation polymorphism. RESULTS: FRP mRNA was overexpressed in synovial tissues from RA patients by 2.3-fold as compared with those from OA. A rheumatoid synovial cell line (E11) transfected with FRP exhibited reduced proliferation, probably mediated by secreted FRP molecule. 16 genomic variations were identified, among which 4 were polymorphisms within the promoter region and exons, and the remainder were either rare variations or intronic polymorphisms. Genotyping of 4 polymorphic sites did not reveal statistically significant association with the susceptibility to RA. CONCLUSION: FRP mRNA is overexpressed in RA synovium, the product of which exerts inhibitory activity on synovial cell growth. Although new polymorphic sites were identified, they were not associated with susceptibility to RA, suggesting that overexpression of FRP is secondarily caused by synovial environment of RA.

Activation of defense-related gene expression and systemic acquired resistance in cucumber mosaic virus-infected tobacco plants expressing the mammalian 2'5'oligoadenylate system.

Tobacco plants expressing the mammalian 2'5'oligoadenylate system (2-5A system) exhibit resistance to cucumber mosaic virus (CMV). Here, to characterize the molecular aspect of the resistance to CMV in 2-5A system-expressing tobaccos, the activation of defense-related genes and systemic acquired resistance (SAR) as the markers for the hypersensitive resistance (HR), were elucidated. Northern hybridization analysis indicated that the expression of four pathogenesis-related (PR) protein genes and five HR-related genes were induced in CMV-infected tobaccos expressing 2-5A system. Furthermore, the induction of SAR against Pseudomonas syringae pv. tabaci as second challenge, was observed on CMV-inoculated tobaccos expressing 2-5A system. These results suggested that the resistance to CMV in tobacco expressing 2-5A system is associated with the establishment of an HR-like response.

A method for forensic identification of vegetable oil stains--rapid analysis of carboxylic acids with methyl esterification using purge-and-trap gas chromatography/mass spectrometry.

A simple method using purge-and-trap gas chromatography/mass spectrometry (P&T-GC/MS) for forensic examination of oil stains was studied. Carboxylic acids, chosen as target components for discrimination of oil samples, were extracted from stains with ether, methyl esterified by tetramethylammonium hydroxide, and analyzed by P&T-GC/MS. Vegetable oils were discriminated according to their carboxylic acid compositions. Carboxylic acid composition was independent of the substrate material of the stain. Although the carboxylic acid composition of the oil changed on exposure to sunlight, identification of oil was possible for oil stains that had been in the shade, if analysis was made within 20 days.

Pitch perception shift: a rare side-effect of carbamazepine.

Carbamazepine-induced pitch perception shifts have rarely been described. Two cases of shifted pitch perception developing during medication with carbamazepine are described. Case 1 possessed absolute pitch. Her pitch perception shift disappeared with the discontinuance of carbamazepine. Case 2 did not have absolute pitch. Even though he experienced a pitch perception shift, he developed a tolerance to the shift. We concluded that carbamazepine was the cause of the pitch perception shift in the first case, while the second case probably became attuned to the change in pitch perception because he did not possess absolute pitch.

Mapping the virus and host genes involved in the resistance response in cucumber mosaic virus-Infected Arabidopsis thaliana.

A yellow strain of cucumber mosaic virus (CMV) [CMV(Y)] induces a resistance response characterized by inhibition of virus systemic movement with development of necrotic local lesions in the virus-inoculated leaves of Arabidopsis thaliana ecotype C24. In this report, the avirulence determinant in the virus genome was defined and the resistance gene (RCY1) of C24 was genetically mapped. The response of C24 to CMV containing the chimeric RNA3 between CMV(Y) and a virulent strain of CMV indicated that the coat protein gene of CMV(Y) determined the localization of the virus in the inoculated leaves of C24. The RCY1 locus was mapped between two CAPS markers, DFR and T43968, which were located in the region containing genetically defined disease resistance genes and their homologues. These results indicate that the resistance response to CMV(Y) in C24 is determined by the combination of the coat protein gene and RCY1 on chromosome 5.

Cardiovascular responses at the onset of passive leg cycle exercise in paraplegics with spinal cord injury.

The purpose of this study was to examine the cardiovascular responses at the onset of passive leg cycle exercise (PLCE) in paraplegics with spinal cord injury (PSCI) to investigate the increase in venous return from the paralyzed lower limbs during PLCE. Six male PSCI having lesions at levels ranging from T8 to L1 and five male able-bodied subjects (ABS) participated in this study. The subjects performed PLCE at pedalling frequencies of 40 rpm for 6 min. Cardiac output (Q(c)), stroke volume (SV) and heart rate (f(c)) were measured before and during PLCE. In the steady state (4th and 5th min) of PLCE, both PSCI and ABS showed a significant increase in Q(c). At the onset of PLCE, however, clear differences in the cardiovascular response were found between PSCI and ABS. The ABS showed a rapid and marked increase in f(c) and consequently Q(c) within 20 s of the onset of PLCE. On the other hand, in PSCI, the Q(c) increased more slowly, compared with that in ABS, because of a smaller increase in f(c) and a delayed increase in SV. The observed delay in the increases of Q(c) and SV at the onset of PLCE in PSCI was presumably due to the absence of afferent reflexes from the lower limbs, and to the additional time needed for venous return to arrive at the heart from the passively moved muscles.

Identification and cloning of an Erwinia carotovora subsp. carotovora bacteriocin regulator gene by insertional mutagenesis.

Avirulent Erwinia carotovora subsp. carotovora CGE234-M403 produces two types of bacteriocin. For the purpose of cloning the bacteriocin genes of strain CGE234M403, a spontaneous rifampin-resistant mutant of this strain, M-rif-11-2, was isolated. By Tn5 insertional mutagenesis using M-rif-11-2, a mutant, TM01A01, which produces the high-molecular-weight bacteriocin but not the low-molecular-weight bacteriocin was obtained. By thermal asymmetric interlaced PCR, the DNA sequence from the Tn5 insertion site and the DNA sequence of a contiguous 1,280-bp region were determined. One complete open reading frame (ORF), designated ORF2, was identified within the sequenced fragment. The 3' end of another ORF, ORF1, was located upstream of ORF2. A noncoding region and a putative promoter were located between ORF1 and ORF2. Downstream from ORF2, the 5' end of another ORF (ORF3) was found. Deduction from the nucleotide sequence indicated that ORF2 encodes a protein of 99 amino acids, which showed high homology with Yersinia enterocolitica Yrp, a regulator of enterotoxin (Y-ST) production; Escherichia coli host factor 1, required for Qbeta-replicase; and Azorhizobium caulinodans NrfA, required for the expression of nifA. ORF2 was designated brg, bacteriocin regulator gene. A fragment containing ORF2 and its promoter was amplified and cloned into pBR322 and pHSG415r, and the recombinant plasmids, pBYL1 and pHYL1, were transferred into E. coli DH5. Plasmid pBYL1 was reisolated and transferred into the insertion mutant TM01A01. Transformants carrying the plasmid, which was reisolated and designated pBYL1, re-produced the low-molecular-weight bacteriocin.

One amino Acid change in cucumber mosaic virus RNA polymerase determines virulent/avirulent phenotypes on cowpea.

ABSTRACT The elicitation of the hypersensitive response (HR) is known to depend on the interaction between a resistance gene of a host plant and a corresponding avirulence gene of a pathogen. The cv. Kurodane-Sanjaku of cowpea (Vigna unguiculata) has the Cry locus that confers resistance against cucumber mosaic virus strain Y (CMV-Y). The resistance is overcome by infection with a legume strain of CMV (CMV-L). RNA 2, which codes for the 2a protein, a subunit of the viral replicase components, has been known to control virulent/avirulent phenotypes. We generated chimeric constructs of full-length cDNA clones of RNA 2 of both strains and inoculated infectious transcripts to delimit the domain controlling symptoms. A 243-base pair fragment containing a coding region for the GDD RNA-dependent RNA polymerase core sequence was shown to be responsible for the phenotypic differences. From sequence alignment analysis, two amino acids (Phe631 and Ala641) of the HR-type 2a protein encoded in this fragment were specifically exchanged to Tyr and Ser, respectively, in the 2a proteins of resistance-breaking strains. Point mutations introduced into RNA 2 backgrounds of both strains that were designed to change the amino acid at position 631 resulted in a change of symptoms, indicating that a single nucleotide change determines the reactions elicited by both strains. Analysis for one additional mutant RNA 2 showed that symptom determination may be correlated with the nature of the lateral chain of amino acid 631.

Relationship between physical characteristics and physiological responses during maximal arm cranking in paraplegics.

The purpose of this study was to elucidate the main physical factor(s) affecting cardiorespiratory responses during maximal arm cranking exercise in patients with paraplegia. Peak oxygen uptake (peak Vo2), peak pulmonary ventilation (peak VE) and peak heart rate (peak HR) were measured during maximal arm cranking exercise in 28 Japanese male patients. A cluster analysis was applied to the data for peak Vo2, peak HR and peak VE, and then the subjects were classified into four groups (A, B, C and D). Group A showed high peak VE and peak HR and low peak Vo2, Group B low peak Vo2 and high values for other parameters, Group C the greatest physiological values for all measurements among the groups and, in contrast, Group D showed low peak Vo2 and the lowest peak VE and peak HR among the groups. The subjects in Group C had low level of spinal cord injury and were sports participants. On the other hand, duration since injury was not related to the cardiorespiratory responses during maximal arm cranking. These findings indicate that the effects on these responses of years since injury are subordinate to those of the level of spinal cord injury and training.

Zinc stimulates protein synthesis in the femoral-metaphyseal tissues of normal and skeletally unloaded rats.

The effect of zinc on protein synthesis in the femoral-metaphyseal tissues of normal and skeletally unloaded rats was investigated. Skeletal unloading was designed using the model of hindlimb suspension in rats. Animals were fed for 2 or 4 days during the unloading. [3H]Leucine was added to the reaction mixture containing the 5500 g supernatant fraction of the homogenate prepared from the femoral-metaphyseal tissues. In vitro protein synthesis was significantly decreased in the bone tissues from the rats which had undergone unloading for 2 or 4 days. When the metaphyseal tissues were cultured for 24 h in the presence of zinc sulfate (10(-5) M) or beta-alanyl-L-histidinato zinc (AHZ, 10(-5) M), zinc compounds clearly stimulated protein synthesis in the metaphyseal tissues from the 4-day unloaded rats. The zinc effect was also seen in the metaphyseal tissues from normal rats. The addition of zinc sulfate (10(-5) M) or AHZ (10(-7) to 10(-5) M) into the reaction mixture containing the 5500 g supernatant fraction of metaphyseal homogenate from normal or unloaded rats produced a significant increase in protein synthesis. This increase was clearly inhibited in the presence of cycloheximide (10(-7) M). The present result demonstrates that protein synthesis is impaired in the femoral-metaphyseal tissues of rats with skeletal unloading, and that this impairment is clearly restored by zinc supplementation.

Effect of essential trace metal on bone metabolism in the femoral-metaphyseal tissues of rats with skeletal unloading: comparison with zinc-chelating dipeptide.

The effect of essential trace metals on bone metabolism was investigated in the femoral-metaphyseal tissues obtained from skeletal-unloaded rats. Skeletal unloading was designed by using the model of hindlimb suspension in rats; the animals were fed for 4 days with the unloading. Femoral-metaphyseal tissues were cultured for 24 hours in a medium containing either vehicle (control), nickel, manganese, cobalt, copper, zinc, or zinc-chelating dipeptide (beta-alanyl-L-histidinato zinc; AHZ) in the concentration range of 10(-6) to 10(-4) M. Bone biochemical components (alkaline phosphatase activity, glucose consumption, and DNA content) were significantly decreased by skeletal unloading. The presence of zinc sulfate or AHZ (10(-5) and 10(-4) M) caused a significant increase of alkaline phosphatase activity in the bone tissues from unloaded rats. This effect was not seen by nickel, manganese, cobalt and copper (10(-6) to 10(-4) M). The culture medium glucose was clearly consumed by the bone tissues. This consumption was inhibited by nickel, manganese, or copper (10(-5) and 10(-4) M), while cobalt, zinc, and AHZ had no effect. DNA content in the bone tissues from unloaded rats was significantly increased by all metal compounds (10(-5) M). The effect of AHZ on bone components was greater than zinc sulfate. The AHZ (10(-5) M)-increased alkaline phosphatase activity in the bone tissues from unloaded rats was clearly blocked by the presence of cycloheximide (10(-6) M), staurosporine (10(-7) M), dibucaine (10(-4) M), or okadaic acid (10(-7) M). The present study demonstrates that, of various essential trace metals, zinc compounds have an unique anabolic effect on bone metabolism in the femoral-metaphyseal tissues of rats with skeletal unloading. Zinc-chelating dipeptide may stimulate bone protein synthesis through the mechanism that is involved in protein kinases.

Histomorphological confirmation of bone loss in the femoral-metaphyseal tissues of rats with skeletal unloading.

The alteration of mineral content in the femoral metaphysis of rats with skeletal unloading was investigated using a model of hindlimb suspension. Animals were fed for 4 days during the unloading. The femoral length, femoral dry weight and femoral mineral density were significantly decreased by the unloading. The calcium, phosphorus and zinc contents in the femoral metaphysis were appreciably reduced by the unloading, although, except for zinc, similar decreases were not seen in the femoral diaphysis. Moreover, the trabecular bone at the femoral metaphysis was clearly reduced by the unloading. Skeletal unloading caused a decrease in osteoid tissue in the primary and secondary spongiosa, indicating that osteoblastic bone formation may be inhibited. The present results clearly demonstrate that skeletal unloading can induce bone loss in the femoral metaphysis.

Effect of vitamin K2 (menaquinone-7) on bone metabolism in the femoral-metaphyseal tissues of normal and skeletal-unloaded rats: enhancement with zinc.

The effect of vitamin K2 (menaquinone-7) on bone metabolism in the femoral-metaphyseal tissues of normal and skeletal-unloaded rats was investigated. Skeletal unloading was designed using a model of hindlimb suspension; the rats were fed for the 4 days of unloading. The metaphyseal tissues obtained from normal and skeletal-unloaded rats were cultured for 48 h in medium containing either vehicle or vitamin K2 (10(-6) and 10(-5) M). The presence of vitamin K2 (10(-5) M) caused a significant increase in alkaline phosphatase activity and calcium content in the metaphyseal tissues from normal rats. Such an effect was not seen in the bone tissues from skeletal-unloaded rats. Additionally, the presence of zinc sulfate (10(-5) M) in effective concentration produced a significant increase in alkaline phosphatase activity and calcium content in the metaphyseal tissues from normal and skeletal-unloaded rats. In the presence of vitamin K2 (10(-5) M), the stimulatory effect of zinc sulfate on bone calcium content was appreciably enhanced; although this effect was completely abolished by cycloheximide (10(-6) M), an inhibitor of protein synthesis. This study demonstrates that the effect of vitamin K2 (menaquinone-7) on trabecular bone calcification in rats with skeletal unloading-induced osteopenia is enhanced by zinc in vitro. The enhancement with zinc may be based on a newly synthesized protein in the bone tissues.

Effect of maximal arm exercise on skin blood flux in the paralyzed lower limbs in persons with spinal cord injury.

The purpose of the present study was to examine the effect of maximal arm exercise on the skin blood circulation of the paralyzed lower limbs in persons with spinal cord injury (PSCI). Eight male PSCI with complete lesions located between T3 and L1 performed graded maximal arm-cranking exercise (MACE) to exhaustion. The skin blood flux at the thigh (SBFT) and that at the calf (SBFC) were monitored using laser-Doppler flowmeter at rest and for 15 s immediately after the MACE. The subject's mean peak oxygen uptake and peak heart rate was 1.41 +/- 0.22 l.min-1 and 171.6 +/- 19.2 beats.min-1, respectively. No PSCI showed any increase in either SBFT or SBFC after the MACE, when compared with the values at rest. These results suggest that the blood circulation of the skin in the paralyzed lower limbs in PSCI is unaffected by the MACE.

Cardiovascular and respiratory responses to passive leg cycle exercise in people with spinal cord injuries.

The purpose of this study was to determine the effect of passive leg cycle exercise (PLE) on cardiovascular and respiratory responses in people with spinal cord injuries (PSCI). Eight PSCI with lesions from T8 to L1 and five control subjects (CS) performed PLE at pedalling frequencies of 20 or 40 rpm for 7 min at room temperature of about 25 degrees C. We measured, at rest and during PLE, the pulmonary ventilation (VE), oxygen uptake (VO2), cardiac output (Q), stroke volume (SV), heart rate (HR) and arterial blood pressure, as well as the skin blood flow (SBF) in the lower limb after PLE. An increase in pedalling frequency promoted an increase in VE and VO2 in both groups. Compared with the CS, the PSCI showed significantly smaller increases in VO2 (P < 0.05). The Qc was significantly elevated during PLE at 20 and 40 rpm in CS, and at 40 rpm in PSCI (P < 0.05). In CS, it resulted from increases in both SV and HR, whereas in PSCI, it was contributed to by a greater increase in SV without a rise in HR. In CS, the increase in pedalling frequency promoted the increases in SV and HR and consequently in Qc. In PSCI, however, the values remained constant irrespective of pedalling frequency. The arterial blood pressure and SBF in the lower limbs were unchanged by PLE in both groups. These results would suggest that passive leg exercise promotes venous return from the paralyzed lower limbs in PSCI.

Zinc decrease and bone metabolism in the femoral-metaphyseal tissues of rats with skeletal unloading.

Whether the decrease of zinc content in the femoral-metaphyseal tissues of rats with skeletal unloading is involved in the alteration of bone metabolism was investigated. Skeletal unloading was designed using the model of hindlimb suspension in rats. Animals were fed for 4 days with the unloading. The metaphyseal zinc content were significantly decreased by the unloading. Zinc accumulation in the metaphyseal tissues by a single oral administration of zinc sulfate (20 mg Zn/100 g body weight) was partially depressed by the unloading, although serum zinc concentration was higher than that in normal rats, suggesting an impaired movement of zinc from serum into bone tissues by the unloading. Skeletal unloading caused a significant decrease of alkaline phosphatase activity and deoxyribonucleic acid (DNA) content in the metaphyseal tissues. These decreases were completely restored by addition of zinc sulfate (10(-4) M) or beta-alanyl-L-histidinato zinc (AHZ; 10(-5) M) in a culture medium with the metaphyseal tissues in vitro. The effects of zinc compounds were abolished by the presence of cycloheximide (10(-8) M), suggesting that the zinc effect is based on a newly synthesized protein. Dipicolinate (10(-4) and 10(-5) M), a potent zinc-chelating agent, caused an appreciable decrease of zinc content and alkaline phosphatase activity in the metaphyseal tissues. This decrease was restored by zinc supplement. The present results suggest that the skeletal unloading-induced decrease of zinc content in the femoral-metaphyseal tissues plays a role in the deterioration of bone metabolism in the unloaded rats.

Severe chlorotic spot symptoms in cucumber mosaic virus strain Y-infected tobaccos are induced by a combination of the virus coat protein gene and two host recessive genes.

To investigate the genetic determinants of severe chlorotic spot symptoms in cucumber mosaic virus (CMV[Y])-inoculated tobacco (Nicotiana tabacum 'Ky57') leaves, symptom expression was compared among tobaccos (N. tabacum 'Ky57', N. tabacum 'Xanthi nc', and their hybrids) inoculated with CMV(Y), CMV(O), or pseudorecombinants of both strains of CMV. Xanthi nc showed very mild chlorotic spot symptoms in CMV(Y)-inoculated leaves. Both CMV(O)-inoculated Ky57 and Xanthi nc leaves showed no symptoms. In pseudorecombinant-inoculated Ky57 leaves, severe symptoms were determined by chimeric CMV(O) and CMV(Y) RNA3 transcripts, which contained most of the coat protein gene of CMV(Y). Genetic analysis of severe symptom formation in CMV(Y) or pseudorecombinant-inoculated tobaccos indicated that two recessive nuclear-coded host genes determined severe symptom formation in CMV(Y)-inoculated Ky57. The two recessive host genetic loci completely cosegregated with the two recessive burley loci (yb1 and yb2 genes) of Ky57, whose character is an apparent reduction in chlorophyll of the stems. When several cultivars of N. tabacum were inoculated with CMV(Y), all burley tobaccos clearly showed severe symptoms, but most other cultivars showed very mild symptoms. These results suggest that severe chlorotic spot symptom formation in CMV(Y)-inoculated Ky57 is induced by a combination of the virus coat protein gene and two recessive host genes that are closely linked to or identical to yb genes.