Escherichia coli ribosome-associated protein SRA, whose copy number increases during stationary phase.

Protein D has previously been demonstrated to be associated with Escherichia coli ribosomes by the radical-free and highly reducing method of two-dimensional polyacrylamide gel electrophoresis. In this study, we show that protein D is exclusively present in the 30S ribosomal subunit and that its gene is located at 33.6 min on the E. coli genetic map, between ompC and sfcA. The gene consists of 45 codons, coding for a protein of 5,096 Da. The copy number of protein D per ribosomal particle varied during growth and increased from 0.1 in the exponential phase to 0.4 in the stationary phase. For these reasons, protein D was named SRA (stationary-phase-induced ribosome-associated) protein and its gene was named sra. The amount of SRA protein within the cell was found to be controlled mainly at the transcriptional level: its transcription increased rapidly upon entry into the stationary phase and was partly dependent on an alternative sigma factor (sigma S). In addition, global regulators, such as factor inversion stimulation (FIS), integration host factor (IHF), cyclic AMP, and ppGpp, were found to play a role either directly or indirectly in the transcription of sra in the stationary phase.

Dietary diacylglycerol suppresses high fat and high sucrose diet-induced body fat accumulation in C57BL/6J mice.

Diacylglycerol (DG) comprises up to approximately 10% of various edible oils. In the present study, we examined the effects of dietary DG consisting mainly of 1,3-species on body weight, body fat accumulation, and mRNA levels of various genes involved in energy homeostasis in obesity-prone C57BL/6J mice. Five-month feeding with the high triacylglycerol (TG) diet (30% TG + 13% sucrose) resulted in significant increases in body weight, visceral fat accumulation, and circulating insulin and leptin levels compared with mice fed the control diet (5% TG). Compared with mice fed the high TG diet, body weight gain and visceral fat weight were reduced by 70% and 79%, respectively, in those fed the high DG diet (30% DG + 13% sucrose). In addition, circulating leptin and insulin levels were reduced to the respective control levels. Compared with high TG feeding, high DG feeding suppressed the elevation of leptin mRNA expression in adipose tissue, and up-regulated acyl-coenzyme (Co)A oxidase and acyl-CoA synthase mRNA expression in the liver. These results indicate that dietary DG is beneficial for suppression of high fat diet-induced body fat accumulation. Furthermore, it is suggested that structural differences in DG and TG, but not the composition of fatty acid, markedly affect nutritional behavior of lipids. -- Murase, T., T. Mizuno, T. Omachi, K. Onizawa, Y. Komine, H. Kondo, T. Hase, and I. Tokimitsu. Dietary diacylglycerol suppresses high fat and high sucrose diet-induced body fat accumulation in C57BL/6J mice. J. Lipid Res. 2001. 42: 372--378.

Expression of Hoxb13 and Hoxc10 in developing and regenerating Axolotl limbs and tails.

The expression of Hox complex genes in correct spatial and temporal order is critical to patterning of the body axis and limbs during embryonic development. In order to understand the role such genes play in appendage regeneration, we have compared the expression of two 5' Hox complex genes: Hoxb13 and Hoxc10 during development and regeneration of the body axis and the limbs of axolotls. In contrast to higher vertebrates, Hoxb13 is expressed not only in the tip of the developing tail, but also in the distal mesenchyme of developing hind limbs, and at low levels in developing forelimbs. Hoxc10 is expressed as two transcripts during both development and regeneration. The short transcript (Hoxc10S) is expressed in the tip of the developing tail, in developing hind limbs, and at low levels in developing forelimbs. The long transcript (Hoxc10L) is expressed in a similar pattern, with the exception that no expression in developing forelimbs could be detected. Hoxb13 and both transcripts of Hoxc10 are expressed at high levels in the regenerating spinal cord during tail regeneration, and in both regenerating hind limbs and forelimbs. The up-regulation of expression of these genes during forelimb regeneration, relative to the very low levels of expression during forelimb development, suggests that they play a critical and perhaps unique role in regeneration. This is particularly true for Hoxc10L, which is not expressed during forelimb development, but is expressed during forelimb regeneration; thus making it the first truly "regeneration-specific" gene transcript identified to date.

[Giant-cell myocarditis without a symptom of heart failure seen in a patient with myasthenia gravis and concurrent Hashimoto's disease].

A 69-year-old Japanese woman initially noticed difficulty in squatting in the last two years, followed by nasal voice, fatiguability in mastication, and blepharoptosis. On admission to our hospital, in addition to these neurological findings, we detected ectopic arrhythmia and Levine II systolic murmur at the apex region, without any subjective symptoms. The serum titer of anti-acetylcholine receptor antibody was elevated to 28 nmol/l (normal: < 0.2), and she responded to repetitive nerve stimulation at a frequency of 3 Hz showing 13% waning, she had positive test for edrophonium administration, and was diagnosed as having myasthenia gravis (MG). There was no thymoma by radiographic examination. She also had Hashimoto's disease confirmed by the laboratory findings. Chest X-p revealed dilatation of the heart, and 24-hour Holter ECG revealed non-sustained ventricular tachcardia (VT). Ventriculography revealed prominent dilatation of the left ventricle and diffuse hypokinetic ventricular wall motility. Endomyocardial biopsy revealed muscle fiber degeneration, cellular infiltration, and scattered multinucleated giant cells, confirming a diagnosis of giant cell myocarditis (GCM). Quadriceps muscle biopsy revealed a small number of muscle fibers with giant nuclei, but no giant cells were seen. Immunological study revealed elevation of CD4/CD8 ratio and memory CD4 cells. Antibody to anti-cardiac and anti-striate muscle were strongly positive in the serum. Four months later, she developed dyspnea on effort and hypoxia, accompanied by severe bradycardia leading to sinus arrest. For acute cardiac deterioration, steroid pulse therapy was started followed by oral predonisolone and azathioprine, which aggravated myasthenic symptoms. The patient was ventilated for respiratory hypercapnia. During immunoabsobent therapy, she developed VT which caused a cardiac arrest, leading to fatal outcome. In case of MG, especially overlapped with other autoimmune diseases, evaluation of cardiac function should be conducted to detect GCM.

How can Ca2+ selectively activate recoverin in the presence of Mg2+? Surface plasmon resonance and FT-IR spectroscopic studies.

We investigated the relationship between metal ion selective conformational changes of recoverin and its metal-bound coordination structures. Recoverin is a 23 kDa heterogeneously myristoylated Ca(2+)-binding protein that inhibits rhodopsin kinase. Upon accommodating two Ca(2+) ions, recoverin extrudes a myristoyl group and associates with the lipid bilayer membrane, which was monitored by the surface plasmon resonance (SPR) technique. Large changes in SPR signals were observed for Sr(2+), Ba(2+), Cd(2+), and Mn(2+) as well as Ca(2+), indicating that upon binding to these ions, recoverin underwent a large conformational change to extrude the myristoyl group, and thereby interacted with lipid membranes. In contrast, no SPR signal was induced by Mg(2+), confirming that even though it accommodates two Mg(2+) ions, recoverin does not induce the large conformational change. To investigate the coordination structures of metal-bound Ca(2+) binding sites, FT-IR studies were performed. The EF-hands, Ca(2+)-binding regions each comprising 12 residues, arrange to coordinate Ca(2+) with seven oxygen ligands, two of which are provided by a conserved bidentate Glu at the 12th relative position in the EF-hand. FT-IR analysis confirmed that Sr(2+), Ba(2+), Cd(2+), and Mn(2+) were coordinated to COO(-) of Glu by a bidentate state as well as Ca(2+), while coordination of COO(-) with Mg(2+) was a pseudobridging state with six-coordinate geometry. These SPR and FT-IR results taken together reveal that metal ions with seven-coordinate geometry in the EF-hands induce a large conformational change in recoverin so that it extrudes the myristoyl group, while metal ions with six-coordinate geometry in the EF-hands such as Mg(2+) remain the myristoyl group sequestered in recoverin.

Preserved sympathetic skin response at the distal phalanx in patients with carpal tunnel syndrome.

OBJECTIVE: To assess the viability of sympathetic sudomotor fibers in carpal tunnel syndrome (CTS). METHODS: We recorded sympathetic skin response (SSR) with a multichannel recording system. Forty-four patients with CTS (51 hands), 7 patients (7 hands) with asymptomatic median mononeuropathy at the wrist (MMW), and 20 normal subjects (20 hands) were studied. We classified the patients into 4 grades of increasing severity. RESULTS: In the hands of normal subjects, SSR was evoked easily at all recorded sites. SSR at the wrist in patients with asymptomatic MMW decreased in amplitude. SSR was markedly distorted at the wrist in severe grades of CTS. The SSR amplitude ratio (wrist/distal phalanx) decreased significantly with more severe grade (rh=-0.4; P<0.05), but the sensitivity was lower than that of other electrodiagnostic criteria. A patient with persistent allodynia at the wrist after surgery showed the slight recovery of SSR amplitude ratio; the other two patients without allodynia showed substantial recovery of SSR amplitude ratio within 24 weeks after surgery. CONCLUSION: SSR amplitude ratio is a poor indicator of CTS diagnosis, but may be useful in assessing the viability of sympathetic sudomotor fibers and may assist in evaluating the response to surgery.

Vacuolar granules in Chlamydomonas reinhardtii: polyphosphate and a 70-kDa polypeptide as major components.

The alga Chlamydomonas reinhardtii contains cytoplasmic vacuoles that are often filled with a dense granule that is released from the cell by exocytosis. Purified granules contained polyphosphate, complexed with calcium and magnesium, as the predominant inorganic components. Antiserum was raised against the major 70-kDa protein in granules purified from wall-deficient (cw15) mutants, which reacted on immunoblots with larger glycoprotein complexes in purified cell wall fractions from wild-type cells. Confocal fluorescence microscopy detected binding of these antibodies predominantly at the periphery of wall-containing C. reinhardtii y1 cells but primarily to loci in the interior of cells of the cw15 strain. Immunoelectron microscopy demonstrated that the 70-kDa protein was localized in vacuolar granules and the trans-Golgi network in sections of cw15 cells but not in the cytosol or chloroplast. Treatment of cells with a dye, fluorescent in its protonated form, indicated that the pH within vacuoles was lower than that in the cytosol, which suggested that the vacuoles are similar to lysosomes. Thus, the vacuoles may serve a dual function to provide an environment for degradation within the cell and also serve as a vehicle for secretion of specific proteins.

Polyadenylation of three classes of chloroplast RNA in Chlamydomonas reinhadtii.

Three classes of RNA, represented by atpB and petD mRNAs, Arg and Glu tRNAs, and 5S rRNA, were found to exist in polyadenylated form in Chlamydomonas reinhardtii chloroplasts. Sequence analysis of cDNA clones derived from reverse transcriptase-polymerase chain reaction protocols used to select polyadenylated RNAs revealed that, at least for the mRNAs and tRNAs, there are three apparent types of polyadenylation. In the first case, the poly(A) tail is added at or near the mature 3' end, even when this follows a strong secondary structure. In the second case, the tail is added to pre-mRNA or pre-tRNA, suggesting a possible competition between polyadenylation and RNA-processing pathways. Finally, in all cases, the poly(A) tail can be added internally, possibly as a part of an RNA-decay pathway. The tails found in Chlamydomonas chloroplasts differ from those of spinach chloroplasts in adenine content, being nearly homopolymeric (>98% adenine) versus 70% in spinach, and are similar in length to those of Escherichia coli, being mostly between 20 and 50 nt. In vitro assays using a Chlamydomonas chloroplast protein extract showed that a 3' end A25 tail was sufficient to stimulate rapid degradation of atpB RNA in vitro, with a lesser effect for petD, and only minor effects on trnE. We therefore propose that polyadenylation contributes to mRNA degradation in Chlamydomonas chloroplasts, but that its effect may vary.

Predominant subpopulations of T lymphocytes in the mammary gland secretions during lactation and intraepithelial T lymphocytes in the intestine of dairy cows.

Lymphocytes obtained from mammary gland secretions (MGS) during lactation or the dry period of dairy cows were simultaneously analyzed and compared to ileal intraepithelial lymphocytes (IEL) and peripheral blood lymphocytes (PBL) using monoclonal antibodies (mAb) specific for bovine leukocyte differentiation antigens. The T-lymphocytes of MGS during lactation and those in IEL were predominantly CD8(+), while T-cells in MGS during the dry period were predominantly CD4(+). In addition, the proportion of gamma delta T-cells in MGS during lactation and IEL was fairly high. A large percentage of CD8(+) cells and T-cells coexpressed the activation molecule, ACT2, yielding a high proportion of ACT2(+) CD8 T-cells and ACT2(+) gamma delta T-cells, in MGS during lactation and IEL. However, both types of cells were found at an extremely low level in MGS during the dry period and in PBL. Thus, the predominant T-cell populations in MGS during lactation are phenotypically similar to those in IEL in the intestine.

Standard radial bone mineral density and physical factors in ordinary Japanese women.

To prevent osteoporosis, which is expected to increase in incidence in this rapidly aging society, in recent years bone mineral density (BMD) has frequently been measured as a predisposition index. However, these measurements are made on different sites with different apparatus, and the results are independently studied by different institutions. In our present investigation, to establish the standard radius BMD as determined by dual-energy X-ray absorptiometry (DXA), we carried out a general population survey in 29 municipalities and prefectures on 11,252 locally residing females aged 15 to 83 years (mean, 35.61 +/- 12.85 years). Their YAM (young adult mean) BMD was estimated at 0.664 +/- 0.054 g/cm2, which was almost the same as the figure given in the 1996 version of the diagnostic criteria for primary osteoporosis. We further studied the relationships of BMD to age and physical factors known to be influential to BMD. It was found that BMD was correlated negatively to age and positively to body mass index (BMI). The average values we obtained for age and physique groups appeared to have provided reliable indices for the primary prevention of osteoporosis.

Localized scleroderma associated with progressing ischemic stroke.

We present a 73 year-old Japanese woman with localized scleroderma involving the right side of the scalp accompanied by continuous tingling pain, who developed insidiously progressive left hemiparesis. In magnetic resonance imaging of the brain, an infarct first appeared in the watershed region of the right middle cerebral artery territory and subsequently extended to deep white matter accompanied by scattered hemorrhages. Focal stenosis in the M2 portion of the right middle cerebral artery was revealed on magnetic resonance angiography, and the distal vessels were only shown faintly. A biopsy specimen from the sclerotic scalp lesion showed obvious thickening of vessel walls and mild mononuclear cell infiltration. We believe that the progressing ischemic stroke was caused by hemodynamic disturbances from localized sclerotic obstruction of the middle cerebral artery, with an autoimmune pathogenesis.

A novel type of non-coding RNA expressed in the rat brain.

We have characterized a novel type of non-coding RNA which consists of tandem repeats of similar sequences, approximately 0.9 kb in size. This RNA, termed Bsr (brain specific repetitive) RNA, is encoded at a single locus (6 q31-->q32) in the rat genome, where 100 to 150 copies of the 0.9 kb sequences are repeated in tandem. Bsr RNA is preferentially expressed in the rat central nervous system (CNS), especially in phylogenetically old structures, such as the pareo- and archicortex, amygdala, thalamus and hypothalamus. In the developing brains, Bsr RNA is expressed in the subsets of differentiating cells but not in proliferating cells. Despite the finding that Bsr RNA appears to be conserved only among the Rattus species, the specific expression pattern of Bsr RNA suggests that it might have some role in the rat CNS.

Atypical rigid form of Huntington's disease: a case with peripheral amyotrophy and congenital defects of a lower limb.

We describe a patient showing an atypical phenotype of Huntington's disease (HD), including prominent generalized dystonia, peripheral amyotrophy of the legs with an inverted champagne bottle configuration and pes equinus. The patient also had congenital defects of the lower left leg. Chorea and psychiatric symptoms were not prominent. Polymerase chain reaction assessment revealed 51 CAG repeats in gene IT 15. Magnetic resonance imaging of the brain demonstrated mild atrophy of the pons and cerebellum, and hyperintensity of the transverse pontine fibers and neostriatum on spin-echo images. Peripheral amyotrophy in this case might have resulted from axonal degeneration related to neuronal damage in the central nervous system, although at the present time we cannot confirm it as a new HD phenotype.

Sympathetic skin responses of the hand in normal subjects: shorter latency at distal phalanx.

We studied the conduction of the sympathetic skin response (SSR) in the hands of 35 normal subjects by simultaneous recording at five sites. The mean latency of the SSR in all subjects increased from the wrist (W) to the middle phalanx (M), but the SSR latency at the distal phalanx (D) was shorter than that at the middle phalanx. The mean conduction time and conduction velocity from W to M was 197.5 ms and 0.87 m/s, respectively, and that from W to D was 48.8 ms and 2.34 m/s, respectively. For evaluation of the cause of the shorter latency at D, digital nerve blocking was performed in two subjects. The blocking of the volar digital nerves at the proximal phalanx abolished SSR at M and D. It was postulated that the initiation or conduction of sudomotor nerve impulse to the distal phalanx would be facilitated compared with those to the other proximal sites in the hand. The SSR conduction time between W and M may be a means of detecting alteration of sympathetic sudomotor nerve activity.

Solitary colonic metastasis of renal cell carcinoma seven years after nephrectomy: a case report.

An 83-year-old man, who had undergone right radical nephrectomy for renal cell carcinoma about 7 years previously, experienced melena and abdominal mass. Barium enema, colonoscopy, computed tomography, and arteriography showed a hypervascular mass on the transverse colon, and a partial transverse colectomy was performed. The postoperative histologic examination revealed that the tumor was a metastatic clear cell carcinoma.

10Sa RNA is associated with 70S ribosome particles in Escherichia coli.

The intracellular distribution of 10Sa RNA in Escherichia coli was investigated in cell extracts. Northern hybridization revealed that a large fraction of 10Sa RNA cosediments with 70S ribosomes. When 70S ribosomes were dissociated into 50S and 30S subunits in the presence of low levels of Mg2+ ions, almost all of the 10Sa RNA disappeared from both subunits. The extent of the association of the 10Sa RNA with ribosomes was much enhanced during the growth phase of the cells. These results suggest the possibility that 10Sa RNA might function on the ribosomes in E. coli cells.

Characterization of vitronectins in atherosclerotic lesions.

Vitronectin is one of the major extracellular matrix proteins that accumulates in atherosclerotic lesions. A monoclonal antibody (EMR1a/212D) specifically stained the extracellular regions in thickened intima which colocalized well with lipid deposition. The antigenic glycoprotein with a molecular weight of 66KDa was revealed to be rabbit vitronectin. When homogenates of WHHL rabbit atheroma were subjected to immunoblot analysis using EMR1a/212D, four molecules with molecular weight 66, 56, 50 and 47KDa were detected. To confirm whether these smaller immunopositive bands were derived from mature vitronectin, another monoclonal antibody (EMR1b/244H) recognizing the polypeptide region of vitronectin was prepared. All four molecules were detected by EMR1b/244H as well as by EMR1a/212D. Two smaller vitronectins (56KDa and 50KDa) were found in atherosclerotic lesions and increased markedly during the development of atherosclerosis. On the other hand, the vitronectin detected in normal rabbit aorta was mainly of the mature type, while 56KDa and 47KDa forms were not detected. The total amount of the four vitronectins in atherosclerotic lesions was 38.5 +/- 5.0 ng/mg wet weight tissue, a value approximately 9.5 fold higher than that found in normal aorta. In conclusions, we found massive accumulation of these vitronectins concomitant with atherosclerotic development in rabbit aorta.

Localization of light-harvesting complex apoproteins in the chloroplast and cytoplasm during greening ofChlamydomonas reinhardtii at 38°C.

Assembly of the major light-harvesting complex (LHC II) and development of photosynthetic function were examined during the initial phase of thylakoid biogenesis inChlamydomonas reinhardtii cells at 38°C. Continuous monitoring of LHC II fluorescence showed that these processes were initiated immediately upon exposure of cells to light. However, mature-size apoproteins of LHC II (Lhcb) increased in amount in an alkali-soluble (non-membrane) fraction in parallel with the increase in the membrane fraction. Alkali-soluble Lhcb were not integrated into membranes when protein synthesis was inhibited, suggesting that they were not active intermediates in LHC II assembly, nor were they recovered in a purified chloroplast preparation. Immunocytochemical analysis of greening cells revealed Lhcb inside the chloroplast near the envelope and in clusters deeper in the organelle. Antibody binding also detected Lhcb in granules within vacuoles in the cytosol, and Lhcb were recovered in granules purified from greening cells. Our results suggest that the cytosolic granules serve as receptacles of Lhcb synthesized in excess of the amount that can be accommodated by thylakoid membrane formation within the plastid envelope.

[Two siblings with adult-onset sialidosis type I (cherry-red spot-myoclonus syndrome)].

Cases of two Japanese siblings with adult-onset sialidosis type I are reported. A 38-year-old man had gradually developed involuntary movement of the extremities from the age of 31. On admission, he had no skeletal abnormalities and hepatosplenomegaly, but showed myoclonus of the extremities and dyskinesia in the perioral region. We found cherry-red spots and a giant potential in a somatosensory evoked potential (SEP) study. Then, the diagnosis of sialidosis type I was confirmed by low activity of white blood cell sialidase. MRI (SE, TR 2,000/TE 100, 40) of the brain revealed a small high intensity are in the cerebral white matter adjacent to the posterior horn of the right cerebral ventricle. To our knowledge, no report on MRI findings of the brain in sialidosis type I has been reported. So far, it is uncertain whether or not such a lesion is caused by sialidosis. He was treated with clonazepam, sodium valproate, diphenylhydantoin, or haloperidol. The former two improved the symptoms, but SEP findings did not change. The subject's 43-year-old brother had also myoclonus and epilepsy since the age of 31, and low activity of sialidase. Their mother had no symptoms, but her sialidase activity level was as low as that of a carrier. These two are the eighth and ninth cases of sialidosis type I in Japan to be confirmed by enzyme activity.