Sarcopenic obesity is associated with a faster decline in renal function in people with type 2 diabetes.

AIM: To evaluate the association between sarcopenic obesity and the decline in estimated GFR in people with type 2 diabetes. METHODS: We enrolled 745 people with type 2 diabetes (mean age 64.6 years, 53.6% men). Body composition was evaluated using dual-energy X-ray absorptiometry. Skeletal muscle index, calculated as appendicular non-fat mass (kg) divided by height squared (m2 ), was used to determine sarcopenia. Sarcopenic obesity was defined as the coexistence of sarcopenia and a ratio of android to gynoid fat mass greater than the median values in each gender. The association of sarcopenic obesity both with the annual rate of decline in estimated GFR and a >30% decline in estimated GFR was evaluated using multivariate linear regression models and Cox proportional hazard models, respectively. RESULTS: Participants with sarcopenic obesity were at an increased risk of a high annual rate of decline in estimated GFR, even after adjustment for the confounding variables (standardized ß = -0.228, P <0.001). Sarcopenic obesity was also significantly associated with risk of a >30% decline in estimated GFR (hazard ratio 4.52, 95% CI 2.16-9.47; P < 0.01) in multivariate model. CONCLUSIONS: Sarcopenic obesity evaluated by dual energy X-ray absorptiometry is associated with a faster decline in renal function in people with type 2 diabetes.

Insulin receptor-related receptor messenger ribonucleic acid in the stomach is focally expressed in the enterochromaffin-like cells.

The insulin receptor-related receptor (IRR) is a member of the insulin receptor family. In contrast to the widespread expression of insulin receptor and insulin-like growth factor-I receptor messenger RNA (mRNA), the expression of IRR mRNA is highly restricted to the kidney and stomach. IRR mRNA in the kidney is focally expressed in the renal distal tubule cells. However, the cellular localization of IRR mRNA in the stomach remains to be elucidated. Here, we examined the cellular localization of IRR mRNA in the rat stomach by in situ hybridization. IRR mRNA in the stomach was abundantly localized in the basal third of the oxyntic glands of the fundic stomach. IRR mRNA in the stomach was colocalized with mRNA for histidine decarboxylase, a marker for the enterochromaffin-like (ECL) cells, indicating that the expression was restricted to ECL cells. ECL cells actively produce and store histamine, which is an important physiological stimulant of acid secretion from the parietal cells. The preferential localization of IRR mRNA in ECL cells suggests that the IRR plays an important role in the function of these cells.

Inhibition of glucocorticoid-induced apoptosis by the expression of antisense gene of mitochondrial ATPase subunit 6(1).

To isolate the apoptosis-linked genes involved in the cell death of thymocytes induced by glucocorticoids, we developed a functional cloning assay. Murine CD4(+)CD8(+) thymic cell line 2-257-20 cells were transfected with cDNA expression libraries obtained from a dexamethasone-resistant cell line. The transfected cells were selected in the presence of dexamethasone, and the plasmids which episomally expanded were then extracted from the surviving cells. One of the rescued cDNAs was found to be an antisense cDNA fragment identical to the mouse mitochondrial ATPase 6 gene. In the stable transfectants with the ATPase 6 antisense gene, the induction of apoptosis by dexamethasone was significantly delayed. Furthermore, the ATP synthesis in these transfectants was also reduced to some extent. ATPase 6 is a subunit of F(o)F(1) ATPase and our results support that ATP synthesis from the mitochondria is necessary for the induction of apoptosis induced by glucocorticoids.

Neurotransmitters of the hypothalamic suprachiasmatic nucleus: immunocytochemical analysis of 25 neuronal antigens.

An immunocytochemical analysis with 33 antisera was undertaken to investigate the localization of 25 different neurotransmitter-related antigens in the hypothalamic suprachiasmatic nucleus in the rat. To obtain estimates of relative densities of immunoreactive axons a stereological approach was used involving counting of intersections of immunoreactive axons with a superimposed semi-circle test grid. All neurotransmitter-related antigens found in perikarya within the suprachiasmatic nucleus, including those stained with antisera against bombesin, gastrin-releasing peptide, neurophysin, vasopressin, somatostatin, gamma-aminobutyrate, glutamate decarboxylase and vasoactive intestinal polypeptide were also found in axons within the nucleus. A greater number of these immunoreactive axons was found within the nucleus than in the adjacent anterior hypothalamus. The size of all immunoreactive axons in the suprachiasmatic nucleus was consistently small; immunoreactive axons were found ramifying widely in the nucleus, often ending with terminal boutons near perikarya immunoreactive for the same antigen. All neurotransmitter-related substances found in perikarya of the suprachiasmatic nucleus were also found in axons crossing over the midline to innervate the contralateral nucleus, providing an anatomical substrate for a high degree of communication between the paired nuclei. Axons immunoreactive for other putative transmitters including serotonin arising outside the nucleus were also found in high densities within the nucleus and crossing over the midline between the nuclei. Immunoreactivity for some transmitters was found in axons of similar densities within and outside the nucleus, including antisera against tyrosine hydroxylase; a small number of dopamine beta-hydroxylase and a few phenylethanolamine N-methyltransferase-immunoreactive axons were found in the SCN, suggesting that dopamine, norepinephrine and epinephrine may occur in a limited number of axons in the nucleus. Small numbers of axons immunoreactive with antisera raised against cholecystokinin, prolactin, substance P, thyrotropin-releasing hormone and choline acetyltransferase were found within the suprachiasmatic nucleus. Axons immunoreactive for luteinizing hormone-releasing hormone, adrenocorticotropic hormone, alpha-melanocyte-stimulating hormone and neurotensin were rarely found within the suprachiasmatic nucleus; axons immunoreactive for luteinizing hormone-releasing hormone, adrenocorticotropic hormone, cholecystokinin and tyrosine hydroxylase were found in both horizontal and coronal sections in the area between the left and right suprachiasmatic nuclei.(ABSTRACT TRUNCATED AT 400 WORDS)

Cancer chemopreventive agents, serratane-type triterpenoids from Picea jezoensis.

Seven serratane-type triterpenoids isolated from the cuticle of Picea jezoensis (Sieb. et Zucc.) Carr. jezoensis (Pinaceae) and the stem bark of Picea jezoensis (Sieb. et Zucc.) Carr. hondoensis (Mayer) Rehder (Pinaceae) were studied their possible inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). All compounds showed strong inhibitory effects on the EBV-EA activation, being stronger than that of oleanolic acid, which exerts on cancer preventive activity in animal carcinogenesis models. Among these compounds, 13alpha, 14alpha-epoxy-3beta-methoxyserratan-21beta-ol and 3beta-methoxy-21alpha-hydroxyserrat-14-en-29-al were investigated for the inhibitory effects in a two-stage mouse skin carcinogenesis test on mouse skin using 7,12-dimethylbenz[a]anthracene as initiator and TPA as promoter. 13alpha,14alpha-Epoxy-3beta-methoxyserratan-21beta-ol was found to exhibit the excellent anti-tumor promoting activity in the in vivo carcinogenesis test.

Expression of insulin receptor-related receptor in the rat brain examined by in situ hybridization and immunohistochemistry.

Insulin receptor-related receptor (IRR) is a member of the insulin receptor family. However, its endogenous ligand and physiological roles are unknown. To elucidate the physiological roles of IRR, an orphan receptor, in the brain, we examined its expression at mRNA and protein levels in the brain by in situ hybridization and immunohistochemistry, respectively. The expression of IRR mRNA in the brain was highly restricted to the forebrain including the nucleus of the diagonal band, medial septal nucleus, ventral pallidum, accumbens nucleus and caudate putamen, and the brainstem including the prepositus hypoglossal nucleus, medial vestibular nucleus, gigantocell reticular nucleus, paragigantocellular nucleus and ventral cochlear nucleus. Most IRR mRNA-positive cells in the forebrain but not in the brainstem were cholinergic neurons. However, most IRR mRNA in the forebrain and brainstem was coexpressed with that of trkA, a high-affinity receptor for nerve growth factor. IRR-immunoreactive cell bodies were also detected in the forebrain and brainstem. The pattern of IRR immunoreactivity was similar to that of IRR mRNA. Its restricted pattern indicates that IRR plays unique roles in the brain, in contrast to insulin and insulin-like growth factor-I receptors, other members of the insulin receptor family, which are widely expressed in the brain.

Cryoprotective effect of the serine-rich repetitive sequence in silk protein sericin.

The silk proteins, fibroin and sericin, are produced in the silk gland of Bombyx mori, and hydrophilic sericin envelops fibroin with successive sticky layers in the formation of a cocoon. To study the biological functions of sericin, we focused on the serine-rich sericin peptide consisting of 38 amino acids, which is a highly conserved and internally repetitive sequence of a sericin protein. The corresponding gene was chemically synthesized, and the PCR-amplified gene was ligated to oligomerize sericin peptide and fused at the amino terminus to a His-tagged and proteolytic cleavage sequence in an inducible expression vector. When the dimers of sericin peptides were overexpressed in Escherichia coli, the transformants showed a prominent increase in cell viability after freezing in medium. Further, the purified dimeric sericin peptide from E. coli was found to be effective in protecting lactate dehydrogenase from denaturation caused by freeze-thaw. Both of these protective effects against freezing stress in cells and proteins were also observed with sericin hydrolysate. These results indicate that this unique sericin peptide, like sericin, has a high cryoprotective activity and will be valuable as a new biomaterial for industrial use.

Expression of insulin receptor-related receptor mRNA in the rat brain is highly restricted to forebrain cholinergic neurons.

Insulin receptor-related receptor (IRR) is a member of the insulin receptor family. However, its endogenous ligand and the physiological roles of IRR are unknown. To elucidate the physiological roles of IRR in the brain, we examined the expression of its mRNA in the rat brain by in situ hybridization. In contrast to the widespread expression of insulin receptor and insulin-like growth factor-1 receptor mRNAs in the brain, the expression of IRR mRNA was highly restricted to the forebrain cholinergic neurons. All the forebrain cholinergic neurons expressed IRR mRNA. The present findings indicate that IRR has a selective role in the brain for forebrain cholinergic function.

Distribution of cellular repopulation and collagen synthesis in a canine anterior cruciate ligament autograft.

Whether the central core of an anterior cruciate ligament autograft reconstruction is nutritionally compromised at a time when revascularization is known to be complete has not been determined by methods that detect matrix synthesis. In a canine model of anterior cruciate ligament reconstruction with patellar tendon autograft, the adequacy of the supply of metabolites for cellular matrix synthesis was determined by autoradiographic analysis. Total collagen synthesis and cellularity were also quantified. Total collagen synthesis was found to be significantly elevated (p = 0.014 by analysis of variance) in the ligament reconstructions as compared with normal anterior cruciate ligaments or patellar tendons but cellularity was not (p = 0.13 by analysis of variance). Autoradiography demonstrated even distribution of [3H]proline incorporation throughout the graft and normal tissue. When revascularization was complete there was an adequate supply of metabolites for cellular synthesis of protein macromolecules within all regions of the ligament reconstruction. At 3 months after reconstruction, the grafts were found to be actively remodeling their collagen matrix. Since the long-term function of an anterior cruciate ligament autograft is dependent on viable fibroblasts to maintain the collagen matrix the canine anterior cruciate ligament reconstruction contains living cells that are able to remodel the matrix under appropriate conditions.

The conformational analysis and photoisomerization of retinochrome analogs with polyenals.

3,7-Dimethyl-2,4,6,8,10-dodecapentaenal was synthesized for reconstitution of the retinochrome analog. Its opsin shift was 1000 cm-1 smaller than that of native retinochrome, whose chromophore contains the same number of double bonds. The conformational change from 6-s-trans to 6-s-cis, as figured in a retinal molecule, plays an important role in the formation of the retinochrome analog, based on the estimation of opsin shifts for retinal analogs locked in the 6-s conformation. Thus the conformation of the 6-7 single bond in the native retinochrome was suggested to be 6-s-cis. Analysis of the circular dichroic spectra of retinochrome analogs revealed that the 6-s conformation is independent of the appearance of the beta-band. The stereoselectivity in the photoisomerization of the retinal analogs by a retinochrome template depends on the hydrophobic binding in the region of the beta-ionone ring.

Non-destructive, ultrasonic evaluation of meat quality in live Japanese Black steers from coloured images produced by a new ultrasound scanner.

An improved colour scanning scope was used for evaluating meat quality (marbling) of live Japanese Black steers. This equipment consisted of a small size ultrasonic probe (2 MHZ) and LCD display. Seventeen fattened Japanese Black cattle were scanned at the region of the 7th rib about one week before slaughter. A picture of the cross-sectional area of the back was obtained immediately after applying the probe and contained 15 colours representing different signal strengths. The time for each scan was 2 seconds. The picture signals were fed into a computer for rapid estimation of fat percentage of the M. longissimus thoracis. After slaughter, the fat content and chemical characteristics were determined on the M. longissimus thoracis obtained from the same rib section. The range of fat content was 7.0 to 23.7% (average 18.47%). A high correlation coefficient (r = 0.90; r.s.d. = 2.01%) was obtained between actual fat percentage of the M. longissimus thoracis and colour-scanning scope SR200 estimates based on the percentage of the weak blue dot(1) in the echo. Estimates of the subcutaneous fat thickness and the cross-sectional area of M. longissimus thoracis from the scans were in good agreement with the actual carcass measurements (r = 0.69; r.s.d=0.52 cm and r = 0.81; r.s.d. = 4.26 cm(2), respectively). These results show that the new colour scanning scope is a useful instrument for estimating meat quality (marbling) in live cattle.

Study on the inhibitory effect of uremic plasma on lipoprotein lipase.

An investigation was undertaken to determine which plasma factors from normal controls and patients with chronic renal failure (CRF) exert have inhibitory effects on the activity of lipoprotein lipase (LPL) purified from heparinized human plasma by using an accurate LPL assay system. Inhibitors of LPL were found to be present in the plasma. The inhibition of the LPL activity was significantly greater in CRF patients than in normal controls. Following hemodialysis (HD), the same concentration of uremic plasma led to less inhibition. The inhibitors existed only in lipoprotein deficient plasma (LPDS), which demonstrated an LPL-inhibitory activity at extremely high concentrations with a significant difference between the patients and normal controls. There was no difference between the two groups at low concentrations. A specific inhibitory effect on LPL in LPDS was noted in the 7S and 4S fractions separated by gel filtration employing Sephacryl S-200 column chromatography. The inhibitory effect of the 7S fraction was found to be dependent on the concentration, and the difference between the two groups was similar to that for LPDS. The results obtained in the present study suggest that the plasma from CRF patients exhibited a strong inhibitory action on the LPL activity as compared to the plasma from normal controls, and the inhibitory action was due primarily due to poor excretion of dialyzable inhibitors.

Mutational analysis of the feedback sites of phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase of Escherichia coli.

In Escherichia coli, aroF, aroG, and aroH encode 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase isozymes that are feedback inhibited by tyrosine, phenylalanine, and tryptophan, respectively. In vitro chemical mutagenesis of the cloned aroG gene was used to identify residues and regions of the polypeptide essential for phenylalanine feedback inhibition.

Radiographic and computed tomographic analysis of the position of the tibial tubercle in recurrent dislocation and subluxation of the patella.

Among the factors that influence the patellofemoral alignment and stability, the position of the tibial tubercle was evaluated using plain radiographs and computed tomography (CT). Radiographs of the Q-angle with the knee in full extension and quadriceps muscle relaxed showed that mean Q-angles in the dislocation or subluxation group and control group were 13.8 degrees and 14.3 degrees, respectively. The major reason for this deceptive measurement was a patella positioned laterally in the dislocation or subluxation group. Better estimation was made with the modified Q-angle measurement by using the most concave point of the intercondylar notch as a reference point instead of the center of the patella. By overshadowing CT images of the levels of the patellofemoral joint and the tibial tubercle, it was possible to evaluate the location of the tibial tubercle in relation to the femoral trochlea. Both the angular measurement (lateral deviation angle [LDA]) and the distance measurement (lateral deviation index [LDI]) showed that the tibial tubercle in the dislocation or subluxation group was rotated externally and shifted laterally (LDA, 36.3+/-7.0 degrees and LDI, 30.1+/-5.6) compared with the control group (LDA, 20.2+/-7.1 degrees and LDI, 15.1+/-5.6). Further study is warranted to elucidate the exact mechanism of recurrent patellar dislocation and subluxation.

Intracellular pH of halobacteria can be determined by the fluorescent dye 2', 7'-bis(carboxyethyl)-5(6)-carboxyfluorescein.

Determination of the internal pH of halobacterial cells grown in 4M salt solution has proven to be a difficult problem. We now report the steady state cytosolic pH of Halobacterium halobium S-9 to be 7.2. Intracellular pH was determined after the cells were loaded with the membrane permeable precursor of the pH sensitive dye 2', 7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein-acetyxymethyl ester - (BCECF/AM). In order to minimize light-scattering in the measurement of the fluorescence, a thin cuvette was newly devised. This method should be suitable for studies of the cytosolic pH in other bacteria.

Antibacterial activity of crinitol and its potentiation.

An acyclic diterpene alcohol, crinitol [1], was identified in a marine brown alga, Sargassum tortile, as the principal antibacterial agent against Gram-positive bacteria, among which Propionibacterium acnes was most sensitive and, Staphylococcus aureus was least. To enhance its activity, crinitol was combined with several antioxidants, which presumably retard oxidative destruction of this molecule which possesses two easily oxidizable allylic alcohol groups. Two synthetic antioxidants, BHA (butylated hydroxyanisole) and BHT (butylated hydroxytoluene), significantly enhanced the activity of crinitol, especially against Streptococcus mutans. Interestingly, crinitol also synergized BHT and BHA against this cariogenic bacterium.

Two truncated forms of rat insulin receptor-related receptor.

The insulin receptor-related receptor (IRR) (1271 amino acids) is expected to have unique functions as a novel member of the insulin receptor family. In this paper, we report two alternatively spliced variants of rat IRR mRNA, which are predicted to encode two truncated forms of IRR, sIRR-1 (410 amino acids) and sIRR-2 (469 amino acids). The amino acid sequence of sIRR-1 is identical to the N-terminal 410-amino acid sequence of IRR. sIRR-2 has an additional 59-amino acid insertion in the C-terminal region. Both truncated forms retain the N-terminal and cysteine-rich domains but lack the transmembrane and intracellular tyrosine kinase domains, indicating that the truncated forms are the secreted forms. The translation products of the truncated form mRNAs were detected in the stomach and kidney by Western analysis. However, the physiological significance of the secreted forms remains to be elucidated.

In-beam electron impact mass spectrometry of N-carbobenzoxy derivatives of oligopeptides composed of leucine and isoleucine.

The characteristics of the in-beam electron impact mass spectra of 13 N-carbobenzoxyoligopeptides (up to pentapeptides) composed mainly of leucine and isoleucine, and their alkyl and N-hydroxysuccinimide esters are described. In the cases of di- and tripeptide derivatives, each in-beam electron impact spectrum exhibited an abundant [M + 1]+ peak as well as amino-acyl fragments. However, in the case of pentapeptides the in-beam spectra measured under the routine conditions corresponded to the conventional electron impact spectra of the cyclopeptide derivatives formed by thermal cyclization in the mass spectrometer and few [M + 1]+ ions of the original peptides were observed. From the studies on temperature spectral change of in-beam electron impact spectra it became clear that rapid heating of the sample over the temperature range 200-300 degrees C gave abundant [M + 1]+ ions as well as fragment ions originating from the thermally intact peptide as the major peaks in the spectrum.

Structure and stereochemistry of epoxyserratanes from the cuticle of Piceajezoensis var. jezoensis.

Three new epoxytriterpenes, 14 beta,15 beta-epoxy-21 beta-hydroxyserratan-3-one (1), 13 alpha,14 alpha-epoxy-21 alpha-methoxyserratan-3-one (2), and 13 alpha,14 alpha-epoxy-3 beta-methoxyserratan-21 beta-ol (3), were isolated together with two known triterpenoids, 21 alpha-methoxyserrat-13-en-3-one (4) and 21 beta-hydroxyserrat-14-en-3-one (5), from the cuticle of Picea jezoensis var. jezoensis. The structures of these new compounds were established on the basis of spectral data (NMR, MS) and single-crystal X-ray analyses (1 and 2) and partial synthesis (2 and 3).

Phosphorylation of an inhibitory subunit of cGMP phosphodiesterase in Rana catesbeiana rod photoreceptors. I. Characterization of the phosphorylation.

Interaction between the inhibitory subunit (P gamma) and catalytic subunits of cGMP phosphodiesterase is essential for the regulation of cGMP phosphodiesterase in vertebrate rod photoreceptors. P gamma phosphorylation in vitro has been studied using a kinase which is extracted from amphibian rod outer segments. Various chromatographies of the kinase preparation using ionic exchange, gel filtration, and heparin-Sepharose columns indicate that a kinase with M(r) 70,000 is responsible for the P gamma phosphorylation. The kinase does not require any of the known activators for protein kinases but is inhibited by cGMP in a concentration-dependent manner. Together with analysis by laser-desorption mass spectrometry, measurement of 32P radioactivity in phosphorylated P gamma indicates that P gamma extracted with GTP-bound transducin alpha subunit is not phosphorylated and that a phosphate is incorporated into more than 80% of the P gamma by the kinase. Phosphoamino acid analysis, sequencing of phosphorylated peptides derived from phosphorylated P gamma, and phosphorylation of synthetic peptides indicate threonine 22 in P gamma is phosphorylated by the kinase. Phosphorylated P gamma has a higher inhibitory activity for active cGMP phosphodiesterase than non-phosphorylated P gamma. These data suggest that threonine 22 in P gamma is phosphorylated by a specific kinase and that the P gamma phosphorylation governs the interaction between P gamma and catalytic subunits of cGMP phosphodiesterase in vertebrate rod photoreceptors.