The prevalence of and factors related to calcium pyrophosphate dihydrate crystal deposition in the knee joint.

OBJECTIVES: The purpose of this study was to reveal the accurate prevalence and related factors to the presence of calcium pyrophosphate dihydrate (CPPD) crystal deposition in cadaveric knee joints. DESIGN: Controlled laboratory study. METHODS: Six hundred and eight knees from 304 cadavers (332 male knees and 276 female knees, formalin fixed, Japanese anatomical specimens) were included in this study. The average age of the cadavers was 78.3 ± 10.7 years. Knees were macroscopically evaluated for the existence of CPPD, and the depth of cartilage degeneration of the femoro-tibial joint following the Outerbridge's classification. CPPD crystal was confirmed under Fourier transform infrared spectroscopy (FTIR) analysis using light microscopy. Statistical analysis was performed to reveal the correlation between the occurrence of CPPD deposition in the knee joint and gender, age, and the depth of cartilage degeneration of the femoro-tibial joint. RESULTS: The prevalence of grossly visible CPPD crystal was 13% (79 knees). In all of these knees, CPPD crystal was confirmed under FTIR analysis. Statistical analysis showed significant correlation between the occurrence of CPPD deposition and gender (P < 0.001), and depth of cartilage degeneration in the femoro-tibial joint (P < 0.001). In the cartilage degeneration positive knees (Over grade 3 in Outerbridge's classification), average age of CPPD deposition knee was significantly higher than CPPD negative knees. CONCLUSIONS: In this study, the prevalence of CPPD deposition disease was evaluated in a relatively large sample size of cadaveric knees. The prevalence of CPPD deposition disease was 13%, and was significantly correlated with the subject's age, gender, and severity of cartilage degeneration in the femoro-tibial joint.

Increased insulin sensitivity and hypoglycaemia in mice lacking the p85 alpha subunit of phosphoinositide 3-kinase.

The hallmark of type 2 diabetes, the most common metabolic disorder, is a defect in insulin-stimulated glucose transport in peripheral tissues. Although a role for phosphoinositide-3-kinase (PI3K) activity in insulin-stimulated glucose transport and glucose transporter isoform 4 (Glut4) translocation has been suggested in vitro, its role in vivo and the molecular link between activation of PI3K and translocation has not yet been elucidated. To determine the role of PI3K in glucose homeostasis, we generated mice with a targeted disruption of the gene encoding the p85alpha regulatory subunit of PI3K (Pik3r1; refs 3-5). Pik3r1-/- mice showed increased insulin sensitivity and hypoglycaemia due to increased glucose transport in skeletal muscle and adipocytes. Insulin-stimulated PI3K activity associated with insulin receptor substrates (IRSs) was mediated via full-length p85 alpha in wild-type mice, but via the p50 alpha alternative splicing isoform of the same gene in Pik3r1-/- mice. This isoform switch was associated with an increase in insulin-induced generation of phosphatidylinositol(3,4,5)triphosphate (PtdIns(3,4,5)P3) in Pik3r1-/- adipocytes and facilitation of Glut4 translocation from the low-density microsome (LDM) fraction to the plasma membrane (PM). This mechanism seems to be responsible for the phenotype of Pik3r1-/- mice, namely increased glucose transport and hypoglycaemia. Our work provides the first direct evidence that PI3K and its regulatory subunit have a role in glucose homeostasis in vivo.

Muscle sympathetic nerve activity during intermittent handgrip exercise.

AIM: This study evaluated whether central command plays an important role in activating muscle sympathetic nerve activity (MSNA) during short-term maximal handgrip contractions. METHODS: The increase in MSNA was examined while influence of minimizing for other factors such as mechanoreflex, metaboreflex and fatigue during repetitive exercise in seven 19- to 26-year-old participants. Maximal voluntary handgrips (15-s contraction with a 45-s relaxation) were performed 10 times with a 15-s pause between alternate hands. MSNA was recorded from the tibial nerve analyzed using the burst frequency (BF) and total sympathetic nerve activity. RESULTS: The BF increased with the first unit, from 14.9±1.8 bursts·min-1 at baseline to 27.7±3.4 bursts·min-1 during contraction. The increase in the MSNA during contractions remained unchanged throughout the repetitions. The BF declined to baseline during the relaxation periods. The peak grip force decreased from 333±25 N for the first grip to 216±20 N for the last contraction. The MSAN increase remained constant despite a possible reduction in mechanoreflex during exercise as indicated from decreased maximal handgrip force. CONCLUSION: We suggested that the MSNA response was induced mainly by central command during short-term maximal handgrip contraction without metaboreflex influence and attenuated mechanoreflex input.

BepiColombo mission confirms stagnation region of Venus and reveals its large extent.

The second Venus flyby of the BepiColombo mission offer a unique opportunity to make a complete tour of one of the few gas-dynamics dominated interaction regions between the supersonic solar wind and a Solar System object. The spacecraft pass through the full Venusian magnetosheath following the plasma streamlines, and cross the subsolar stagnation region during very stable solar wind conditions as observed upstream by the neighboring Solar Orbiter mission. These rare multipoint synergistic observations and stable conditions experimentally confirm what was previously predicted for the barely-explored stagnation region close to solar minimum. Here, we show that this region has a large extend, up to an altitude of 1900 km, and the estimated low energy transfer near the subsolar point confirm that the atmosphere of Venus, despite being non-magnetized and less conductive due to lower ultraviolet flux at solar minimum, is capable of withstanding the solar wind under low dynamic pressure.

Shadowing the actions of a predator: backlit fluorescent microscopy reveals synchronous nonbinary septation of predatory Bdellovibrio inside prey and exit through discrete bdelloplast pores.

The Bdellovibrio are miniature "living antibiotic" predatory bacteria which invade, reseal, and digest other larger Gram-negative bacteria, including pathogens. Nutrients for the replication of Bdellovibrio bacteria come entirely from the digestion of the single invaded bacterium, now called a bdelloplast, which is bound by the original prey outer membrane. Bdellovibrio bacteria are efficient digesters of prey cells, yielding on average 4 to 6 progeny from digestion of a single prey cell of a genome size similar to that of the Bdellovibrio cell itself. The developmental intrabacterial cycle of Bdellovibrio is largely unknown and has never been visualized "live." Using the latest motorized xy stage with a very defined z-axis control and engineered periplasmically fluorescent prey allows, for the first time, accurate return and visualization without prey bleaching of developing Bdellovibrio cells using solely the inner resources of a prey cell over several hours. We show that Bdellovibrio bacteria do not follow the familiar pattern of bacterial cell division by binary fission. Instead, they septate synchronously to produce both odd and even numbers of progeny, even when two separate Bdellovibrio cells have invaded and develop within a single prey bacterium, producing two different amounts of progeny. Evolution of this novel septation pattern, allowing odd progeny yields, allows optimal use of the finite prey cell resources to produce maximal replicated, predatory bacteria. When replication is complete, Bdellovibrio cells exit the exhausted prey and are seen leaving via discrete pores rather than by breakdown of the entire outer membrane of the prey.

Control of lymphopoiesis by p50csk, a regulatory protein tyrosine kinase.

The csk gene encodes a nonreceptor protein tyrosine kinase that acts in part by regulating the activity of src-family protein tyrosine kinases. Since the src-family kinases p56lck and p59fyn play pivotal roles during lymphocyte development, it seemed plausible that p50csk might contribute to these regulatory circuits. Using a gene targeting approach, mouse embryonic stem cell lines lacking functional csk genes were generated. These csknull embryonic stem cells proved capable of contributing to many adult tissues, notably heart and brain. However, although csknull progenitors colonized the developing thymus, T and B cell differentiation were both blocked at very early stages. This represented a relatively selective interdiction of lymphocyte maturation, since csknull hematopoietic progenitors supported the development of normal-appearing MAC-1+ blood leukocytes, and the successful maturation of granulocyte/macrophage-colony-forming units from fetal liver progenitors. We conclude that p50csk regulates normal lymphocyte differentiation, but that it almost certainly does so by acting on targets other than p56lck and p59fyn.

Involvement of Fyn tyrosine kinase in progression of cytokinesis of B lymphocyte progenitor.

We analyzed the role of Fyn tyrosine kinase in cell cycle progression of B lymphocyte progenitor (pro B cell). Whereas there were no substantial defects in the intramarrow B cell genesis in the fyn(-) mouse, and long-term proliferation of fyn(-) pro B cells was maintained in vitro under a serum containing culture condition, the cell cycle was arrested at G2/M upon serum deprivation. Morphological analyses demonstrated that the cytokinesis of fyn(-) pro B cells was retarded in the presence of serum and that the entry of fyn(-) pro B cells into late telophase was completely blocked under the serum-free condition. In contrast, the earlier phases of mitosis of fyn(-) pro B cells proceeded normally without FCS. This failure to initiate late telophase resulted in the accumulation of elliptical binucleated cells that might be the outcome of the nuclear division without cytokinesis. Consistent with this defect in the progression of cytokinesis, Fyn was localized in the midspace of dividing pro B cells at anaphase. These results suggested that Fyn localizes at the midspace of dividing pro B cells and regulates the progression of cytokinesis.

Extracellular matrix survival signals transduced by focal adhesion kinase suppress p53-mediated apoptosis.

In many malignant cells, both the anchorage requirement for survival and the function of the p53 tumor suppressor gene are subverted. These effects are consistent with the hypothesis that survival signals from extracellular matrix (ECM) suppress a p53-regulated cell death pathway. We report that survival signals from fibronectin are transduced by the focal adhesion kinase (FAK). If FAK or the correct ECM is absent, cells enter apoptosis through a p53-dependent pathway activated by protein kinase C lambda/iota and cytosolic phospholipase A2. This pathway is suppressible by dominant-negative p53 and Bcl2 but not CrmA. Upon inactivation of p53, cells survive even if they lack matrix signals or FAK. This is the first report that p53 monitors survival signals from ECM/FAK in anchorage- dependent cells.

Length of the flagellar hook and the capacity of the type III export apparatus.

Length determination in biology generally uses molecular rulers. The hook, a part of the flagellum of motile bacteria, has an invariant length. Here, we examined hook length and found that it was determined not by molecular rulers but probably by the amount of subunit protein secreted by the flagellar export apparatus. The export apparatus shares common features with the type III virulence-factor secretion machinery and thus may be used more widely in length determination of structures other than flagella.

Xid-like immunodeficiency in mice with disruption of the p85alpha subunit of phosphoinositide 3-kinase.

Mice with a targeted gene disruption of p85alpha, a regulatory subunit of phosphoinositide 3-kinase, had impaired B cell development at the pro-B cell stage, reduced numbers of mature B cells and peritoneal CD5+ Ly-1 B cells, reduced B cell proliferative responses, and no T cell-independent antibody production. These phenotypes are nearly identical to those of Btk-/- or xid (X-linked immunodeficiency) mice. These results provide evidence that p85alpha is functionally linked to the Btk pathway in antigen receptor-mediated signal transduction and is pivotal in B cell development and functions.

Supramolecular structure of the Salmonella typhimurium type III protein secretion system.

The type III secretion system of Salmonella typhimurium directs the translocation of proteins into host cells. Evolutionarily related to the flagellar assembly machinery, this system is also present in other pathogenic bacteria, but its organization is unknown. Electron microscopy revealed supramolecular structures spanning the inner and outer membranes of flagellated and nonflagellated strains; such structures were not detected in strains carrying null mutations in components of the type III apparatus. Isolated structures were found to contain at least three proteins of this secretion system. Thus, the type III apparatus of S. typhimurium, and presumably other bacteria, exists as a supramolecular structure in the bacterial envelope.

Role of NADH shuttle system in glucose-induced activation of mitochondrial metabolism and insulin secretion.

Glucose metabolism in glycolysis and in mitochondria is pivotal to glucose-induced insulin secretion from pancreatic beta cells. One or more factors derived from glycolysis other than pyruvate appear to be required for the generation of mitochondrial signals that lead to insulin secretion. The electrons of the glycolysis-derived reduced form of nicotinamide adenine dinucleotide (NADH) are transferred to mitochondria through the NADH shuttle system. By abolishing the NADH shuttle function, glucose-induced increases in NADH autofluorescence, mitochondrial membrane potential, and adenosine triphosphate content were reduced and glucose-induced insulin secretion was abrogated. The NADH shuttle evidently couples glycolysis with activation of mitochondrial energy metabolism to trigger insulin secretion.

Effects of alpha-amylase and its inhibitors on acid production from cooked starch by oral streptococci.

This study evaluated acid production from cooked starch by Streptococcus mutans, Streptococcus sobrinus, Streptococcus sanguinis and Streptococcus mitis, and the effects of alpha-amylase inhibitors (maltotriitol and acarbose) and xylitol on acid production. Streptococcal cell suspensions were anaerobically incubated with various carbohydrates that included cooked potato starch in the presence or absence of alpha-amylase. Subsequently, the fall in pH and the acid production rate at pH 7.0 were measured. In addition, the effects of adding alpha-amylase inhibitors and xylitol to the reaction mixture were evaluated. In the absence of alpha-amylase, both the fall in pH and the acid production rate from cooked starch were small. On the other hand, in the presence of alpha-amylase, the pH fell to 3.9-4.4 and the acid production rate was 0.61-0.92 micromol per optical density unit per min. These values were comparable to those for maltose. When using cooked starch, the fall in pH by S. sanguinis and S. mitis was similar to that by S. mutans and S. sobrinus. For all streptococci, alpha-amylase inhibitors caused a decrease in acid production from cooked starch, although xylitol only decreased acid production by S. mutans and S. sobrinus. These results suggest that cooked starch is potentially acidogenic in the presence of alpha-amylase, which occurs in the oral cavity. In terms of the acidogenic potential of cooked starch, S. sanguinis and S. mitis were comparable to S. mutans and S. sobrinus. Alpha-amylase inhibitors and xylitol might moderate this activity.

Impairment of suckling response, trigeminal neuronal pattern formation, and hippocampal LTD in NMDA receptor epsilon 2 subunit mutant mice.

Multiple epsilon subunits are major determinants of the NMDA receptor channel diversity. Based on their functional properties in vitro and distributions, we have proposed that the epsilon 1 and epsilon 2 subunits play a role in synaptic plasticity. To investigate the physiological significance of the NMDA receptor channel diversity, we generated mutant mice defective in the epsilon 2 subunit. These mice showed no suckling response and died shortly after birth but could survive by hand feeding. The mutation hindered the formation of the whisker-related neuronal barrelette structure and the clustering of primary sensory afferent terminals in the brainstem trigeminal nucleus. In the hippocampus of the mutant mice, synaptic NMDA responses and longterm depression were abolished. These results suggest that the epsilon 2 subunit plays an essential role in both neuronal pattern formation and synaptic plasticity.

Genetic control of the bacterial flagellar regulon.

A number of cis- and trans-acting transcriptional factors in the flagellar regulons of Caulobacter crescentus and Salmonella typhimurium have been identified and characterized to varying degrees over the past year, bringing us closer to understanding the regulations of these complex gene hierarchies.

Interaction of murine granulocyte-macrophage progenitors and supporting stroma involves a recognition mechanism with galactosyl and mannosyl specificities.

To study the molecular basis of "homing" of granulocyte-macrophage progenitors (CFU-C), we synthesized probes by covalent linking of sugars to bovine serum albumin. Long-term marrow cultures were established in the presence and absence of these probes. In the presence of galactosyl and mannosyl probes, total cell and CFU-C production in the supernate as well as the adherent layer were halted, and cobblestones (representing CFU-C bound to stroma) disappeared. Fucosyl probe and diffusible sugars had no effect on these parameters. These studies suggested membrane lectins with specificity for galactosyl and mannosyl residues may be responsible for the binding of CFU-C to supporting stroma. To determine if CFU-C possesses homing receptors with these specificities, we induced agglutination in marrow cell suspensions with these neoglycoprotein probes. Selective agglutination was observed only by galactosyl and mannosyl probes. The results suggest that CFU-C homing receptors are membrane lectins with specificity for galactosyl and mannosyl residues.

Restored insulin-sensitivity in IRS-1-deficient mice treated by adenovirus-mediated gene therapy.

Insulin resistance is commonly observed both in overt diabetes and in individuals prone to, but not yet manifesting, diabetes. Hence the maintenance or restoration of insulin sensitivity may prevent the onset of this disease. We previously showed that homozygous disruption of insulin receptor substrate-1 (IRS-1) in mice resulted in insulin resistance but not diabetes. Here, we have explored the mechanism of systemic insulin resistance in these mice and used adenovirus-mediated gene therapy to restore their insulin sensitivity. Mice expressing the IRS-1transgene showed almost normal insulin sensitivity. Expression of an IRS-1 mutant (IRS-1Deltap85) lacking the binding site for the p85 subunit of phosphatidylinositol 3-kinase (PI3K) also restored insulin sensitivity, although PI3K is known to play a crucial role in insulin's metabolic responses. Protein kinase B (PKB) activity in liver was decreased in null mice compared with the wild-type and the null mice expressing IRS-1 or IRS-1Deltap85. In primary hepatocytes isolated from null mice, expression of IRS-1 enhanced both PI3K and PKB activities, but expression of IRS-1Deltap85 enhanced only PKB. These data suggest that PKB in liver plays a pivotal role in systemic glucose homeostasis and that PKB activation might be sufficient for reducing insulin resistance even without full activation of PI3K.

Diurnal change of thyroid-stimulating hormone mRNA expression in the rat pars tuberalis.

Thyroid-stimulating hormone (TSH)-producing cells (TSH cells), which account for a large fraction of the cells in the rat pars tuberalis (PT), have been found to express MT1 melatonin receptor and mammalian clock genes at high densities. Although these findings suggest that TSH production in the rat PT is regulated by melatonin and/or the biological clock, there have been no studies focusing on the diurnal change and regulation mechanism of TSH production in the rat PT. Therefore, in the present study, we examined diurnal changes of in TSH beta and alpha-glycoprotein subunit (alpha GSU) mRNA expression and TSH immunoreactivity (-ir) in the rat PT, and also examined the relationship between melatonin and TSH production in vivo. Both TSH beta mRNA expression and alpha GSU mRNA expression in the PT showed diurnal variations: the expression levels were lowest at the light phase [Zeitgeber time (ZT)4] and high at the dark phase (ZT12 and ZT20). TSH-ir in the PT showed the lowest level at ZT4, as was found for mRNA expression. Interestingly, TSH-ir, which was confined to the Golgi apparatus at ZT4, spread to the cytoplasm, and most of the TSH cells in the PT were uniformly immunostained in the cytoplasm at ZT20. Despite the fact that chronic administration of melatonin suppressed TSH beta and alpha GSU mRNA expression, TSH-ir in the PT was significantly enhanced. These findings results clearly show that there are diurnal changes in TSH expression and accumulation in rat PT-TSH cells and suggest that these fluctuations are regulated by melatonin.

Introduction of p130cas signaling complex formation upon integrin-mediated cell adhesion: a role for Src family kinases.

Integrin-mediated cell adhesion triggers intracellular signaling cascades, including tyrosine phosphorylation of intracellular proteins. Among these are the focal adhesion proteins p130cas (Cas) and focal adhesion kinase (FAK). Here we identify the kinase(s) mediating integrin-induced Cas phosphorylation and characterize protein-protein interactions mediated by phosphorylated Cas. We found that expression of a constitutively active FAK in fibroblasts results in a consecutive tyrosine phosphorylation of Cas. This effect required the autophosphorylation site of FAK, which is a binding site for Src family kinases. Integrin-mediated phosphorylation of Cas was not, however, compromised in fibroblasts lacking FAK. In contrast, adhesion-induced tyrosine phosphorylation of Cas was reduced in cells lacking Src, whereas enhanced phosphorylation of Cas was observed Csk- cells, in which Src kinases are activated. These results suggest that Src kinases are responsible for the integrin-mediated tyrosine phosphorylation of Cas. FAK seems not to be necessary for phosphorylation of Cas, but when autophosphorylated, FAK may recruit Src family kinases to phosphorylate Cas. Cas was found to form complexes with Src homology 2 (SH2) domain-containing signaling molecules, such as the SH2/SH3 adapter protein Crk, following integrin-induced tyrosine phosphorylation. Guanine nucleotide exchange factors C3G and Sos were found in the Cas-Crk complex upon integrin ligand binding. These observations suggest that Cas serves as a docking protein and may transduce signals to downstream signaling pathways following integrin-mediated cell adhesion.

Immunocytochemical analyses and targeted gene disruption of GTPBP1.

We previously identified a gene encoding a putative GTPase, GTPBP1, which is structurally related to elongation factor 1alpha, a key component of protein biosynthesis machinery. The primary structure of GTPBP1 is highly conserved between human and mouse (97% identical at the amino acid level). Expression of this gene is enhanced by gamma interferon in a monocytic cell line, THP-1. Although counterparts of this molecule in Caenorhabditis elegans and Ascaris suum have also been identified, the function of this molecule remains to be clarified. In the present study, our immunohistochemical analyses on mouse tissues revealed that GTPBP1 is expressed in some neurons and smooth muscle cells of various organs as well as macrophages. Immunofluorescence analyses revealed that GTPBP1 is localized exclusively in cytoplasm and shows a diffuse granular network forming a gradient from the nucleus to the periphery of the cells in smooth muscle cell lines and macrophages. To investigate the physiological role of GTPBP1, we used targeted gene disruption in embryonic stem cells to generate GTPBP1-deficient mice. The mutant mice were born at the expected Mendelian frequency, developed normally, and were fertile. No manifest anatomical or behavioral abnormality was observed in the mutant mice. Functions of macrophages, including chemotaxis, phagocytosis, and nitric oxide production, in mutant mice were equivalent to those seen in wild-type mice. No significant difference was observed in the immune response to protein antigen between mutant mice and wild-type mice, suggesting normal function of antigen-presenting cells of the mutant mice. The absence of an eminent phenotype in GTPBP1-deficient mice may be due to functional compensation by GTPBP2, a molecule we recently identified which is similar to GTPBP1 in structure and tissue distribution.