Identification of a novel type 2 fiber population in mammalian skeletal muscle by combined use of histochemical myosin ATPase and anti-myosin monoclonal antibodies

A novel type of myosin heavy chain (MHC), called 2X, has been recently identified in type 2 fibers of rat skeletal muscles using an immunochemical approach. In the present study, the same panel of anti-MHC monoclonal antibodies was used in immunohistochemistry combined with enzyme histochemistry to identify and compare type 2X fibers in hindlimb skeletal muscles of rat, mouse, and guinea pig. Immunohistochemistry shows that 2X MHC is localized in a large subset of type 2 fibers and is co-expressed with 2A or 2B MHC in a small number of fibers. Enzyme histochemistry shows that type 2X fibers display low myosin ATPase activity after pre-incubation at pH 4.3 and high activity after paraformaldehyde pre-incubation at pH 10.4. After pre-incubation at pH 4.6, myosin ATPase shows intermediate and high activity in rat and mouse 2X fibers, respectively, whereas it is low in guinea pig 2X fibers. Succinate dehydrogenase displays moderate to high activity in 2X fibers of all species. Taken together, these staining patterns allow this novel fiber population to be distinguished from the other type 2 fibers using only enzyme histochemistry. Nevertheless, the combined use of immuno- and enzyme histochemistry prevents incorrect fiber typing due to the interspecies variability of myosin ATPase activity among the correspondent fiber types, and completely modifies the presently used classification of mouse type 2 fibers.     

Mammalian beta-adrenergic receptors. Distinct glycoprotein populations containing high mannose or complex type carbohydrate chains

Mammalian beta-adrenergic receptor binding peptides can be visualized by covalently labeling them with the photoaffinity reagent p-azido-m-[125I]iodobenzylcarazolol followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The receptor peptides migrate as broad bands of Mr approximately equal to 62,000. In the present study, we examined the carbohydrate composition of the mammalian beta receptor through the use of specific exo- and endoglycosidases and lectin affinity chromatography. Treatment of p-azido-m-[125I]iodobenzylcarazolol-labeled beta2-adrenergic receptors from hamster lung or rat erythrocyte with the exoglycosidases neuraminidase and alpha-mannosidase provided evidence for the existence of both high mannose and complex type carbohydrate chains on beta 2-adrenergic receptors. The nonadditivity of the effect of sequential treatments with these enzymes suggested discrete populations of beta-adrenergic receptors containing either complex or high mannose type chains. Deglycosylation of receptor with endoglycosidase F results in a single labeled polypeptide at Mr = 49,000 for both systems. The same two populations of the beta receptors (high mannose or complex type chain) could also be fractionated by lectin affinity chromatography of solubilized p-azido-m-[125I]iodobenzylcarazolol-labeled receptors. The high mannose-containing receptors could be absorbed to and specifically eluted from concanavalin A-agarose. Those containing complex type carbohydrates could be adsorbed to and eluted from wheat germ agglutinin-agarose. Taken together, these data suggest that mammalian beta-adrenergic receptors contain both complex and high mannose type carbohydrate chains and that microheterogeneity of these chains likely explains the broad band pattern typically obtained on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Low-Carbohydrate Diet and Type 2 Diabetes Risk in Japanese Men and Women: The Japan Public Health Center-Based Prospective Study

ObjectiveEvidence is sparse and contradictory regarding the association between low-carbohydrate diet score and type 2 diabetes risk, and no prospective study examined the association among Asians, who consume greater amount of carbohydrate. We prospectively investigated the association of low-carbohydrate diet score with type 2 diabetes risk.MethodsParticipants were 27,799 men and 36,875 women aged 45–75 years who participated in the second survey of the Japan Public Health Center-Based Prospective Study and who had no history of diabetes. Dietary intake was ascertained by using a validated food-frequency questionnaire, and low-carbohydrate diet score was calculated from total carbohydrate, fat, and protein intake. The scores for high animal protein and fat or for high plant protein and fat were also calculated. Odds ratios of self-reported, physician-diagnosed type 2 diabetes over 5-year were estimated by using logistic regression.ResultsDuring the 5-year period, 1191 new cases of type 2 diabetes were self-reported. Low-carbohydrate diet score for high total protein and fat was significantly associated with a decreased risk of type 2 diabetes in women (P for trend <0.001); the multivariable-adjusted odds ratio of type 2 diabetes for the highest quintile of the score were 0.63 (95% confidence interval 0.46–0.84), compared with those for the lowest quintile. Additional adjustment for dietary glycemic load attenuated the association (odds ratio 0.75, 95% confidence interval 0.45–1.25). When the score separated for animal and for plant protein and fat, the score for high animal protein and fat was inversely associated with type 2 diabetes in women, whereas the score for high plant protein and fat was not associated in both men and women.DiscussionLow-carbohydrate diet was associated with decreased risk of type 2 diabetes in Japanese women and this association may be partly attributable to high intake of white rice. The association for animal-based and plant-based low-carbohydrate diet warrants further investigation.     

青春双歧杆菌对2型糖尿病模型大鼠肠道菌群和脂质代谢的影响

目的通过观察青春双歧杆菌对2型糖尿病模型大鼠肠道菌群的变化,和血清中总胆固醇（TC）、甘油三酯（TG）、高密度脂蛋白（HDL-C）、超氧化物歧化酶（SOD）和丙二醛（MDA）的水平,探讨青春双歧杆菌对2型糖尿病模型大鼠肠道功能和脂质代谢的影响。方法采用青春双歧杆菌灌胃2型糖尿病模型大鼠,取粪便检查正常菌群,取血和脏器检测TC、TG、HDL-C、SOD和MDA含量。结果青春双歧杆菌导致肠道内双歧杆菌、乳杆菌的数量增加,而肠杆菌、肠球菌数量下降;TC、TG和MDA水平下降,而HDL-C和SOD水平升高。结论青春双歧杆菌具有改善2型糖尿病模型大鼠肠道功能和降血脂作用,与二甲双胍联合应用效果更佳。     

镁转运体MGT7参与拟南芥对高钙环境的适应

高Ca²⁺环境对许多植物的生长不利, 因此研究植物对高Ca²⁺环境的适应机制非常重要。研究发现, 拟南芥(Ara- bidopsis thaliana)镁转运体MGT7功能缺失突变体mgt7-1和mgt7-2具有高Ca²⁺敏感表型: 在高Ca²⁺培养基上, 相对于野生型Col-0, 突变体叶鲜重显著下降, 但根长无显著差异。高Ca²⁺对MGT7启动子活性和包括MGT7在内的镁转运体基因表达无显著调节作用。Col-0与mgt7突变体之间, 在外加Ca²⁺诱导细胞质Ca²⁺瞬时升高和Ca²⁺含量方面无显著差异; 但是, 在正常和高Ca²⁺培养基上, mgt7突变体的Mg含量均显著低于Col-0。高Ca²⁺显著抑制Col-0和mgt7突变体内Mg的积累。因此我们假设, mgt7突变体的高Ca²⁺敏感表型是由于其体内Mg含量下降导致的。进一步的研究证实, 只有增加培养基中Mg²⁺的含量, 而不是N、P、K和S, 才可以使突变体的高Ca²⁺敏感表型得到恢复。     

Differential production of chirping behavior evoked by electrical stimulation of the weakly electric fish, Apteronotus leptorhynchus

Aperonotus leptorhynchus (Gymnotiformes) produces wave-like electric organ discharges distinguished by a high degree of constancy. Transient frequency and amplitude modulations of these discharges occur both spontaneously and during social interactions, which can be mimicked by external electrical stimulation. The so-called chirps can be divided into four different types. Independent of the type of chirp produced under spontaneous conditions, the fish generate only significant numbers of type-2 chirps under evoked conditions. The rate of production of chirps of this type is largely determined by the frequency relative to the fish's frequency and signal intensity. Frequencies of + 10 Hz of the fish's own discharge frequency most effectively elicit chirps. Type-2 chirps can also be evoked through stimulation at or near the higher harmonic frequencies of the fish's frequency, but the chirp rate decreases with increasing number of the higher harmonic component. Over a certain range, the rate of production of type-2 chirps increases with increasing stimulus intensity. At very high intensities the generation of type-2 chirps is accompanied by the production of a novel type of electrical signal ("abrupt frequency rise") characterized by a frequency increase of approximately 20 Hz and high repetition rates of roughly 10 s(-1). We hypothesize that the different types of electric modulations subserve different behavioral functions.     

Dipeptidyl peptidase-IV expression and activity in human glomerular endothelial cells

Glucagon-like peptide-1 (GLP-1), a meal-stimulated gastrointestinal insulinotropic hormone inactivated by dipeptidyl peptidase-IV (DPP-IV), is reduced in type 2 diabetic patients. The present study shows that 2-week exposure of human glomerular endothelial cells to high glucose (22 mM) determines a highly significant increase in DPP-IV activity and mRNA expression, which cannot be entirely accounted for by hyperosmolarity. On the other hand, incubation of purified DPP-IV in a buffer solution added with high glucose does not affect enzyme activity. These results suggest that high glucose increases expression and activity of DPP-IV, possibly contributing to GLP-1 reduction in type 2 diabetic patients.     

Calpain 10 SNP-44 gene polymorphism affects susceptibility to type 2 diabetes mellitus and diabetic-related conditions

The association of the gene encoding calpain 10 with type 2 diabetes mellitus (T2DM) has been reported. In this study we aimed to evaluate the association of SNP-19,-44, and -63 polymorphisms of calpain 10 with type 2 diabetes and diabetic-related conditions, such as diabetic retinopathy, nephropathy, and neuropathy in a Turkish population. The study group included 202 patients (133 female and 69 male) with T2DM, while the control group included 80 nondiabetic people (44 female and 36 male). Genotyping was done by the polymerase chain reaction and restriction fragment length polymorphism method. Calpain 10 SNP-44 TC genotype was found to be significantly frequent in type 2 diabetic patients with respect to the control group (p < 0.01). Body mass index (BMI) was found to be significantly high in TC genotype with type 2 diabetic patients (p < 0.05). SNP-44 T allele frequency was found to be lower in type 2 diabetic patients compared with the controls (p < 0.01). We conclude that the calpain 10 SNP-44 gene polymorphism may be accepted as a risk factor in the development of T2DM and elevated BMI in type 2 diabetic patients in a Turkish population.     

Muscle fibre composition of the eye muscles of the carp (Cyprinus carpio L.) defined on the base of histochemical observations

A histochemical study has been carried out on the eye muscles of the carp. On the base of the ATP-ase and SDH activity and with regard to the localization and diameter of muscle fibres six types of muscle fibres situated in the defined zones can be distinguished. The type 1 and 2 fibres display a moderately high ATP-ase activity at pH 9.4 and rather low activity after alkaline and acid preincubation. Type 1 shows a high SDH activity in contrast to type 2 with a low SDH activity. The other four types of fibres have the high ATP-ase activity at pH 9.4. Type 3 contains fibres with a moderately high ATP-ase activity after alkaline preincubation with a rather low activity after acid preincubation and with a low SDH activity. The fibres of type 4 characterized by the high ATP-ase activity after alkaline and acid preincubation and by the high SDH activity. The fibres of type 5 display high ATP-ase activity after alkaline and acid preincubation and the low SDH activity. They are situated in the white and intermediate fibre zones. The fibres of type 6 are comparable to the fibres of type 5, however they differ diameter and localization, i.e. they are situated in small diameter fibre zone. Using the electron microscope four types of fibres (A, B, C, and D) are found. They vary in the localization of T system, the organization of Z-line and in M-line appearance. In the type B of muscle fibres two different localizations of T system have been discerned.     

Brain Regional Heterogeneity of pH Effects on GABAA Receptor-Associated [35S]TBPS Binding

We have utilized quantitative autoradiography with the GABA(A) receptor chloride channel blocker [35S]t-butylbicyclophosphorothionate ([35S]TBPS) in rodent brain sections to investigate if differential proton modulation of various GABA(A) receptor subtypes expressed in various brain regions are differentially sensitive to pH alternations. Acidic and basic pHs decreased the binding, the mean values at pH 5.4 and pH 9.4 being 17% and 76% of the binding at pH 7.4, respectively. The regional profiles of the pH effects could be divided into two types. In regions with high basal binding at pH 7.4. the pH profile was usually 'bell-shaped,' with maximal binding at pH 7.4 (type 1). In regions with low basal binding at pH 7.4, the pH profile (type 2) revealed very low binding at pH 5.4, lower sensitivity to high pH, and usually maximal binding at pH 8.4. In brain regions with type 1 pH modulation alpha1 and beta2 subunits are abundantly expressed, whereas alpha2 and beta3 subunits are abundant in type 2 regions. Therefore the alpha1beta2gamma2 and alpha2beta3gamma2 receptor subtypes are suggested to be preferentially responsible for brain regional heterogeneity of the pH modulation of [35S]TBPS binding.     

The effect of challenge and trickle Trichuris muris infections on the polarisation of the immune response

In the field, determination of mechanisms of immunity to geohelminths are problematic due to the variation in infection exposure, host genetics, nutrition and co-infection. This study uses a well defined laboratory model, Trichuris muris in the mouse to study immune responses to challenge and trickle infections. The rationale is thus to study parasite acquisition under more natural antigen dose exposure. Antigen dose has previously been shown in this system to affect the outcome of infection with low antigen doses favouring type 1 responses (and susceptibility) and high antigen doses favouring type 2 responses (and resistance). A high level challenge infection could be established in a normally resistant host but only following priming of the immune response by a low level infection. Once type 2 responses were initiated it was impossible to switch an ongoing type 2 response even using IL-12 which is a potent stimulus of type 1 responses. Trickle infections resulted in no clear polarisation of the immune response. It was possible to build up the level of infection to a threshold level beyond which type 2 responses and expulsion were initiated. This threshold level was dependent upon host genetic background. Our results reveal a complex spectrum of responses and demonstrate that resistance and type 2 responses can be built up with increasing parasite exposure. The data provide compelling evidence to support a role for acquisition of acquired immunity to gastro-intestinal nematodes under complex infection patterns such as those found in the field.     

Consistent linkage of dominantly inherited osteogenesis imperfecta to the type I collagen loci: COL1A1 and COL1A2

The segregation of COL1A1 and COL1A2, the two genes which encode the chains of type I collagen, was analyzed in 38 dominant osteogenesis imperfecta (OI) pedigrees by using polymorphic markers within or close to the genes. This was done in order to estimate the consistency of linkage of OI genes to these two loci. None of the 38 pedigrees showed evidence of recombination between the OI gene and both collagen loci, suggesting that the frequency of unlinked loci in the population must be low. From these results, approximate 95% confidence limits for the proportion of families linked to the type I collagen genes can be set between .91 and 1.00. This is high enough to base prenatal diagnosis of dominantly inherited OI on linkage to these genes even in families which are too small for the linkage to be independently confirmed to high levels of significance. When phenotypic features were compared with the concordant collagen locus, all eight pedigrees with Sillence OI type IV segregated with COL1A2. On the other hand, Sillence OI type I segregated with both COL1A1 (17 pedigrees) and COL1A2 (7 pedigrees). The concordant locus was uncertain in the remaining six OI type I pedigrees. Of several other features, the presence or absence of presenile hearing loss was the best predictor of the mutant locus in OI type I families, with 13 of the 17 COL1A1 segregants and none of the 7 COL1A2 segregants showing this feature.     

Diabetes: A Cardiac Condition Manifesting as Hyperglycemia

ObjectiveTo investigate the reasons for the increased risk of cardiovascular events and mortality in individuals with type 2 diabetes mellitus.MethodsFrom January 1990 to March 2008, literature relevant to low-density lipoprotein (LDL) and highdensity lipoprotein (HDL) cholesterol, hemoglobin A1c, acute hyperglycemia, postprandial hyperglycemia, systolic blood pressure, insulin resistance, endothelial dysfunction, microalbuminuria, diabetic cardiomyopathy, left ventricular hypertrophy, function inhibitors of the renin-angiotensin system and sympathetic nervous system, statins, and antiplatelet therapy as related to cardiac events and mortality in type 2 diabetic patients was reviewed.ResultsIncreased numbers of cardiac events and mortality in type 2 diabetes are associated with low HDL and high LDL cholesterol, high hemoglobin A1c, and high systolic blood pressure. Acute hyperglycemia, postprandial hyperglycemia, and possibly use of traditional sulfonylureas also increase incidence of cardiac events and mortality. The presence of microalbuminuria signifies endothelial dysfunction and an increased risk of cardiac events. Hypertension should be treated to goals that are lower in the diabetic patient with multiple therapies, which include suppressors of the renin-angiotensin and sympathetic nervous systems. Decreased improvement in outcomes for the diabetic patient with cardiovascular disease may be accounted for by the failure to treat insulin resistance and ventricular dysfunction. The high incidence of heart failure in the diabetic patient is due to the toxic triad of diabetic cardiomyopathy, left ventricular hypertrophy, and extensive coronary artery disease.ConclusionHigh risk of cardiovascular events, heart failure, and mortality in type 2 diabetes can be lowered with risk factor reduction and therapies that prevent or improve ventricular function. (Endocr Pract. 2008;14:924-932)     

Genetic basis of acanthosis nigricans in Mexican Americans and its association with phenotypes related to type 2 diabetes

Acanthosis nigricans (AN) is a skin condition associated with hyperinsulinemia and insulin resistance and has been shown to be a risk factor for type 2 diabetes. The influence of genetic factors on AN and the basis of its association with type 2 diabetes and its risk factors are unknown. Using data from 397 participants from two Mexican American family studies, we investigated the heritability of AN and its genetic correlation with other diabetes risk factors. AN was examined as both a continuous trait and a dichotomous trait by means of a previously described validated scale. The results indicated that the heritability (h2) for AN, when examined as a continuous trait, was high (0.58+/-0.10) and statistically significant (P<0.001). The h2 for AN as a dichotomous trait was estimated to be moderate (0.23+/-0.05) and was also significant (P=0.018). The additive genetic correlations between AN (either as a continuous trait or a dichotomous trait) and type 2 diabetes and its risk factors, including body mass index and fasting insulin, were high or moderately high and statistically significant. The random environmental correlations, by contrast, were low and statistically insignificant. These data suggest that genes that influence AN have pleiotropic effects on diabetes and its risk factors.     

Androgen-activating enzymes in the central nervous systemProceedings of Xth International Congress on Hormonal Steroids, Quebec, Canada, 17–21 June 1998.

In the rat brain, several steroids can be converted by specific enzymes to either more potent compounds or to derivatives showing new biological effects. One of the most studied enzyme is the 5-reductase (5-R), which acts on 3keto-Δ4 steroids. In males, testosterone is the main substrate and gives rise to the most potent natural androgen dihydrotestosterone. In females, progesterone is reduced to dihydroprogesterone, a precursor of allopregnanolone, a natural anxiolytic/anesthetic steroid. Other substrates are some gluco- and minero-corticoids. Two isoforms of the 5-R, with limited degree of homology, have been cloned: 5-R type 1 and type 2. The 5-R type 1 possesses low affinity for the various substrates and is widely distributed in the body, with the highest levels in the liver; in the brain, this isoform is expressed throughout life and does not appear to be controlled by androgens. 5-R type 1 in the rat brain is mainly concentrated in myelin membranes, where it might be involved in the catabolism of potentially neurotoxic steroids. The 5-R type 2 shows high affinity for the various substrates, a peculiar pH optimum at acidic values and is localized in androgen-dependent structures. In the rat brain, the type 2 isoform is expressed at high levels only in the perinatal period and is controlled by androgens, at least in males. In adulthood, the type 2 gene appears to be specifically expressed in localised brain regions, like the hypothalamus and the hippocampus.

The 5-R type 2 is present in the GT1 cells, a model of LHRH-secreting neurons. These cells also contain the androgen receptor, which is probably involved in the central negative feedback effect exerted by androgens on the hypothalamic–pituitary–gonadal axis. The physiological significance of these and additional data will be discussed.     

Growth factors for sequential cellular de- and re-differentiation in tissue engineering

A model system for the in vitro generation of cartilaginous constructs was used to study a tissue engineering paradigm whereby sequentially applied growth factors promoted chondrocytes to first de-differentiate into a proliferative state and then re-differentiate and undergo chondrogenesis. Early cultivation in medium with supplemental TGF-β1/FGF-2 doubled cell fractions in 2-week constructs compared to unsupplemented controls. Subsequent culture with supplemental IGF-I yielded large 4-week constructs with high fractions of cartilaginous extracellular matrix (ECM) and high compressive moduli, whereas prolonged culture with supplemental FGF-2 yielded small 4-week constructs with low ECM fractions and moduli. Sequential supplementation with TGF-β1/FGF-2 and then IGF-I yielded 4-week constructs with type-specific mRNA expression and protein levels that were high for type II and negligible for type I collagen, in contrast to other growth factor regimens studied. The data demonstrate that structural, functional, and molecular properties of engineered cartilage can be modulated by sequential application of growth factors.     

Mechanisms of oleic acid deterioration in insulin secretion: role in the pathogenesis of type 2 diabetes

Obesity is highly associated with type 2 diabetes where free fatty acids (FFAs) may be a trigger factor. To examine this hypothesis, in this study, we investigated the role of FFAs in the pathogenic development of type 2 diabetes. The release of insulin, the expression of preproinsulin (PPI), glucose transporter2 (GLUT2) and pancreatic duodenal homeobox-1 (PDX-1), and levels of intracellular free Ca++([Ca++]i) were measured in rat pancreatic islets treated with or without high concentrations of FFA (0.1 and 1.0 mM oleic acid) for 24 h. In comparison with untreated control, islets exposed to oleic acid showed an increase in basal insulin release and a decrease in glucose induced insulin secretion (GSIS). Elevated expression of PPI, PDX-1 and GLUT2 was also observed after treatment of the islets with oleic acid, which may partially contribute to the increased basal insulin secretion. Moreover, [Ca++]i levels increased after oleic acid exposure, which most likely accounts for the decrease of GSIS. Our findings, thus strongly suggest, that the increased levels of basal insulin secretion involved in glucose sensing, insulin producing and insulin secreting induced by high levels of FFAs may cause hyperinsulinemia in patients with type 2 diabetes, and thus long-term hyperinsulinemia could desensitize insulin receptors. We hypothesize that hyperinsulinemia may be a primary and independent event in the pathogenesis of diabetes. If proven, it may be possible to create novel and effective approaches for the prevention and treatment of type 2 diabetes.     

An alternative form of IL-18 in human blood plasma: complex formation with IgM defined by monoclonal antibodies

Monoclonal Abs 21 and 132 were raised against human functionally inactive rIL-18, and plasma IL-18 levels were determined by the sandwich ELISA established with these mABS: Plasma IL-18, designated type 2, was detected by this ELISA, and the levels found were not consistent with those obtained with the commercially available kit for determination of functionally active IL-18 (type 1). Type 1 was detected in all volunteers, whereas type 2 was detected in approximately 30% of healthy subjects, and the levels of type 2 in their blood plasma were high (25-100 ng/ml) compared with those of type 1 (0.05-0.3 ng/ml). We purified IL-18 type 2 from blood plasma of volunteers with high IL-18 type 2 concentrations, and its M(r) was determined to be 800 kDa by SDS-PAGE and molecular sieve HPLC. The purified 800-kDa protein, either caspase-1-treated or untreated, expressed no or marginal IL-18 function in terms of potentiation of NK-mediated cytolysis and IFN-gamma induction, and it barely bound IL-18R-positive cells. N-terminal amino acid analysis indicated that the purified protein was IgM containing a minimal amount of IL-18 proform and its fragment. Again, the purified IgM from IL-18 type2-positive volunteers exhibited cross-reaction with mAb 21 against IL-18. This band was not detected with 125-2H, an mAb against functionally active IL-18. Hence, human IgM carries functionally inactive IL-18 forming a disulfide-bridged complex, and this IL-18 moiety is from 10- to 100-fold higher than the conventional type 1 IL-18 in blood circulation in approximately 30% normal subjects.     

Allosteric and non-allosteric E. coli phosphofructokinases: effects on growth.

In wild type $\text{Escherichia coli}$ K-12 ca. 90% of phosphofructokinase is known to be the allosteric enzyme Pfk-1, and the rest is Pfk-2, a non-allosteric enzyme. An isogenic strain series has now been constructed with varying combinations and amounts of Pfk-1 and Pfk-2 (e.g., no Pfk-1, high level of Pfk-2; normal level Pfk-1, high level Pfk-2, etc.). In minimal medium with glucose, glucose-6-P, and glycerol, aerobically and anaerobically, provided there is adequate total amount of enzyme, what allosteric type it is does not make much difference to growth rate or yield of this organism.