Systemic Deletion of ARRDC4 Improves Cardiac Reserve and Exercise Capacity in Diabetes.

BACKGROUND: Exercise intolerance is an independent predictor of poor prognosis in diabetes. The underlying mechanism of the association between hyperglycemia and exercise intolerance remains undefined. We recently demonstrated that the interaction between ARRDC4 (arrestin domain-containing protein 4) and GLUT1 (glucose transporter 1) regulates cardiac metabolism. METHODS: To determine whether this mechanism broadly impacts diabetic complications, we investigated the role of ARRDC4 in the pathogenesis of diabetic cardiac/skeletal myopathy using cellular and animal models. RESULTS: High glucose promoted translocation of MondoA into the nucleus, which upregulated Arrdc4 transcriptional expression, increased lysosomal GLUT1 trafficking, and blocked glucose transport in cardiomyocytes, forming a feedback mechanism. This role of ARRDC4 was confirmed in human muscular cells from type 2 diabetic patients. Prolonged hyperglycemia upregulated myocardial Arrdc4 expression in multiple types of mouse models of diabetes. We analyzed hyperglycemia-induced cardiac and skeletal muscle abnormalities in insulin-deficient mice. Hyperglycemia increased advanced glycation end-products and elicited oxidative and endoplasmic reticulum stress leading to apoptosis in the heart and peripheral muscle. Deletion of Arrdc4 augmented tissue glucose transport and mitochondrial respiration, protecting the heart and muscle from tissue damage. Stress hemodynamic analysis and treadmill exhaustion test uncovered that Arrdc4-knockout mice had greater cardiac inotropic/chronotropic reserve with higher exercise endurance than wild-type animals under diabetes. While multiple organs were involved in the mechanism, cardiac-specific overexpression using an adenoassociated virus suggests that high levels of myocardial ARRDC4 have the potential to contribute to exercise intolerance by interfering with cardiac metabolism through its interaction with GLUT1 in diabetes. Importantly, the ARRDC4 mutation mouse line exhibited greater exercise tolerance, showing the potential therapeutic impact on diabetic cardiomyopathy by disrupting the interaction between ARRDC4 and GLUT1. CONCLUSIONS: ARRDC4 regulates hyperglycemia-induced toxicities toward cardiac and skeletal muscle, revealing a new molecular framework that connects hyperglycemia to cardiac/skeletal myopathy to exercise intolerance.

Kinesins Modify ERR1-Dependent Transcription Using a Conserved Nuclear Receptor Box Motif.

Kinesin family motors are microtubule (MT)-stimulated ATPases known best as transporters of cellular cargoes through the cytoplasm, regulators of MT dynamics, organizers of the mitotic spindle, and for insuring equal division of DNA during mitosis. Several kinesins have also been shown to regulate transcription by interacting with transcriptional cofactors and regulators, nuclear receptors, or with specific promotor elements on DNA. We previously showed that an LxxLL nuclear receptor box motif in the kinesin-2 family motor KIF17 mediates binding to the orphan nuclear receptor estrogen related receptor alpha (ERR1) and is responsible for the suppression of ERR1-dependent transcription by KIF17. Analysis of all kinesin family proteins revealed that multiple kinesins contain this LxxLL motif, raising the question as to whether additional kinesin motors contribute to the regulation of ERR1. In this study, we interrogate the effects of multiple kinesins with LxxLL motifs on ERR1-mediated transcription. We demonstrate that the kinesin-3 family motor KIF1B contains two LxxLL motifs, one of which binds to ERR1. In addition, we show that expression of a KIF1B fragment containing this LxxLL motif inhibits ERR1-dependent transcription by regulating nuclear entry of ERR1. We also provide evidence that the effects of expressing the KIF1B-LxxLL fragment on ERR1 activity are mediated by a mechanism distinct from that of KIF17. Since LxxLL domains are found in many kinesins, our data suggest an expanded role for kinesins in nuclear receptor mediated transcriptional regulation.

Molecular characterisation of varicella zoster virus genotypes in Sri Lanka.

INTRODUCTION: Genotyping of wild type of varicella zoster virus (VZV) in Sri Lanka would help to distinguish the VZV wild type infection from varicella vaccine associated infections. METHODS: PCR-RFLP analysis of VZV ORF 38, 54 and 62 was used for genotyping in VZV from blood or vesicular fluid from 31 patients with chickenpox or herpes zoster. The PstI restriction site of ORF 38, BglI restriction site of ORF 54 and SmaI restriction site of ORF 62 were analyzed using RFLP to determine the genotype. RESULTS: Except for one strain, all other VZV isolates had the genotype characteristic of the wild type VZV strain PstI+BglI+ SmaI-, which was characteristic of the Asian strain. None of the isolates had the American or the European VZV profile (PstI+BglI-) but were similar to isolates from Africa and Asia (PstI+BglI+). Interestingly, one of the VZV strains isolated from a patient with chickenpox had the characteristic genotype of the vaccine strain PstI- BglI+ SmaI+. CONCLUSIONS: The genotype of the VZV in Sri Lanka is similar to the Asian VZV genotype and can be easily distinguished from the VZV vaccine strain by using the polymorphisms in ORF 38, ORF 54 and ORF 62.

Direct-reversible binding of small molecules to G protein ßγ subunits.

Heterotrimeric guanine nucleotide-binding proteins (G proteins) composed of three subunits α, ß, γ mediate activation of multiple intracellular signaling cascades initiated by G protein-coupled receptors (GPCRs). Previously our laboratory identified small molecules that bind to Gßγ and interfere with or enhance binding of select effectors with Gßγ. To understand the molecular mechanisms of selectivity and assess binding of compounds to Gßγ, we used biophysical and biochemical approaches to directly monitor small molecule binding to Gßγ. Surface plasmon resonance (SPR) analysis indicated that multiple compounds bound directly to Gßγ with affinities in the high nanomolar to low micromolar range but with surprisingly slow on and off rate kinetics. While the k(off) was slow for most of the compounds in physiological buffers, they could be removed from Gßγ with mild chaotropic salts or mildly dissociating collision energy in a mass-spectrometer indicating that compound-Gßγ interactions were non-covalent. Finally, at concentrations used to observe maximal biological effects the stoichiometry of binding was 1:1. The results from this study show that small molecule modulation of Gßγ-effector interactions is by specific direct non-covalent and reversible binding of small molecules to Gßγ. This is highly relevant to development of Gßγ targeting as a therapeutic approach since reversible, direct binding is a prerequisite for drug development and important for specificity.

Organic causation of morbid jealousy.

This article describes the organic contribution to morbid jealousy. Although the true prevalence of morbid jealousy is unknown, organic factors contribute significantly to its development. We present an assortment of five case histories to highlight the importance of organic causation in this phenomenon. The first two cases portray organic delusional disorder arising as an aftermath of cerebral infarcts. They are both associated with left sided brain lesions. Though organic processes generally respond poorly to treatment, case 3 (patient with head injury), is unusual as it describes a young man whose symptoms resolve on recovering from the effects of a head injury. Likewise, case 4 (patient with a meningioma) who made a complete recovery following surgery, emphasizes the need for early detection of reversible causes. The difficulty in identifying the common substrate for a phenomenon with such a wide variety of causations is amply displayed by the abundance of theories forwarded. The blurred demarcation between normal jealousy and pathological jealousy leads to further uncertainty. The excess representation of morbid jealousy in organic conditions is not enlightened by these theories. Organic pathology, by affecting the higher centers of the brain, may remove the control over instinctual behaviour. Evidence for this is hard to establish but the evolutionary perspective of jealousy akin to that of the animal kingdom alludes to possible explanations.

Identification and characterization of unique proline-rich peptides binding to the mitochondrial fission protein hFis1.

Mammalian mitochondrial fission requires at least two proteins, hFis1 and the dynamin-like GTPase DLP1/Drp1. The mitochondrial protein hFis1 is anchored at the outer membrane by a C-terminal transmembrane domain. The cytosolic domain of hFis1 contains six alpha helices [alpha1-alpha6] out of which [alpha2-alpha5] form tetratricopeptide repeat (TPR)-like motifs. DLP1 and possibly other proteins are thought to interact with the hFis1 TPR region during the fission process. It has also been suggested that the alpha1-helix regulates protein-protein interactions at the TPR. We performed random peptide phage display screening using the hFis1[alpha2-alpha6] as the target and identified ten different peptide sequences. Phage ELISA using mutant hFis1 indicates that the peptide binding requires the alpha2 and alpha3 helices and the intact TPR structure. Competition experiments and surface plasmon resonance analyses confirmed that a subset of free peptides enriched with proline residues directly bind to the target. Two of these peptides bind to the alpha1-containing intact cytosolic domain of hFis1 with decreased affinity. Peptide microinjection into cells abolished the mitochondrial swelling induced by overexpression of alpha1-deleted hFis1, and significantly decreased cytochrome c release from mitochondria upon apoptotic induction. Our data demonstrate that hFis1 can bind to multiple amino acid sequences selectively, and that the TPR constitutes the main binding region of hFis1, providing a first insight into the hFis1 TPR as a potential therapeutic target.

Small molecule disruption of G protein beta gamma subunit signaling inhibits neutrophil chemotaxis and inflammation.

G protein betagamma subunit-dependent signaling is important for chemoattractant-dependent leukocyte chemotaxis. Selective small molecule targeting of phosphoinositide 3-kinase (PI3-kinase) gamma catalytic activity is a target of interest for anti-inflammatory pharmaceutical development. In this study, we examined whether small-molecule inhibition of Gbetagamma-dependent signaling, including Gbetagamma-dependent activation of PI3-kinase gamma and Rac1, could inhibit chemoattractant-dependent neutrophil migration in vitro and inflammation in vivo. Small-molecule Gbetagamma inhibitors suppressed fMLP-stimulated Rac activation, superoxide production, and PI3-kinase activation in differentiated HL60 cells. These compounds also blocked fMLP-dependent chemotaxis in HL60 cells and primary human neutrophils. Systemic administration inhibited paw edema and neutrophil infiltration in a mouse carrageenan-induced paw edema model. Overall, the data demonstrate that targeting Gbetagamma-regulation may be an effective anti-inflammation strategy.

Augmentation of tendon healing in an intraarticular bone tunnel with use of a bone growth factor.

We hypothesized that an exogenous bone growth factor could augment healing of a tendon graft in a bone tunnel in a rabbit anterior cruciate ligament-reconstruction model. Seventy rabbits underwent bilateral anterior cruciate ligament reconstructions with a semitendinosus tendon graft. One limb received a collagen sponge carrier vehicle containing a mixture of bone-derived proteins while the contralateral limb was treated with either no sponge or a sponge without bone-derived proteins. The reconstruction was evaluated at 2, 4, or 8 weeks with histologic, biomechanical, and magnetic resonance imaging analysis. Histologic analysis demonstrated that specimens treated with bone-derived proteins had a more consistent, dense interface tissue and closer apposition of new bone to the graft, with occasional formation of a fibrocartilaginous interface, when compared with control specimens. The treated specimens had significantly higher load-to-failure rates than did control specimens. Treatment with bone-derived proteins resulted in an average increase in tensile strength of 65%. The treated specimens were stronger than control specimens at each time point, but the difference was greatest at 8 weeks. On the basis of signal characteristics and new bone formation, magnetic resonance imaging was useful for predicting which limb was treated, the site of failure, and the limbs with higher load-to-failure values. This study demonstrates the potential for augmenting tendon healing in an intraarticular bone tunnel using an osteoinductive growth factor.

Histological analysis of human meniscal allografts. A preliminary report.

BACKGROUND: Little is known about the biology of meniscal allograft transplantation in humans. In particular, little information is available about the phenotype of the cells that repopulate the allograft, whether an immune response is elicited against the graft, and whether the repopulating cells synthesize normal extracellular matrix components. METHODS: A small biopsy specimen of the meniscal allograft (twenty-eight menisci in twenty-five patients) and the adjacent synovial membrane (sixteen patients) was harvested during follow-up arthroscopy in patients who had undergone meniscal allograft transplantation at a mean of sixteen months earlier. Seventeen patients had undergone concomitant reconstruction of the anterior cruciate ligament with an allograft. Normal menisci (unimplanted allografts) and synovial specimens from age-matched controls were examined as well. All twenty-eight meniscal allografts were examined histologically. Immunohistochemical analysis was carried out on ten menisci and nine synovial specimens with use of monoclonal antibodies to class-I and class-II major histocompatibility complex antigens, CD-8, CD-11b, and CD-19 epitopes, as well as other epitopes, to demonstrate immunogenic macromolecules, cytotoxic T-lymphocytes, activated macrophages, and B-lymphocytes. RESULTS: Most of the specimens demonstrated incomplete repopulation with viable cells. The repopulating cells stained positively with phenotype markers for both synovial cells and fibroblasts. Polarized light microscopy demonstrated evidence of active remodeling of the matrix. The cells in frozen, unimplanted menisci stained positively for class-I and class-II human leukocyte antigens, indicating immunogenicity at the time of transplantation. Overall, nine of twelve specimens contained immunoreactive cells (B-lymphocytes or cytotoxic T-cells) in the meniscus or synovial tissue. However, only a small number of these cells was present. There was no evidence of frank immunological rejection. The clinical outcome (success or failure of the transplant) was not related to the overall histological score or to the presence of an immune response in the meniscal or synovial biopsy specimen. CONCLUSIONS: Human meniscal allograft transplants are repopulated with cells that appear to be derived from the synovial membrane; these cells appear to actively remodel the matrix. Although there is histological evidence of an immune response directed against the transplant, this response does not appear to affect the clinical outcome. The presence of histocompatibility antigens on the meniscal surface at the time of transplantation (even after freezing) indicates the potential for an immune response against the transplant. CLINICAL RELEVANCE: Despite the absence of frank immunological rejection, a subtle immune reaction may affect the healing, incorporation, and revascularization of the graft. It is possible that the structural remodeling associated with cellular repopulation may render the meniscus more susceptible to injury.

Determination of ticarcillin in human plasma and in urine by reversed-phase LC.

A liquid chromatographic assay for ticarcillin (ticar.) in plasma and urine is described. For analysis, the internal standard cefoperazone (cfp) is dissolved in acetonitrile, which is used for precipitating the protein. The supernatant is evaporated, reconstituted in running mobile phase and injected directly onto the reversed-phase C18 column, with detection at 205 nm. The mobile phase is composed of water-acetonitrile-o-phosphoric acid-tetramethylammonium chloride (TMA). Coefficients of variation for reproducibility were in the range of 2.2-15.5% for extra-low, low, medium and high controls. Limits of detection were 0.5 microgram ml-1 for plasma and 1 microgram ml-1 for urine. No interference from other cephalosporins or other antibiotics was noted. This liquid chromatographic assay is simple, accurate, requires no extraction and overcomes previous problems related to the drug's peak splitting due to isomerization.

Paired ion reversed-phase HPLC assay for the determination of iothalamic acid and para aminohippuric acid in urine.

A paired ion reversed-phase high performance liquid chromatographic method for simultaneous determination of iothalamic acid (Io) and para aminohippuric acid (PAH) in urine is described. The method uses a single internal standard for both drugs. The only sample preparation required is dilution of urine (1:100 or 1:500) with deionized water. The internal standard is added to a small aliquot of the diluted specimen and injected. For HPLC, a C8 column and a mobile phase consisting of potassium phosphate buffer with dodecyl triethylammonium phosphate IP reagent, 25% organic modifier with UV detection at 254 nm was used. Within day and between day variation for the assay were in the range of 1.48-9.46% for iothalamic acid and 1.84-10.36% for para aminohippuric acid for four levels of concentration. Limits of quantitation were 50.0 micrograms ml-1 for iothalamic acid and 75.0 micrograms ml-1 for para aminohippuric acid. Mean recovery was 98.55% for Io and 97.79% for PAH. This isocratic HPLC assay is simple, rapid and relatively inexpensive.

Gastric Pepsin in an Anuran Larva: (Lepidobatrachus laevis/pepsinogen).

Larval stomach development was studied in the obligate carnivorous larva of the frog Lepidobatrachus laevis. Pepsin producing cells of the larval stomach were identified using rabbit anti-porcine pepsin and immunohistochemical techniques. Pepsin production was detected at a very early stage of development (stage 24: during opercular development) when the larvae were first competent for feeding. Peptic activity in isolated larval stomachs was demonstrated in a microassay using acid denatured hemoglobin at pH 1.7. The total activity per stomach increased 5,400 fold through the beginning of metamorphosis and the specific activity increased 345 fold through the same period. Electrophoretic analysis of the larval pepsinogens, using a caseinolytic assay revealed the presence of one major pepsinogen at stage 24; two additional isozymes were observed during later larval development. The molecular weight of the isopepsinogens was 34,800.