In vitro and in vivo studies of the ALS-FTLD protein CHCHD10 reveal novel mitochondrial topology and protein interactions.

Mutations in coiled-coil-helix-coiled-coil-helix-domain containing 10 (CHCHD10), a mitochondrial twin CX9C protein whose function is still unknown, cause myopathy, motor neuron disease, frontotemporal dementia, and Parkinson's disease. Here, we investigate CHCHD10 topology and its protein interactome, as well as the effects of CHCHD10 depletion or expression of disease-associated mutations in wild-type cells. We find that CHCHD10 associates with membranes in the mitochondrial intermembrane space, where it interacts with a closely related protein, CHCHD2. Furthermore, both CHCHD10 and CHCHD2 interact with p32/GC1QR, a protein with various intra and extra-mitochondrial functions. CHCHD10 and CHCHD2 have short half-lives, suggesting regulatory rather than structural functions. Cell lines with CHCHD10 knockdown do not display bioenergetic defects, but, unexpectedly, accumulate excessive intramitochondrial iron. In mice, CHCHD10 is expressed in many tissues, most abundantly in heart, skeletal muscle, liver, and in specific CNS regions, notably the dopaminergic neurons of the substantia nigra and spinal cord neurons, which is consistent with the pathology associated with CHCHD10 mutations. Homozygote CHCHD10 knockout mice are viable, have no gross phenotypes, no bioenergetic defects or ultrastructural mitochondrial abnormalities in brain, heart or skeletal muscle, indicating that functional redundancy or compensatory mechanisms for CHCHD10 loss occur in vivo. Instead, cells expressing S59L or R15L mutant versions of CHCHD10, but not WT, have impaired mitochondrial energy metabolism. Taken together, the evidence obtained from our in vitro and in vivo studies suggest that CHCHD10 mutants cause disease through a gain of toxic function mechanism, rather than a loss of function.

Molecular characterization and antibiotic resistance of group G streptococci in Israel: comparison of invasive, non-invasive and carriage isolates.

Beta-hemolytic group G streptococci (GGS) are increasingly recognized as a source of substantial morbidity, causing mild to severe sporadic infections as well as outbreaks. The purpose of this study was to determine the genetic diversity and antibiotic resistance of GGS in Israel in order to aid in prevention and control. A total of 325 GGS isolates were collected in Israel between 2007 and 2011 from three determined settings: (1) carriage (n = 60), an observational longitudinal carriage study in the IF, (2) non-invasive (n = 166), clinical sporadic and epidemic non-invasive cases in the IDF, and (3) invasive (n = 99) cases of bacteremia collected during this period in Israel from a similar age group, at the national Streptococcal Reference Center. All isolates were characterized genetically and by their antibiotic-resistance profile. emm typing revealed 35 distinct types and subtypes among 228 S. dysgalactiae subsp. equisimilis (SDSE) isolates, with high genetic diversity. An additional 97 GGS were identified as Streptococcus anginosus (SAG). The proportion of SDSE was higher in the invasive (100 %) and non-invasive (63.8 %) isolates compared to the carriage ones (38.3 %). Clindamycin, erythromycin, azithromycin and tetracycline resistance was detected in 6.6 %, 8.6 %, 9.7 % and 37.6 % of isolates, respectively. Overall, the most resistant isolates were in the invasive group and the fewest were in the SAG group. Considerable genetic diversity and common antibiotic resistance were revealed among GGS strains which differed according to the epidemiologic settings. Further clinical, epidemiological and basic research of GGS as a pathogen is warranted.

Emotional stressors trigger cardiovascular events.

AIMS: To describe the relation between emotional stress and cardiovascular events, and review the literature on the cardiovascular effects of emotional stress, in order to describe the relation, the underlying pathophysiology, and potential therapeutic implications. MATERIALS AND METHODS: Targeted PUBMED searches were conducted to supplement the authors' existing database on this topic. RESULTS: Cardiovascular events are a major cause of morbidity and mortality in the developed world. Cardiovascular events can be triggered by acute mental stress caused by events such as an earthquake, a televised high-drama soccer game, job strain or the death of a loved one. Acute mental stress increases sympathetic output, impairs endothelial function and creates a hypercoagulable state. These changes have the potential to rupture vulnerable plaque and precipitate intraluminal thrombosis, resulting in myocardial infarction or sudden death. CONCLUSION: Therapies targeting this pathway can potentially prevent acute mental stressors from initiating plaque rupture. Limited evidence suggests that appropriately timed administration of beta-blockers, statins and aspirin might reduce the incidence of triggered myocardial infarctions. Stress management and transcendental meditation warrant further study.

Cannabinoids inhibit testosterone secretion by mouse testes in vitro.

Addition of delta-9-tetrahydrocannabinol or cannabinol to an incubation medium containing decapsulated mouse testes caused a significant reduction in the accumulation of testosterone in the medium. This result suggests that the reported effects of cannabis on male sexual and reproductive function may result from direct inhibition of testicular steroidogenesis by both psychoactive and nonpsychoactive constituents of marihuana.

Isolation and characterization of two major urinary metabolites of 1 -tetrahydrocannabinol.

Two of the major metabolites which appear in rabbit urine after the administration of Delta(1)-tetrahydrocannabinol have been isolated and their structures have been tentatively established. The available evidence indicates that they are 7-carboxy-Delta(1)-tetrahydrocannabinols with an additional hydroxyl group on the side chain. The substances occur both free and as conjugates.

Human recombinant erythropoietin promotes differentiation of murine megakaryocytes in vitro.

To determine if erythropoietin affects megakaryocytopoiesis, we measured acetylcholinesterase (AchE) activity, a marker of the murine megakaryocytic lineage, after the addition of human recombinant erythropoietin to serumless murine bone marrow cultures. Erythropoietin increased AchE activity substantially. Moreover, when the hormone was added to serumless cultures of 426 isolated single megakaryocytes derived from megakaryocytic colonies, erythropoietin induced a significant increase in the diameters of these cells. From a Bayesian analysis of the likelihood that some megakaryocytes increased in DNA content during the culture period, we estimate that 61% of the cells increased in ploidy. These data indicate that the action of erythropoietin is not restricted to the erythroid lineage.

Anandamide synthesis is induced by arachidonate mobilizing agonists in cells of the immune system.

The hypothesis that the capability of agents to mobilize arachidonic acid (AA) could predict increased anandamide (ANA) synthesis in a macrophage cell line has been examined. Lipopolysaccharide (LPS), platelet-activating factor (PAF) and cannabinoids such as Delta9-tetrahydrocannabinol (THC) and anandamide were all found to be agonists for the release of AA and led to increased ANA synthesis in RAW264.7 mouse macrophage cells. Nitric oxide, in contrast, stimulated AA release without raising ANA levels. ANA stimulation of its own synthesis indicates the existence of a positive feedback mechanism. The possible involvement of the CB2 receptor in THC-mediated AA release and ANA synthesis is addressed using the antagonist SR144528. ANA synthesis is also increased by the combination of calcium ionophore and indomethacin, suggesting that ANA is metabolized by a cyclooxygenase in this system. The data imply that ANA could play a role in the response of the immune system to cannabinoids and bacterial endotoxins and that AA mobilization is a predictor for increased ANA synthesis.

Stimulation of prostaglandin synthesis in WI-38 human lung fibroblasts following inhibition of phospholipid acylation by p-hydroxymercuribenzoate.

The release of arachidonic acid and its metabolites, prostaglandin E2 and thromboxane A2, from WI-38 human lung fibroblasts was modulated by p-hydroxymercuribenzoate. Exposure to the inhibitor resulted in a dose-dependent decrease in [1-14C]arachidonic acid uptake and incorporation into phospholipids and neutral lipid pools. Activities of lung fibroblast arachidonyl-CoA synthetase and lysolecithin acyltransferase were inhibited by 100 microM p-hydroxymercuribenzoate. [14C]Arachidonic acid labelled fibroblasts exhibited an increased release of [14C]arachidonate and [14C]prostaglandin E2 of 54% and 112%, respectively, when exposed to 100 microM of inhibitor. The stimulatory effects of 8.0 microM delta 1-tetrahydrocannabinol on arachidonate release and prostaglandin E synthesis (Burstein, S., Hunter, S.A., Sedor, C. and Shulman, S. (1982) Biochem. Pharmacol. 31, 2361-2365) were modified by the inclusion of inhibiting agent, resulting in a 608% stimulation in arachidonic acid release, while prostaglandin E2 and thromboxane A2 synthesis increased 894% and 390%, respectively, over levels obtained by untreated cells. The levels of arachidonate metabolites were altered by inhibitor when compared to cells treated with cannabinoid alone. No significant inhibition by delta 1-tetrahydrocannabinol was found on arachidonic uptake in these cells. In unlabelled studies, p-hydroxymercuribenzoate resulted in a profound, dose-dependent stimulation of prostaglandin E synthesis of 1490% at 150 microM inhibitor concentration. These results provide evidence that free arachidonate is reincorporated via acylation, thereby implicating this pathway as a possible control mechanism for the synthesis of arachidonic acid metabolites.

Rapid synthesis of 26-hydroxycholesterol from mevalonic acid in the Syrian hamster.

Using isotope-ratio mass spectrometry, newly synthesized 26-hydroxycholesterol and its derivative, 3 beta-hydroxy-5-cholenoic acid, were detected in the liver and bile of animals within 2 h following intravenous administration of [5-13C]mevalonic acid. The findings indicate that 26-hydroxycholesterol merits further consideration as an in vivo modulator of HMG-CoA reductase activity.

Positive inotropic and lusitropic effects of intravenous flosequinan in patients with heart failure.

OBJECTIVES: This study was designed to assess the direct effects of flosequinan on myocardial function. BACKGROUND: Flosequinan has been shown to improve symptoms and exercise tolerance in patients with heart failure. Although previous studies have established that flosequinan is a vasodilator, it is not known to what extent direct actions of the drug on myocardial contractility or diastolic properties contribute to its beneficial hemodynamic effects. METHODS: Nitroprusside and intravenous flosequinan were administered sequentially to 18 patients with severe heart failure (New York Heart Association functional class III or IV, left ventricular ejection fraction 0.14 +/- 0.02). Micromanometer left ventricular pressure and radionuclide volume data were combined to construct pressure-volume loops during 1) a baseline period, 2) nitroprusside infusion, 3) a second baseline period, and 4) flosequinan infusion. RESULTS: The peak rate of left ventricular pressure development increased from 899 +/- 84 to 1,070 +/- 94 mm Hg/s (p less than 0.05) with flosequinan. The baseline left ventricular end-systolic pressure-volume relation was constructed in 15 patients from the two baseline pressure-volume loops and from that obtained during afterload manipulation with nitroprusside. During flosequinan administration, the relation between end-systolic pressure and volume was shifted upward and leftward, indicating enhanced contractility, in 14 of 15 patients (p less than 0.001). The maximal rate of decrease in left ventricular pressure during isovolumetric relaxation increased in magnitude with flosequinan from 882 +/- 63 to 1,026 +/- 68 mm Hg/s (p less than 0.05). CONCLUSIONS: These results indicate that intravenous flosequinan has positive inotropic and lusitropic effects in patients with heart failure. Further studies are needed to assess the direct myocardial effects of oral flosequinan.

Percutaneous balloon pericardiotomy for the treatment of cardiac tamponade and large pericardial effusions: description of technique and report of the first 50 cases.

OBJECTIVES: This study describes the technique, clinical characteristics and results of the first 50 patients undergoing percutaneous balloon pericardiotomy as part of a multicenter registry. BACKGROUND: Percutaneous balloon pericardiotomy involves the use of a percutaneous balloon dilating catheter to create a nonsurgical pericardial window. METHODS: Patients eligible for percutaneous balloon pericardiotomy had either cardiac tamponade (n = 36) or a moderate to large pericardial effusion (n = 14). In addition to clinical follow-up, serial echocardiograms and chest X-ray films were obtained. RESULTS: The procedure was considered successful in 46 patients after a mean follow-up period of 3.6 +/- 3.3 months. Two patients required an early operation, one for bleeding from a pericardial vessel and one for persistent pericardial catheter drainage. Two patients required a late operation for recurrent tamponade. Minor complications of the procedure included fever in 6 of the first 37 patients (studied before the prophylactic use of antibiotic agents), thoracentesis or chest tube placement in 8 and a small spontaneously resolving pneumothorax in 2. Despite the short-term success of this procedure, the long-term prognosis of the 44 patients with malignant pericardial disease remained poor (mean survival time 3.3 +/- 3.1 months). CONCLUSIONS: Percutaneous balloon pericardiotomy is successful in helping to manage large pericardial effusions, particularly in patients with a malignant condition. It may become the preferred treatment to avoid a more invasive procedure for patients with pericardial effusion and a limited life expectancy.

The cannabinoid acids: nonpsychoactive derivatives with therapeutic potential.

The discovery of carboxylic acid metabolites of the cannabinoids (CBs) dates back more than three decades. Their lack of psychotropic activity was noted early on, and this resulted in a total absence of further research on their possible role in the actions of the CBs. More recent studies have revealed that the acids possess both analgesic and anti-inflammatory properties and may contribute to the actions of the parent drug. A synthetic analog showed similar actions at considerably lower doses. In this review, a brief survey of the extensive literature on metabolism of delta 9-tetrahydrocannabinol to the acids is presented, while more emphasis is given to the recent findings on the biological actions of this class of CBs. A possible mechanism involving effects on eicosanoids for some of these actions is also suggested. Finally, an analogy with a putative metabolite of anandamide, an endogenous CB, is discussed.

Canine megakaryocytopoiesis: analysis utilizing a monoclonal antibody to a 140-kd dog platelet protein.

The dog, a convenient and relatively large animal whose size permits repetitive blood and marrow sampling, marrow biopsy, and mechanical apheresis, would be a suitable experimental model for the study of in vivo megakaryocytopoiesis. A monoclonal antibody to a 140-kd dog platelet membrane protein has been developed that reacts with canine megakaryocytes. Using the fluoresceinated derivative of this antibody to identify megakaryocytes and propidium iodide staining to measure relative DNA content, the DNA distribution of megakaryocytes in dog bone marrow or in cultured dog marrow cells could be rapidly assessed by flow cytometry. Normal dogs showed a modal ploidy of 16N (54%), with 17% 8N and 16% 32N. In contrast, dogs made thrombocytopenic by plateletpheresis showed a shift in distribution to higher ploidy cells (36% 32N). The data show that use of a specific marker of megakaryocytes in combination with flow cytometric analysis is an accurate and reproducible method of assessing megakaryocytopoiesis in a convenient and easily manipulable animal model.

Characteristics of a novel rat anti-mouse platelet monoclonal antibody: application to studies of megakaryocytes.

Using murine platelets as an immunogen, a rat monoclonal antibody (designated 4A5) that recognizes only murine blood platelets and marrow megakaryocytes was developed. The extent of binding of 4A5 to platelets was dependent upon their state of activation. Following phorbol ester, ionophore, or thrombin stimulation of resting platelets, a decrease of > 50% in the binding of 4A5 was observed by flow cytometry. This decrease in antibody binding to the platelets was accompanied by an increase in antibody released into the platelet-free supernatant following platelet activation. When platelets were first radioiodinated, followed by activation and incubation of the platelet-free supernatant with 4A5-derivatized beads, no precipitable counts were observed compared with control resting platelets. This suggests that antibody release was related to an activation-dependent conformational change in the 4A5 epitope. Following solubilization of biotinylated platelets, 4A5 bound to an 80-kd membrane protein. Immunohistochemical studies with 4A5 showed that megakaryocytes could be identified both in vitro and ex vivo. When marrow was first stained histochemically with 4A5 followed by staining for acetylcholinesterase, the distribution of stained cells was similar. Flow cytometric analysis using 4A5 and propidium iodide showed that the antibody could be used to identify megakaryocytes for ploidy analysis in vivo or in vitro. 4A5 was capable of inducing a moderate thrombocytopenia in mice compared with polyclonal anti-platelet serum. When bound to plastic or to magnetic beads, 4A5 could be used to purify murine megakaryocytes to homogeneity. The data suggest that monoclonal antibody 4A5 will be useful in quantitative studies of murine platelets and megakaryocytes.

Regulation of interleukin 6 expression in murine bone marrow stromal cells.

The regulation of interleukin 6 (IL-6) expression in the B-lymphocyte-supporting murine stromal cell line BMS2 has been examined in response to exogenous cytokines and chemical agents. Kinetic analyses of IL-6 mRNA induction and decay are presented together with analysis of the IL-6 biological activity. The cytokines tumor necrosis factor, interleukin 1 (alpha and beta), and transforming growth factor beta, as well as forskolin and dibutyryl cyclic AMP, all induce a transient rise in the steady-state level of IL-6 mRNA and an increased release of IL-6 protein. To study its regulation at the chromatin level, the murine IL-6 genomic gene has been cloned. Induction of IL-6 expression correlates with increased DNA nicking, consistent with increased topoisomerase I and endogenous nuclease activity. This finding is supported by kinetic analyses using camptothecin, a topoisomerase I inhibitor. We conclude that IL-6 regulation in murine stromal cells capable of supporting B-lymphopoiesis is comparable to that observed in human diploid fibroblasts.

Quantitation of platelet life span in splenectomized dogs.

Platelet life spans in dogs were measured pre- and post- splenectomy utilizing in vitro whole blood biotinylation. Four splenectomized dogs were found to have significantly lengthened platelet life spans, 193 +/- 7 hours (mean +/- 1 SD;n=4) postsurgery vs. control life spans of 131 +/- 15 hours (n=6; p< 0.01) when analyzed with the multiple-hit model. Additionally, platelets from normal dogs transfused into splenectomized dogs were found to have convex survival curves with extended life spans approximating that of the splenectomized dog. These data indicate that the spleen is a significant determinant of platelet life span in dogs, with survivals increasing approximately 47% upon splenectomy.

Expression of a foreign protein in human megakaryocytes and platelets by retrovirally mediated gene transfer.

Recent progress in the culture of human megakaryocytes (MKs) has led to the capacity to produce platelets in vitro. This capability enables investigation into the possibility of modifying platelet structure and/or function by genetically altering the MK. To this end, a cDNA for the murine CD9 (mCD9) cell surface protein was introduced into MK progenitors by retrovirally mediated gene transfer and subsequently detected in cultured MKs with a monoclonal antibody (MoAb) that specifically recognizes the murine protein. CD34+ human peripheral blood or marrow progenitors, enriched by immunomagnetic bead selection, were cultured for 5 days in the presence of growth factors, including stem cell factor and thrombopoietin, to induce MK progenitors into the cell cycle. The stimulated cells were then cocultured with the mCD9 retroviral producer cell line for 3 days, followed by culture in serum-depleted medium for 3 to 7 additional days. Flow cytometry analysis using the anti-CD9 MoAb and TAB, a MoAb recognizing human GPIIb, revealed that a large proportion (40-100%) of the MKs expressed mCD9. To ascertain whether these cells were capable of producing mCD9+ platelets, flow cytometry analysis was performed at a time when proplatelets were observed in the culture. mCD9 was detected in up to 59% of the TAB+ platelet-sized particles. Because deteriorating MKs can produce platelet-sized particles in vitro, experiments were performed to determine whether mCD9+ TAB+ particles were functionally active. Addition of phorbol myristate acetate resulted in the redistribution of P-selectin (CD62) from the alpha granule to the platelet surface as detected by MoAbs S12 and G5 in three-color flow cytometry analyses. These studies showed that up to 76% of the mCD9+ TAB+ particles were functionally active. The data show that retrovirally mediated gene transfer is a viable approach for genetically altering MK progenitors, resulting in platelets that express heterologous proteins.

Immunoreactive somatostatin and luteinizing hormone releasing hormone in median eminence synaptosomes of the rat: detection by immunohistochemistry and quantification by radioimmunoassay.

Somatostatin and LHRH were detected by radioimmunoassay in the synaptosome fraction obtained by homogenization and differential centrifugation of the rat median eminence. Both somatostatin and LHRH were demonstrated by electron microscopic immunocytochemistry in secretory granules within synaptosomes.