Molecular identification of nicotinic acid receptor

Nicotinic acid and its derivative, Acipimox, have been widely used in the treatment of hyperlipidemia. Pharmacological studies have demonstrated that they exert the beneficial effect through the activation of a Gi-protein-coupled receptor on adipocyte, which has remained elusive to date. Here we show that a novel GPCR, designated HM74b because of its high similarity to HM74, is a receptor for nicotinic acid. HM74b mRNA is found in human, murine, and rat adipose tissues. Nicotinic acid and Acipimox inhibit forskolin-stimulated intracellular cAMP accumulation in human HM74b-expressing cells and activate GTP gamma S binding in a dose-dependent manner. [3H]Nicotinic acid specifically binds to HM74b-expressing membrane and its binding is replaced by Acipimox. This finding will open a new phase of research on the physiological role of nicotinic acid and will be a clue to develop novel antihyperlipidemic drugs.     

Phosphorylation of Drosophila heat shock transcription factor.

The role that phosphorylation plays in regulating heat shock factor (HSF) function and activity has been the subject of several studies. Here, we demonstrate that Drosophila melanogaster HSF (DmHSF) is a phosphoprotein that is multiply phosphorylated at some sites and is dephosphorylated at others upon heat shock. However, the steady-state level of phosphorylation of Drosophila HSF remains unchanged after heat shock. Phosphoamino-acid analysis reveals that predominantly serine residues are phosphorylated for both the non-shocked and heat shocked molecules. Gel mobility shift assays using extracts from SL2 cells treated with a variety of phosphatase and kinase inhibitors show little or no effect on the heat shock induced DNA binding activity of HSF or on its recovery. We conclude that phosphorylation plays no significant role in regulating the heat induced DNA binding activity of Drosophila HSF.     

The measurement of shock waves following heel strike while running

A non-invasive method for demonstrating the shock wave which propagates through the skeletal system following heel strike is described. This wave was not seen in force plate studies where adequate shock absorption was provided by running shoes. In the present study six subjects ran across a force plate without shoes before and after they were fatigued on a treadmill to demonstrate possible changes in the heel strike transient. Most of the parameters measured were not altered by fatigue, and a relationship between the shock wave and height, but not the weight of the runner was demonstrated. The different mechanisms leading to this phenomenon, and its implication in the areas of osteoarthritic degeneration and running mechanics are discussed.     

Induction of the 72-kilodalton heat shock protein and protection from ultraviolet B-induced cell death in human keratinocytes by repetitive exposure to heat shock or 15-deoxy-delta(12,14)-prostaglandin J2

It has been demonstrated that hyperthermia protects keratinocytes from ultraviolet B (UVB)-induced cell death in culture and in vivo. This effect is mediated by the antiapoptotic effect of heat shock proteins that are transiently induced after exposure to heat at sublethal temperatures. Consequently, induction of Hsp has been proposed as a novel means of photoprotection. However, in the face of daily UVB exposure of human skin in vivo, this approach would not be useful if keratinocytes become less sensitive to Hsp induction with repeated exposure to the inducing agent. The aim of this study was to investigate whether repeated exposure to hyperthermia or to the stress protein activating cyclopentenone prostaglandin 15-deoxy-delta(12,14)-prostaglandin J2 (15dPGJ2) leads to adaptation of the cells, attenuation of the heat shock response, and abrogation of the protective effect. Normal human epidermal keratinocytes (NHEK) and the carcinoma-derived cell line A431 were exposed to either 42 degrees C or to 15dPGJ2 for 4 hours at 24-hour intervals for 4 consecutive days. The intracellular level of the 72-kDa heat shock protein (Hsp72) was determined by enzyme-linked immunosorbent assay (ELISA). Cells were exposed to UVB from a metal halide source after the last heat or 15dPGJ2 treatment, and survival was determined 24 hours after exposure by a MTT assay. Our results demonstrate that (1) heat shock and 15dPGJ2 are potent inducers of Hsp72 expression and lead to increased resistance to UVB-induced cell death in human keratinocytes; (2) re-exposure to heat shock leads to a superinduction without attenuation of the absolute increase in Hsp72 and of its UVB-protective effect; (3) the UVB tolerance induced by 15dPGJ2 is enhanced by repeated exposure without a further increase of Hsp72; (4) repeated heat shock and 15dPGJ2 up to a concentration of 1 microg/mL have no influence on cell growth over a period of 4 days. We conclude that through repeated exposure to Hsp-inducing factors, stress tolerance can be maintained without additional toxicity in human keratinocytes. These results provide a basis for the development of nontoxic Hsp inducers that can be repeatedly applied without loss of effect.     

Small doses of capsaicin given intragastrically inhibit gastric basal acid secretion in healthy human subjects.

Although the direct inhibitory effect of small dose of capsaicin on gastric secretory responses was proved in animal observations, the role of capsaicin-sensitive afferent nerves (CSAN) and the effect of capsaicin applied in small and high doses on gastric secretion in human has not been clarified yet. In this study we investigated the influence of different small doses (100-800 microg) of capsaicin given intragastrically through an orogastric tube on gastric basal secretory responses in 10 healthy human subjects. Gastric basal secretory responses (volume, H+-concentration, H+-output) were measured from the suctions of gastric juice for a 1-h period. It has been found that: a) capsaicin dose-dependently inhibited the volume and H+-output of gastric juice; b) ID50 was found to be about 400 microg for capsaicin on gastric acid secretion; c) the time interval for capsaicin-induced gastric inhibition existed for about 1 h indifferently from the higher dose (800 microg) of capsaicin given after. It has been concluded that the capsaicin (given in small doses) inhibits the gastric basal acid output via stimulation of the inhibition of capsaicin sensitive afferent nerves.     

Correlation between steroid sex hormones, egg laying capacity and cercarial shedding in Biomphalaria alexandrina snails after treatment with Haplophyllum tuberculatum

Schistosomiasis is considered the second most pre-valiant worldwide parasitic disease ranked next to malaria. It has significant economic and public health consequences in many developing countries. Several ways have been practiced in order to bring the disease under an adequate control through the breakage of the life cycle of the parasite. Snail control could be regarded as a rapid and efficient of reducing or eliminating transmission and remains among the methods of choice for schistosomiasis control. The aim of this work is to evaluate the role of Haplophyllum tuberculatum (family Rutaceae) as a plant molluscicide. The mortality rate of Biomphalaria alexandrina snails were monitored after treatment with three extracts of the plant aerial parts; petroleum ether, chloroform and ethanol. Chloroform extract that recorded the most potent effect was further evaluated through measuring the toxicity pattern against B. alexandrina snails, egg laying capacity, cercarial shedding, phenol oxidase enzyme and the levels of steroid sex hormones. Histopathological examination of hepatopancreas and ovotestis of treated snails were also done for result confirmation. Treatment of snails by chloroform extract recorded reduction in egg laying capacity, decrease in cercarial shedding, diminution in phenol oxidase enzyme, disturbance in steroid sex hormones and sever alternation of the histopathological picture of snails tissue. In conclusion, H. tuberculatum recorded molluscicidal potency against B. alexandrina snails. Further studies are needed for its environmental applications.     

Exploiting T cells specific for human minor histocompatibility antigens for therapy of leukemia

Minor histocompatibility (H) antigens are major targets of a graft-versus-leukemia (GVL) effect mediated by donor CD8(+) and CD4(+) T cells following allogeneic hematopoietic cell transplantation (HCT) between human leukocyte antigen identical individuals. In the 15 years since the first molecular characterization of human minor H antigens, significant strides in minor H antigen discovery have been made as a consequence of advances in cellular, genetic and molecular techniques. Much has been learned about the mechanisms of minor H antigen immunogenicity, their expression on normal and malignant cells, and their role in GVL responses. T cells specific for minor H antigens expressed on leukemic cells, including leukemic stem cells, can be isolated and expanded in vitro and infused into allogeneic HCT recipients to augment the GVL effect to prevent and treat relapse. The first report of the adoptive transfer of minor H antigen-specific T-cell clones to patients with leukemic relapse in 2010 illustrates the potential for the manipulation of alloreactivity for therapeutic benefit. This review describes the recent developments in T-cell recognition of human minor H antigens, and efforts to translate these discoveries to reduce leukemia relapse after allogeneic HCT.     

Kappa- opioid receptor regulates human sperm functions via SPANX-A/D protein family

The kappa-opioid receptor (KOR) is involved in the regulation of the fertilizing capacity of human sperm. Recently, a testicular-specific protein family, SPANX-A/D, has also been found to be involved in regulating this process. In order to determine if KOR has a role in the regulation of sperm fertility through the SPANX-A/D protein family, we activated the kappa opioid receptor adding its selective agonist, U50488H to normozoospermic human spermatozoa. Then, we performed immunofluorescence assays and immunoprecipitation experiments followed by LC-MS/MS. According to our results, KOR activation may cause the translocation of SPANX-A/D into the nucleus of human spermatozoa. Phosphoproteomic studies show that KOR does not cause phosphorylation changes in SPANX-A/D residues. However, interactome assays demonstrate that KOR activation provokes changes in SPANX-A/D potential interactors involved in sperm motility, energy metabolism and nuclear processes. Taking these results into account, KOR may regulate human sperm fertility through SPANX-A/D protein family, modifying its subcellular location and interactions. Although further studies are needed, this finding could help us describing the molecular mechanisms underlying sperm fertility as well as developing new strategies for treating infertility.     

Helicobacter pylori infection and skin diseases.

There is increasing evidence for systemic effects of gastric H. pylori infection which may result in extragastrointestinal disorders. This review summarizes the available medical literature up to September 1999, identified through a MEDLINE research including own studies, regarding H. pylori and skin diseases. Due to current knowledge best evidence for a potential link of H. pylori infection exists for chronic urticaria although the data are still conflicting. Thus, the search for H. pylori should be included in the diagnostic management of chronic urticaria. With regard to other skin diseases such as rosacea, hereditary or acquired angioedema due to C1-esterase inhibitor deficiency, systemic sclerosis, Sch?nlein-Henoch purpura, Sj?gren's syndrome, sweet's syndrome, and atopic dermatitis only single of few cases have been reported so far. Thus, we clearly need further randomized, double-blind and placebo-controlled studies including adequate diagnostic schedules, sufficient eradication treatment protocols, confirmation of eradication, and adequate control groups to establish a role of H. pylori in skin diseases. Caution must be taken not to accuse H. pylori as the infectious agent responsible for every disease, particularly since H. pylori infection is very common. Although from an epidemiological and morphological view the skin diseases to which H. pylori has been linked seem to be completely different it is striking that in most of them an autoimmune pathogenesis is suspected or considerable vascular impairment can be found.     

Inducible heat shock protein 70 and its role in preconditioning and exercise

Heat shock proteins (Hsp) are well known to be expressed in response to a range of cellular stresses. They are known to convey protection against protein denaturation and a subsequent immediate stress. Inducible heat shock protein 70 (Hsp70) is among the most studied of these stress proteins and its role and function are discussed here in terms of thermal and in particular exercise preconditioning. Preconditioning has been shown to confer cellular protection via expression Hsp, which may be of benefit in preventing protein damage following subsequent periods of exercise. Many studies have used animal models to gather data on Hsp70 and these and the most recent human studies are discussed.     

Development of a Multiple-Locus Variable number of tandem repeat Analysis (MLVA) for Leptospira interrogans and its application to Leptospira interrogans serovar Australis isolates from Far North Queensland, Australia

The human gastric pathogen Helicobacter pylori causes chronic gastritis, peptic ulcer disease, gastric carcinoma, and mucosa-associated lymphoid tissue (MALT) lymphoma. It infects over 50% of the worlds' population, however, only a small subset of infected people experience H. pylori-associated illnesses. Associations with disease-specific factors remain enigmatic years after the genome sequences were deciphered. Infection with strains of Helicobacter pylori that carry the cytotoxin-associated antigen A (cagA) gene is associated with gastric carcinoma. Recent studies revealed mechanisms through which the cagA protein triggers oncopathogenic activities. Other candidate genes such as some members of the so-called plasticity region cluster are also implicated to be associated with carcinoma of stomach. Study of the evolution of polymorphisms and sequence variation in H. pylori populations on a global basis has provided a window into the history of human population migration and co-evolution of this pathogen with its host. Possible symbiotic relationships were debated since the discovery of this pathogen. The debate has been further intensified as some studies have posed the possibility that H. pylori infection may be beneficial in some humans. This assumption is based on increased incidence of gastro-oesophageal reflux disease (GERD), Barrett's oesophagus and adenocarcinoma of the oesophagus following H. pylori eradication in some countries. The contribution of comparative genomics to our understanding of the genome organisation and diversity of H. pylori and its pathophysiological importance to human healthcare is exemplified in this review.     

Hsp70 protects mitotic cells against heat-induced centrosome damage and division abnormalities

The effect of heat shock on centrosomes has been mainly studied in interphase cells. Centrosomes play a key role in proper segregation of DNA during mitosis. However, the direct effect and consequences of heat shock on mitotic cells and a possible cellular defense system against proteotoxic stress during mitosis have not been described in detail. Here, we show that mild heat shock, applied during mitosis, causes loss of dynamitin/p50 antibody staining from centrosomes and kinetochores. In addition, it induces division errors in most cells and in the remaining cells progression through mitosis is delayed. Expression of heat shock protein (Hsp)70 protects against most heat-induced division abnormalities. On heat shock, Hsp70 is rapidly recruited to mitotic centrosomes and normal progression through mitosis is observed immediately after release of Hsp70 from centrosomes. In addition, Hsp70 expression coincides with restoration of dynamitin/p50 antibody staining at centrosomes but not at kinetochores. Our data show that during mitosis, centrosomes are particularly affected resulting in abnormal mitosis. Hsp70 is sufficient to protect against most division abnormalities, demonstrating the involvement of Hsp70 in a repair mechanism of heat-damaged mitotic centrosomes.     

Protein Kinase C and Melanocyte Transformation

Melanoma cells which have been isolated from metastatic melanoma tissue are able to survive and proliferate in serum supplemented media. In contrast, normal human melanocytes require the presence of growth stimulators if they are to survive in culture. A tumor promotor, 12–0-tetradecanoyl-phorbol-13-acetate (TPA) and substances that increase intracellular levels of cyclic-adenosine-monophosphate (cAMP), such as cholera toxin or isobutylmethyl xanthine, have been widely used for this purpose. The phorbol diester receptor was shown in 1982 to be the phospholipid- and calcium-dependent enzyme protein kinase C (PKC). We therefore directed our studies to the role of PKC regulation in the growth of normal human melanocytes and their transformation. Our studies show that while melanoma cells are inhibited by TPA, the growth of normal melanocytes is stimulated in a dose-dependent manner. The inhibitor, 1-(5-isoquinolinesulfonyl)-2-methyl-piperizine dihydrochloride (H7), which has been found to be the most specific for PKC, had no effect on the growth of normal melanocytes, but inhibited the growth of melanoma cells in a dose-dependent manner. PKC was isolated from the membrane and cytosol of normal melanocytes and melanoma cells. The basal (resting) levels of PKC activity in normal melanocytes was low compared to that measured in melanoma cells, and after short-term (1 hour) treatment with TPA the PKC activity was greatest at the membrane, with the activity decreasing the cytosol. Upon prolonged (48 hours) treatment with TPA, this redistribution of activity continued in normal melanocytes and the total activity increased. In melanoma cells, however, the total PKC activity decreased, particularly in the membrane fraction. A difference in activity and distribution of the enzyme was also seen after short-term (1 hour) treatment with H7. There was very little effect seen on PKC in normal melanocytes; however, the effect on melanoma cells was similar to that seen after 48 hours of exposure to TPA with a decrease in total activity, particularly in the membrane fraction. These results indicate that the regulation of PKC, in particular its activation by TPA, is altered during the transformation of normal human melanocytes     

Reduced activity of the interferon-induced double-stranded RNA-dependent protein kinase during a heat shock stress

Previous studies have shown that the antiviral response induced by interferon in murine cells could be degraded after a heat shock. Here we have confirmed that a similar effect occurs also in interferon-treated human HeLa cells subjected to a heat shock. In addition, we have investigated the fate of the interferon-induced, double-stranded RNA-dependent protein kinase in heat-shocked cells. This protein kinase is a Mr 68,000 protein (p68 kinase) which, when autophosphorylated, catalyzes phosphorylation of the protein synthesis eukaryotic initiation factor-2, thus mediating inhibition of protein synthesis. After heat shock of interferon-treated HeLa cells, the double-stranded RNA-dependent autophosphorylation of p68 kinase in cytoplasmic extracts is greatly reduced whereas the phosphorylation of other cellular proteins is not affected. In vivo, autophosphorylation of p68 kinase is also reduced in heat-shocked cells whereas there is no apparent effect on the phosphorylation state of other proteins. In such cells, the interferon-mediated antiviral response becomes modified according to the virus challenge, i.e. these cells remain resistant to vesicular stomatitis virus but become partially sensitive to encephalomyocarditis virus (EMCV) infection. The reduction in the activity of p68 kinase is due to its reduced nonionic detergent solubility occurring during the heat shock period. The resultant reduced detergent extractibility of p68 kinase is dependent on the intensity of the thermal stress. In contrast to the effect after a heat shock, arsenite treatment of interferon-treated HeLa cells induces heat shock proteins, but neither modifies the antiviral response nor affects the extractibility of p68 kinase. These results indicate that the degradation of the anti-EMCV response and reduced p68 kinase activity occur in response to heat treatment independently of the induction of heat shock proteins. The role of p68 kinase in the mechanism of the antiviral response against EMCV and vesicular stomatitis virus is discussed.     

Heat shock protein 27 as a neuroprotective biomarker and a suitable target for stem cell therapy and pharmacotherapy in ischemic stroke

Ischemic stroke is a major common cause of death and long‐term disability worldwide. Several pathophysiological events including excitotoxicity, oxidative/nitrative stress, inflammation, and apoptosis are involved in ischemic injuries. Recently, the molecular mechanisms involved in cerebral ischemia through a focus on a member of small heat shock proteins family, Hsp27, has been developed. Notably, following exposure to ischemia, Hsp27 expression in the brain could be increased rather than the normal condition and it may play an important role in neuroprotection after ischemic stroke. The neuroprotection effects of Hsp27 may arise from its anti‐oxidant, anti‐inflammatory, anti‐apoptotic, and chaperonic properties. Moreover, some therapeutic strategies such as stem cell therapy and pharmacotherapy have been developed with Hsp27 targeting. In this review, we describe the function and structure of Hsp27 and its possible role in neuroprotection after ischemic stroke. Finally, we present current studies in stroke therapy, which focused on Hsp27 targeting.