Cloning of chromosomal beta-lactamase genes from Yersinia enterocolitica

Two beta-lactamase genes present in the chromosome of Yersinia enterocolitica have been cloned individually into the plasmid pACY184 and expressed in Escherichia coli. The gene for broad-spectrum beta-lactamase I ('A') was cloned from a strain belonging to the O:3 serotype, and the gene for (cephalosporinase) beta-lactamase II ('B') was cloned from a strain of the O:5b serotype. The properties of the beta-lactamases expressed in E. coli are similar to those previously described in Y. enterocolitica.     

Capsaicin as an inducer of damage-associated molecular patterns (DAMPs) of immunogenic cell death (ICD) in human bladder cancer cells

Few conventional cytotoxic anticancer therapeutics induce immunogenic cell death (ICD). This means that they induce tumor cells to undergo apoptosis while eliciting the emission of a spatiotemporal-defined combination of damage-associated molecular patterns (DAMPs) decoded by the immune system to activate antitumor immunity effective for long-term therapeutic success. The neurotoxin capsaicin (CPS) can induce both cancer cell apoptosis and immune-mediated tumor regression. In the present study, we investigated whether CPS is capable of eliciting the emission of ICD hallmarks in human bladder cancer cell lines undergoing apoptosis. We demonstrated that CPS induces pre- and early apoptotic cell surface exposure of calreticulin (CRT), HSP90, and HSP70 as well as ATP release. Moreover, CRT exposure was prevented by inhibition of endoplasmic reticulum–Golgi traffic by brefeldin A. Furthermore, high-mobility group box 1, HSP90, and HSP70 were passively released at late apoptotic stages. We provide the first evidence that CPS is an inducer of ICD hallmarks, suggesting CPS as a novel potential immunogenic cytotoxic agent.     

Cytochrome c dysregulation induced by HIV infection of astrocytes results in bystander apoptosis of uninfected astrocytes by an IP3 and calcium‐dependent mechanism

HIV entry into the CNS is an early event after peripheral infection, resulting in neurologic dysfunction in a significant number of individuals despite successful anti‐retroviral therapy. The mechanisms by which HIV mediates CNS dysfunction are not well understood. Our group recently demonstrated that HIV infection of astrocytes results in survival of HIV infected cells and apoptosis of surrounding uninfected astrocytes by the transmission of toxic intracellular signals through gap junctions. In the current report, we characterize the intracellular signaling responsible for this bystander apoptosis. Here, we demonstrate that HIV infection of astrocytes results in release of cytochrome C from the mitochondria into the cytoplasm, and dysregulation of inositol trisphosphate/intracellular calcium that leads to toxicity to neighboring uninfected astrocytes. Blocking these dysregulated pathways results in protection from bystander apoptosis. These secondary messengers that are toxic in uninfected cells are not toxic in HIV infected cells, suggesting that HIV protects these cells from apoptosis. Thus, our data provide novel mechanisms of HIV mediated toxicity and generation of HIV reservoirs. Our findings provide new potential therapeutic targets to reduce the CNS damage resulting from HIV infection and to eradicate the generation of viral reservoirs.

     

FNDC5/Irisin Is Not Only a Myokine but Also an Adipokine

Exercise provides clear beneficial effects for the prevention of numerous diseases. However, many of the molecular events responsible for the curative and protective role of exercise remain elusive. The recent discovery of FNDC5/irisin protein that is liberated by muscle tissue in response to exercise might be an important finding with regard to this unsolved mechanism. The most striking aspect of this myokine is its alleged capacity to drive brown-fat development of white fat and thermogenesis. However, the nature and secretion form of this new protein is controversial. The present study reveals that rat skeletal muscle secretes a 25 kDa form of FNDC5, while the 12 kDa/irisin theoretical peptide was not detected. More importantly, this study is the first to reveal that white adipose tissue (WAT) also secretes FNDC5; hence, it may also behave as an adipokine. Our data using rat adipose tissue explants secretomes proves that visceral adipose tissue (VAT), and especially subcutaneous adipose tissue (SAT), express and secrete FNDC5. We also show that short-term periods of endurance exercise training induced FNDC5 secretion by SAT and VAT. Moreover, we observed that WAT significantly reduced FNDC5 secretion in fasting animals. Interestingly, WAT of obese animals over-secreted this hormone, which might suggest a type of resistance. Because 72% of circulating FNDC5/irisin was previously attributed to muscle secretion, our findings suggest a muscle-adipose tissue crosstalk through a regulatory feedback mechanism.     

Set Configuration in Resistance Exercise: Muscle Fatigue and Cardiovascular Effects

Purpose

Cardiovascular responses of traditional resistance (TS) training have been extensively explored. However, the fatigue mechanisms associated with an intra-set rest configuration (ISR) have not been investigated. This study compares two modalities of set configurations for resistance exercise that equates work to rest ratios and measures the central and peripheral fatigue in combination with cortical, hemodynamic and cardiovascular measures.

Methods

11 subjects performed two isometric knee extension training sessions using TS and ISR configurations. Voluntary activation (VA), single twitch amplitude, low frequency fatigue (LFF), Mwave, motor evoked potential (MEP), short intracortical inhibition (SICI), intracortical facilitation (ICF) and heart rate variability were evaluated before and after each training session. During each session beat to beat heart rate, blood pressure and rate pressure product (RPP) were also evaluated.

Results

After exercise VA decreased significantly for TS but not for ISR (P < 0.001), single twitch amplitude and LFF values were lower for TS than ISR (P < 0.004), and SICI was reduced only for the TS configuration (P = 0.049). During exercise RPP values were significantly higher for the TS than for ISR (P = 0.001). RPP correlated with VA for TS (r = -.85 P < 0.001) suggesting a relationship between central fatigue and cardiovascular stress.

Conclusions

We conclude that ISR induced lower central and peripheral fatigue as well as lower cardiovascular stress in comparison with TS configuration. Our study suggests that set configuration is a key factor in the regulation of the neuromuscular and cardiovascular responses of resistance training.     

Genotoxic ability of cadmium, chromium and nickel salts studied by kinetochore staining in the cytokinesis-blocked micronucleus assay

The aneugenic and clastogenic ability of cadmium chloride(II), cadmium sulfate(II), nickel chloride(II), nickel sulfate(II), chromium chloride(III) and potassium dichromate(IV) have been evaluated through kinetochore-stained micronucleus test. Traditional genotoxicity assays evaluate DNA damage, gene mutations and chromosome breakage. However, these tests are not adequate to detect aneugenic agents that do not act directly on DNA. Staining kinetochores in the cytokinesis-blocked micronucleus assay is a useful way to discriminate between clastogens and aneuploidogens and may allow a rapid identification of aneuploidy-inducing environmental compounds.Human diploid fibroblasts (MRC-5) were employed. All compounds increased micronuclei frequency in a statistically significant way. However, increases in kinetochore-positive micronuclei frequencies were higher than in kinetochore-negative ones. The present work demonstrates the genotoxic ability of the cadmium and chromium salts studied. Aneugenic as well as clastogenic ability could be observed with this assay. Nickel salts, as it was expected because of their known weak mutagenicity, showed lower genotoxic effects than the other metal salts studied. As the test employed only allows the detection of malsegregation, it is proposed that this mechanism is at least one of those by which the tested metal salts induced aneuploidy. On the other hand, visualization of kinetochores in all experiments suggests that the compounds studied did not act by damaging these structures.     

Kinetic study of phenol hydroxylase and catechol 1,2-dioxygenase biosynthesis by <Emphasis Type="Italic">Candida tropicalis</Emphasis> cells grown on different phenolic substrates

When Candida tropicalis was grown on phenol, catechol or resorcinol, the highest levels of specific activity of phenol hydroxylase (EC. 1.14.13.7) and catechol 1,2-dioxygenase (EC. 1.13.11.1) were attained with phenol. With the three aromatic compounds tested, the yeast cells exhibited sharp peaks of specific activity of both enzymes at particular incubation times. Phenol-induced cells containing high levels of both enzymes were capable of degrading rapidly and without delay 4-chlorophenol and 2,6-dichlorophenol, and to a lesser extend pentachlorophenol. However, the yeast could not grow on chlorophenols as major carbon and energy source.     

Integrated control of Boophilus microplus ticks in Cuba based on vaccination with the anti-tick vaccine Gavac TM

Boophilus microplus has developed resistance against a range of chemical acaricides which has stimulated the development of alternative methods such as vaccination against ticks. In Cuba, the Bm86-based recombinant vaccine Gavac^TM has been successfully used in a number of controlled laboratory and field trials in cattle against B. microplus. In this paper, we have evaluated Gavac^TM in a large scale field trial wherein 588,573 dairy cattle were vaccinated with the aim to reduce the number of acaricidal treatments. It was found that the number of acaricidal treatments could be reduced by 87% over a period of 8 years (1995–2003). Prior to the introduction of the vaccine, 54 clinical cases of babesiosis and six fatal cases were reported per 1000 animals. Six years later, the incidence of babesiosis was reduced to 1.9 cases per 1000 cattle and mortality reduced to 0.18 per 1000. The national consumption of acaricides in Cuba could be reduced by 82% after the implementation of the integrated anti-B. microplus control program.