Biochemical mechanism of combined effect of ethambutol and sparfloxacin against Mycobacterium smegmatis

M. smegmatis cells grown in the presence of combination of ethambutol (EMB) and sparfloxacin (SPX) had decreased level of total cellular lipids as compared to control as well as cells grown in the presence of sub-inhibitory concentration (MIC50) of individual drugs. Amongst various phospholipids analyzed, maximum decrease was observed in the content of phosphatidylinositolmannosides (PIMs) of the cells grown in combination of EMB and SPX. In contrast, the subcellular distribution of phospholipids revealed a significant increase in PIMs content of both cell wall and cell membrane of the cells grown in the presence of combination of drugs as compared to control as well as individual drugs. Mycolic acids of M. smegmatis cells were found to be main targets as combination of drugs resulted in significant decrease in total cellular as well as cell wall mycolic acids as compared to control and individual drugs. Changed lipid composition of M. smegmatis cells grown in the presence of MIC50 of EMB, SPX and combination resulted in significant surface changes as was evident from decreased limiting fluorescence (Fmax) intensity of 1-anilinonaphthalene-8-sulfonate (ANS). Thus, the results of this study suggested that ethambutol and sparfloxacin in combination exerted their antimycobacterial effect principally due to their action on phosphatidylinositolmannosides (PIMs) and mycolic acids, which form the permeability barrier of mycobacteria.     

Factors associated with birth rate and live birth rate in multi-male breeding groups of rhesus monkeys

Reproductive records of 284 female rhesus monkeys housed in six multimale corrals at the California Primate Research Center were examined for the birth seasons 1977–1982 to determine possible associations between the probability of birth or live birth and female age, parity, origin, parturition in the previous season, infant birth date, and infant birth date in previous season. Multiple logistic regression analysis was used to identify and quantitate the effects of factors on the probability of birth or live birth, while controlling for the possibly confounding effects of other factors in the model. Females who had infants early in the previous season were 2.5 times as likely to give birth as those who had infants late in the previous season. Females with two or three previous births were 2.1 times as likely to give birth, and those with four or five previous births were 6.7 times as likely to give birth as were females with no or one previous birth. Controlling for other factors (age, parity, and timing of birth in the previous season), corralborn females were 3.3 times as likely to give birth as either wild-caught or domestic-born monkeys not native to the corrals. Domestic-born females who were not corral natives were 0.3 times as likely to have live births as wild-caught females. Births late in the season were 1.8 times as likely to result in live infants as births early in the season.     

Natural solution to antibiotic resistance: bacteriophages ‘The Living Drugs’

Antibiotics have been a panacea in animal husbandry as well as in human therapy for decades. The huge amount of antibiotics used to induce the growth and protect the health of farm animals has lead to the evolution of bacteria that are resistant to the drug’s effects. Today, many researchers are working with bacteriophages (phages) as an alternative to antibiotics in the control of pathogens for human therapy as well as prevention, biocontrol, and therapy in animal agriculture. Phage therapy and biocontrol have yet to fulfill their promise or potential, largely due to several key obstacles to their performance. Several suggestions are shared in order to point a direction for overcoming common obstacles in applied phage technology. The key to successful use of phages in modern scientific, farm, food processing and clinical applications is to understand the common obstacles as well as best practices and to develop answers that work in harmony with nature.     

SLX4: multitasking to maintain genome stability

The SLX4/FANCP tumor suppressor has emerged as a key player in the maintenance of genome stability, making pivotal contributions to the repair of interstrand cross-links, homologous recombination, and in response to replication stress genome-wide as well as at specific loci such as common fragile sites and telomeres. SLX4 does so in part by acting as a scaffold that controls and coordinates the XPF–ERCC1, MUS81–EME1, and SLX1 structure-specific endonucleases in different DNA repair and recombination mechanisms. It also interacts with other important DNA repair and cell cycle control factors including MSH2, PLK1, TRF2, and TOPBP1 as well as with ubiquitin and SUMO. This review aims at providing an up-to-date and comprehensive view on the key functions that SLX4 fulfills to maintain genome stability as well as to highlight and discuss areas of uncertainty and emerging concepts.     

The use of probiotics in shrimp aquaculture

Shrimp aquaculture, as well as other industries, constantly requires new techniques in order to increase production yield. Modern technologies and other sciences such as biotechnology and microbiology are important tools that could lead to a higher quality and greater quantity of products. Feeding and new practices in farming usually play an important role in aquaculture, and the addition of various additives to a balanced feed formula to achieve better growth is a common practice of many fish and shrimp feed manufacturers and farmers. Probiotics, as 'bio-friendly agents' such as lactic acid bacteria and Bacillus spp., can be introduced into the culture environment to control and compete with pathogenic bacteria as well as to promote the growth of the cultured organisms. In addition, probiotics are nonpathogenic and nontoxic microorganisms without undesirable side-effects when administered to aquatic organisms. These strains of bacteria have many other positive effects, which are described in this article.     

Sphingolipid players in the leukemia arena

Sphingolipids function as bioactive mediators of different cellular processes, mostly proliferation, survival, differentiation and apoptosis, besides being structural components of cellular membranes. Involvement of sphingolipid metabolism in cancerogenesis was demonstrated in solid tumors as well as in hematological malignancies. Herein, we describe the main biological and clinical aspects of leukemias and summarize data regarding sphingolipids as mediators of apoptosis triggered in response to anti-leukemic agents and synthetic analogs as inducers of cell death as well. We also report the contribution of molecules that modulate sphingolipid metabolism to development of encouraging strategies for leukemia treatment. Finally we address how deregulation of sphingolipid metabolism is associated to occurrence of therapy resistance both in vitro and in vivo. Sphingolipids can be considered promising therapeutic tools alone or in combination with other compounds, as well as valid targets in the attempt to eradicate leukemia and overcome drug resistance.     

Comet assay: a reliable tool for the assessment of DNA damage in different models

New chemicals are being added each year to the existing burden of toxic substances in the environment. This has led to increased pollution of ecosystems as well as deterioration of the air, water, and soil quality. Excessive agricultural and industrial activities adversely affect biodiversity, threatening the survival of species in a particular habitat as well as posing disease risks to humans. Some of the chemicals, e.g., pesticides and heavy metals, may be genotoxic to the sentinel species and/or to non-target species, causing deleterious effects in somatic or germ cells. Test systems which help in hazard prediction and risk assessment are important to assess the genotoxic potential of chemicals before their release into the environment or commercial use as well as DNA damage in flora and fauna affected by contaminated/polluted habitats. The Comet assay has been widely accepted as a simple, sensitive, and rapid tool for assessing DNA damage and repair in individual eukaryotic as well as some prokaryotic cells, and has increasingly found application in diverse fields ranging from genetic toxicology to human epidemiology. This review is an attempt to comprehensively encase the use of Comet assay in different models from bacteria to man, employing diverse cell types to assess the DNA-damaging potential of chemicals and/or environmental conditions. Sentinel species are the first to be affected by adverse changes in their environment. Determination of DNA damage using the Comet assay in these indicator organisms would thus provide information about the genotoxic potential of their habitat at an early stage. This would allow for intervention strategies to be implemented for prevention or reduction of deleterious health effects in the sentinel species as well as in humans. IITR Communication No. 2656     

Oligonucleotide directed mutagenesis of the human beta-globin gene: a general method for producing specific point mutations in cloned DNA.

A nonadecanucleotide has been used both as a site specific mutagen to introduce a T leads to A transversion mutation in the human beta-globin gene cloned in pBR322 as well as a probe to screen transformed colonies for the desired mutant. The specificity of the oligonucleotide as a mutagen and as a hybridization probe provide a general method for producing site specific mutations in DNA cloned in plasmid vectors such as pBR322.     

Abundance of RNase4 and RNase5 mRNA and protein in host defence related tissues and secretions in cattle

Members of the RNaseA family are present in various tissues and secretions but their function is not well understood. Some of the RNases are proposed to participate in host defence. RNase4 and RNase5 are present in cows' milk and have antimicrobial activity. However, their presence in many tissues and secretions has not been characterised. We hypothesised that these two RNases are present in a range of tissues and secretions where they could contribute to host defence. We therefore, determined the relative abundance of RNase4 and RNase5 mRNA as well as protein levels in a range of host defence related and other tissues as well as a range of secretions in cattle, using real time PCR and western blotting. The two RNases were found to be expressed in liver, lung, pancreas, mammary gland, placenta, endometrium, small intestine, seminal vesicle, salivary gland, kidney, spleen, lymph node, skin as well as testes. Corresponding proteins were also detected in many of the above tissues, as well as in seminal fluid, mammary secretions and saliva. This study provides evidence for the presence of RNase4 and RNase5 in a range of tissues and secretions, as well as some major organs in cattle. The data are consistent with the idea that these proteins could contribute to host defence in these locations. This work contributes to growing body of data suggesting that these proteins contribute to the physiology of the organism in a more complex way than acting merely as digestive enzymes.     

Prospects for the use of sulfated polysaccharides from brown seaweeds as vaccine adjuvants

The data from the Russian and foreign literature on the effects of brown seaweed-derived sulfated polysaccharides (fucoidans) used as potential vaccine adjuvants to enhance the anti-infection and anti-tumor immune response are discussed in the present review. Due to their low toxicity, high biocompatibility, safety, and good tolerability by macroorganisms, as well as their mechanisms of immunomodulatory activity, fucoidans can be considered as promising adjuvants to administer in the composition of prophylactic and therapeutic vaccines. Fucoidans are agonists to receptors of innate immunity and are potent inducers of the cellular and humoral immune response, which is an important factor to be taken into account in the development of vaccines against various pathogens, including viruses, as well as anti-tumor vaccines. The results of numerous studies in which sulfated polysaccharides were tested as components of experimental vaccines, as presented in this review, show that these substances can be used as safe and effective adjuvants.     

Sphingolipid players in the leukemia arena

Sphingolipids function as bioactive mediators of different cellular processes, mostly proliferation, survival, differentiation and apoptosis, besides being structural components of cellular membranes. Involvement of sphingolipid metabolism in cancerogenesis was demonstrated in solid tumors as well as in hematological malignancies. Herein, we describe the main biological and clinical aspects of leukemias and summarize data regarding sphingolipids as mediators of apoptosis triggered in response to anti-leukemic agents and synthetic analogs as inducers of cell death as well. We also report the contribution of molecules that modulate sphingolipid metabolism to development of encouraging strategies for leukemia treatment. Finally we address how deregulation of sphingolipid metabolism is associated to occurrence of therapy resistance both in vitro and in vivo. Sphingolipids can be considered promising therapeutic tools alone or in combination with other compounds, as well as valid targets in the attempt to eradicate leukemia and overcome drug resistance.     

Effects of successional status, habit, sexual systems, and pollinators on flowering patterns in tropical rain forest trees

Based on data from observations of 302 tree species at La Selva, Costa Rica, we tested a range of hypotheses about the relationship between flowering parameters such as time, frequency, and duration and ecological features such as successional status, habit, sexual systems, and pollen vectors with and without considering the effect of family membership. We predicted that early successional species would flower any time of the year, but species pollinated by different vectors as well as dioecious species would flower nonrandomly across seasons. However, there was little evidence that flowering time varied with successional status, pollen vectors, and sexual systems. As we predicted, supra-annual flowering was proportionately less common in early successional species as compared to late ones, in understory species as compared to canopy species, and in dioecious species as compared to those with hermaphroditic flowers. When considering phylogeny, however, supra-annual flowering in the understory was not as rare as predicted. Our prediction of longer flowering in the early successional species as compared to late successional species was also supported. Predictions about longer flowering of dioecious species as compared to hermaphroditic species and of species pollinated by generalist vectors as compared to the specialists were not supported, though there was a trend in the expected direction.     

Peptide synthesis catalyzed by polyethylene glycol-modified chymotrypsin in organic solvents

Chymotrypsin modified with polyethylene glycol was successfully used for peptide synthesis in organic solvents. The benzene-soluble modified enzyme readily catalyzed both aminolysis of N-benzoyl-L-tyrosine p-nitroanilide and synthesis of N-benzoyl-L-tyrosine butylamide in the presence of trace amounts of water. A quantitative reaction was obtained when either hydrophobic or bulky amides of L- as well as D-amino acids were used as acceptor nucleophiles, while almost no reaction occurred with free amino acids or ester derivatives. The acceptor nucleophile specificity of modified chymotrypsin as a catalyst in the formation of both amide and peptide bonds in organic solvents was quite comparable to that in aqueous solution as well as to that of the leaving group in hydrolysis reactions. By contrast, the substrate specificity of modified chymotrypsin in organic solvents was different from that in water since arginine and lysine esters were found to be as effective as aromatic amino acids to form the acyl-enzyme with subsequent synthesis of a peptide bond.     

Acute adaptation of mice to hypoxic hypoxia.

Tolerance to hypoxia in vivo and in vitro was significantly increased by acute and repetitive exposure of mice to autoprogressive hypoxia. The average tolerance times of the successive 2nd, 3rd, 4th and 5th runs of exposure were, respectively, 2, 4, 6 and 8 times as long as that of the first exposure. The survival times under hypobaric chamber and cyanide toxification in the 4th exposure were, respectively, 10 (and even as much as 86) and 4 times those in control mice without exposure to hypoxia. Mandibular respiration and spinal reflex in vitro in hypoxia-resistant animals lasted 5-6 times as long as in control animals not previously exposed to hypoxia. Animals that received brain homogenate from hypoxia-resistant mice remained alive in a hypobaric chamber 2 times as long as those that received homogenate from controls and those that received saline. These results indicate that a kind of quickly developing adaptation with increased tolerance is achieved by acute and repetitive exposure of mice to progressive autohypoxia and some plastic or adaptive changes occur in the brain of hypoxia-resistant animals, including the production of some kind of water-soluble antihypoxic factors.     

Long-lasting ill-effects of neonatal qualitative and/or quantitative dysnutrition in the human

Qualitative dysnutrition (pure bottle-feeding) as well as quantitative dysnutrition (overnutrition as well as undernutrition) during the first trimenon of postnatal life was found to lead to long-lasting mental, psychic and/or physical ill-effects in the human. Females who were completely bottlefed during neonatal life showed significantly decreased school achievements as well as significantly decreased learning capacity and social adaptability at 16 years of age as compared to females who were purely breastfed or breast- plus bottlefed in neonatal life. Males who were artificially overfed during neonatal life also showed significantly decreased school achievements and significantly decreased learning capacity in adolescence as compared to males with normal weight development in neonatal life.     

Morphological and biochemical characterization of macrophages activated by carrageenan and lipopolysaccharide in vivo

Macrophages are able to recognize, internalize and destroy a large number of pathogens, thus restricting the infection until adaptive immunity is initiated. In this work our aim was to analyze the surface charge of cells activated by carrageenan (CAR) and lipopolysaccharide (LPS) through light and electron microscopy approaches as well as the release of inflammatory mediators in vitro. The ultrastuctural analysis and the light microscopy data showed that in vivo administration of CAR represents a potent inflammatory stimulation for macrophages leading to a high degree of spreading, an increase in their size, in the number of the intracellular vacuoles and membrane projections as compared to the macrophages collected from untreated animals as well as mice submitted to LPS. Our data demonstrated that CAR stimulated-macrophages displayed a remarkable increase in nitric oxide production and PGE2 release as compared to the cells collected from non-stimulated and stimulated mice with LPS in vivo. On the other hand, non-stimulated macrophages as well as macrophages stimulated by LPS produce almost the same quantities of TNF-alpha, while in vivo stimulation by CAR leads to a 30-40% increase of cytokine release in vitro compared to the other groups. In conclusion, our morphological and biochemical data clearly showed that in vivo stimulation with CAR induces a potent inflammatory response in macrophages representing an interesting model to analyze inflammatory responses.     

Industrial and biotechnological applications of laccases: a review

Laccases have received much attention from researchers in last decades due to their ability to oxidise both phenolic and non-phenolic lignin related compounds as well as highly recalcitrant environmental pollutants, which makes them very useful for their application to several biotechnological processes. Such applications include the detoxification of industrial effluents, mostly from the paper and pulp, textile and petrochemical industries, use as a tool for medical diagnostics and as a bioremediation agent to clean up herbicides, pesticides and certain explosives in soil. Laccases are also used as cleaning agents for certain water purification systems, as catalysts for the manufacture of anti-cancer drugs and even as ingredients in cosmetics. In addition, their capacity to remove xenobiotic substances and produce polymeric products makes them a useful tool for bioremediation purposes. This paper reviews the applications of laccases within different industrial fields as well as their potential extension to the nanobiotechnology area.     

Tissue microarrays as a platform for proteomic investigation

Tissue microarrays have become an essential tool in translational pathology. They are used to confirm results from other experimental platforms, such as expression microarrays, as well as a primary tool to explore the expression profile of proteins by immunohistochemical analysis. Tissue microarrays are routinely used molecular epidemiology, drug development and determining the diagnostic, prognostic and predictive value of new biomarkers. By applying traditional protein based assays, as well as novel assays to the platform, tissue microarrays have gained a new utility as a proteomic tool for both basic science as well as clinical investigation. This article will explore the new approaches that are being applied to tissue microarrays to, characterize the human proteome, and new technologies that allow tissue microarrays to function as a protein array. The U.S. Government's right to retain a non-exclusive, royalty-free license in and to any copyright is acknowledged     

Dendritic cells: limited potential in immunotherapy

Dendritic cells (DC) represent the most potent antigen-presenting cells (APC) of the immune system for their unique capability of presenting antigen to T-cells. Their use as cellular vaccines after charging with antigen ex vivo has been shown to induce protective and therapeutic anti-tumor immunity with regression of tumor manifestations in animal models of experimental cancer therapy. Human monocyte-derived DC (MoDC) generated in vitro in the presence of GM-CSF and IL-4 are regarded equivalent to immature DC. They can be induced to mature under various experimental conditions. MoDC, in their immature as well as mature state have been widely used for experimental as well as for clinical purposes. However, unequivocal proof for the clinical efficiency of MoDC-based anti-tumor vaccinations is still missing. There is now increasing experimental evidence demonstrating that MoDC may be hampered in their ability to migrate in response to inflammatory as well as homeostatic chemataxins. We therefore suggest that MoDC may not represent the equivalent of migratory DC in vivo limiting their use as magic bullets in tumor immunotherapy.     

Three-dimensional adipose tissue model using low shear bioreactors

Summary Presented here are techniques developed to culture and analyze three-dimensional (3-D) adipose-like tissues as a means to bridge the gap between current liminations in culturing preadipocytes (PAs) and that of providing clinically relevant volumes of adipose tissue useful for soft tissue engineering stratgies in reconstructive surgery. Pilot studies were performed to determine techniques to visualize and analyze 3-D PA-like tissues as well as to develop successful strategies to culture 3T3-L1 cells in a high aspect ratio vessel rotating-wall bioreactor both with and without microcarriers. Next, a series of cultures were accessed to verify these techniques as well as to compare the culture of the cells with and without microcarriers. Finally, a perfused rotating-wall bioreactor was used to further investigate the nature of the aggregates or tissues being generated. The aggregates that formed in the perfused system were analyzed via histology and in vivo animal studies. PA-like tissues as large as 4–5 mm in diameter without microcarriers that were capable of lipid-loading and composed of viable cells were achieved. We have successfully demonstrated that large tissue aggregates can be grown in bioreactor culture systems.