Cell population indexes of spermatogenic yield and testicular sperm reserves in adult jaguars (Panthera onca)

The intrinsic yield of spermatogenesis and supporting capacity of Sertoli cells are the desirable indicators of sperm production in a species. The objective of the present study was to quantify intrinsic yield and the Sertoli cell index in the spermatogenic process and estimate testicular sperm reserves by histological assessment of fragments obtained by testicular biopsy of five adult jaguars in captivity. The testicular fragments were fixed in 4% glutaric aldehyde, dehydrated at increasing alcohol concentrations, included into hydroxyethyl methacrylate, and were cut into 4 μm thickness. In the seminiferous epithelium of the jaguar, 9.2 primary spermatocytes in pre-leptotene were produced by “A” spermatogonia. During the meiotic divisions only 3.2 spermatids were produced by a primary spermatocyte. The general spermatogenic yield of the jaguar was about 23.4 cells and each Sertoli cell was able to maintain about 19.2 germ cells, 11 of them were round spermatids. In each seminiferous epithelium cycle about 166 million spermatozoa were produced by each gram of testicular tissue. In adult jaguars, the general spermatogenic yield was similar to the yield observed in pumas, greater than that observed for the domestic cat, but less compared to most domestic animals.     

Angiogenesis and Inflammation Signaling Are Targets of Beer Polyphenols on Vascular Cells

Emerging evidence indicates that chronic inflammation and oxidative stress cluster together with angiogenic imbalance in a wide range of pathologies. In general, natural polyphenols present health‐protective properties, which are likely attributed to their effect on oxidative stress and inflammation. Hops used in beer production are a source of polyphenols such as xanthohumol (XN), and its metabolites isoxanthohumol (IXN) and phytoestrogen 8‐prenylnaringenin (8PN). Our study aimed to evaluate XN, IXN, and 8PN effects on angiogenesis and inflammation processes. Opposite in vitro effects were observed between 8PN, stimulating endothelial and smooth muscle cell (SMC) growth, motility, invasion and capillary‐like structures formation, and XN and IXN, which inhibited them. Mouse matrigel plug and rat skin wound‐healing assays confirmed that XN and IXN treatments reduced vessel number as well as serum macrophage enzymatic activity, whereas 8PN increased blood vessels formation in both assays and enzyme activity in the wound‐healing assay. A similar profile was found for serum inflammatory interleukin‐1β quantification, in the wound‐healing assay. Our data indicate that whereas 8PN stimulates angiogenesis, XN and IXN manifested anti‐angiogenic and anti‐inflammatory effects in identical conditions. These findings suggest that the effects observed for individual compounds on vascular wall cells must be carefully taken into account, as these polyphenols are metabolized after in vivo administration. The modulation of SMC proliferation and migration is also of special relevance, given the role of these cells in many pathological conditions. Furthermore, these results may provide clues for developing useful therapeutic agents against inflammation‐ and angiogenesis‐associated pathologies. J. Cell. Biochem. 111: 1270–1279, 2010. © 2010 Wiley‐Liss, Inc.     

Genetic transformation of Rangpur lime (Citrus limonia osbeck) with thebO (bacterio-opsin) genen and its initial evaluation forPhytophthora nicotianae resistance

Transgenic plants expressing the bacterio-opsin (bO) gene can spontaneously activate programmed cell death (ped) and may enhance broad-spectrum pathogen resistance by activating an intrinsic defense pathway in plant species such as tobacco and potato. In this work, we produced transgenic Rangpur lime plants with thebO gene, viaAgrobacterium tumefaciens-mediated transformation, and evaluated these plants forPhytophthora nicotianae resistance. Two transgenic lines were successfully regenerated and transformation was confirmed by GUS activity assay, PCR analysis, Southern, Northern and Western blot analyses, in addition to detecting the expressed bO protein by an immunological approach. Evaluation forPhytophthora nicotianae resistance was carried out by plant inoculations with the pathogen and quantification of the affected area. One of the two transgenic lines showed greater tolerance to the fungal pathogen as compared to the control, with significantly smaller stem lesions after pathogen challenge. This increase in pathogen tolerance is correlated with a significantly higher level of transgene expression in this line when compared with the other transgenic line. This is the first report of the introduction of a potentially important gene into Rangpur lime to provide novel pathogen tolerance.     

A specific short dextrin-hydrolyzing extracellular glucosidase from the thermophilic fungus Thermoascus aurantiacus 179-5

The thermophilic fungus Thermoascus aurantiacus 179-5 produced large quantities of a glucosidase which preferentially hydrolyzed maltose over starch. Enzyme production was high in submerged fermentation, with a maximal activity of 30 U/ml after 336 h of fermentation. In solid-state fermentation, the activity of the enzyme was 22 U/ml at 144 h in medium containing wheat bran and 5.8 U/ml at 48 h when cassava pulp was used as the culture medium. The enzyme was specific for maltose, very slowly hydrolyzed starch, dextrins (2-7G) and the synthetic substrate (alpha-PNPG), and did not hydrolyze sucrose. These properties suggest that the enzyme is a type II alpha-glucosidase. The optimum temperature of the enzyme was 70 degrees . In addition, the enzyme was highly thermostable (100% stability for 10 h at 60 degrees and a half-life of 15 min at 80 degrees), and stable within a wide pH range.     

Adhesion of water stressed Helicobacter pylori to abiotic surfaces

AIM: The main aim of this work was to study and compare the adhesion of water exposed Helicobacter pylori to six different substrata and correlate any changes in morphology, physiology, ability to form aggregates and cultivability when in the planktonic or in the sessile phase. METHODS AND RESULTS: The number of total cells adhered for different water exposure times and modifications in the cell shape were evaluated using epifluorescence and scanning electron microscopy, and physiology assessed using Syto9 and propidium iodide (PI) cellular uptake. All abiotic surfaces were rapidly colonized by H. pylori, and colonization appeared to reach a steady state after 96 h with levels ranging from 2.3 x 10(6) to 3.6 x 10(6) total cells cm(-2). Cell morphology was largely dependent on the support material, with spiral bacteria, associated with the infectious form of H. pylori, subsisting in a higher percentage on nonpolymeric substrata. Also, sessile bacteria were generally able to retain the spiral shape for longer when compared with planktonic bacteria, which became coccoid more quickly. The formation of large aggregates, which may act as a protection mechanism against the negative impact of the stressful external environmental conditions, was mostly observed on the surface of copper coupons. However, Syto9 and PI staining indicates that most of H. pylori attached to copper or SS304 have a compromised cell membrane after only 48 h. Cultivability methods were only able to detect the bacteria up to the 2 h exposure-time and at very low levels (up to 500 CFU cm(-2)). CONCLUSIONS: The fact that the pathogen is able to adhere, retain the spiral morphology for longer and form large aggregates when attached to different plumbing materials appeared to point to pipe materials in general, and copper plumbing in particular, as a possible reservoir of virulent H. pylori in water distribution systems. However, the Syto9/PI staining results and cultivability methods indicate that the attached H. pylori cells quickly enter in a nonviable physiological state. SIGNIFICANCE AND IMPACT OF THE STUDY: This represents the first study of H. pylori behaviour in water-exposed abiotic surfaces. It suggests that co-aggregation with the autochthonous heterotrophic consortia present in water is necessary for a longer survival of the pathogen in biofilms associated to drinking water systems.     

Influence of Hydrological Pulse on Bacterial Growth and DOC Uptake in a Clear-Water Amazonian Lake

This study was conducted to evaluate: (1) the bacterial growth and the dissolved organic carbon (DOC) uptake in an Amazonian lake (Lake Batata) at high-water and low-water periods of the flood pulse; (2) the influence of nitrogen and phosphorus (NP) additions on bacterial growth and DOC uptake in Lake Batata at two flood pulse periods; and (3) the bioavailability of the main DOC sources in Lake Batata. Lake Batata is a typical clear-water Amazonian lake, located in the watershed of Trombetas River, Central Amazon, Brazil. Bacterial batch cultures were set up with 90% 0.2-μm filtered water and 10% inoculum from Lake Batata. N-NH₄NO₃ and P-KH₂PO₄, with final concentrations of 50 and 5 μM, respectively, were added to the cultures, except for controls. Extra sources of DOC (e.g., algal lysate, plant leachates) were added to constitute six distinct treatments. Bacterial response was measured by maximum bacterial abundance and rates of bacterial production, respiration, DOC uptake, and bacterial growth efficiency (BGE). Bacterial growth and DOC uptake were higher in NP treatments than in controls, indicating a consistent nutrient limitation in Lake Batata. The composition of DOC also seems to be an important regulating factor of bacterial growth in Lake Batata. Seasonally, bacterial growth and DOC bioavailability were higher at low-water period, when the phytoplankton is a significant extra source of DOC, than at high-water period, when the forest is the main source of DOC. DOC bioavailability was better estimated based on the diversity and the diagenetic stage of carbon compounds than on single classes of labile compounds. Changes in BGE were better related to CNP stoichiometry in the water, and the “excess” of organic substrates was oxidized in catabolism, despite the quality of these compounds for bacterial growth. Finally, we conclude that bacterial growth and DOC uptake vary throughout the flood pulse in clear-water Amazonian ecosystems as a result of changes in nutrient concentration and in DOC composition.     

Lysine and threonine biosynthesis in sorghum seeds: characterisation of aspartate kinase and homoserine dehydrogenase isoenzymes

Aspartate kinase (AK, EC 2.7.2.4) and homoserine dehydrogenase (HSDH, EC 1.1.1.3) have been partially purified and characterised from immature sorghum seeds. Two peaks of AK activity were eluted by anion‐exchange chromatography [diethylaminoethyl (DEAE)‐Sephacel] with 183 and 262 mM KCl, and both activities were inhibited by lysine. Similarly, two peaks of HSDH activity were eluted with 145 and 183 mM KCl; the enzyme activity in the first peak in elution order was shown to be resistant to threonine inhibition, whereas the second was sensitive to threonine inhibition. However, following gel filtration chromatography (Sephacryl S‐200), one peak of AK activity co‐eluted with HSDH and both activities were sensitive to threonine inhibition, suggesting the presence of a bifunctional threonine‐sensitive AK–HSDH isoenzyme with a molecular mass estimated as 167 kDa. The activities of AK and HSDH were studied in the presence of lysine, threonine, methionine, valine, calcium, ethylene glycol bis(2‐aminoethylether)‐N,N,N′N′‐tetraacetic acid, calmodulin, S‐adenosylmethionine (SAM), S‐2‐aminoethyl‐l ‐cysteine (AEC) and increasing concentrations of KCl. AK was shown to be inhibited by threonine and lysine, confirming the existence of two isoenzymes, one sensitive to threonine and the other sensitive to lysine, the latter being predominant in sorghum seeds. Methionine, SAM plus lysine and AEC also inhibited AK activity; however, increasing KCl concentrations and calcium did not produce any significant effect on AK activity, indicating that calcium does not play a role in AK regulation in sorghum seeds. HSDH also exhibited some inhibition by threonine, but the majority of the activity was not inhibited, thus indicating the existence of a threonine‐sensitive isoenzyme and a second predominant threonine‐insensitive isoenzyme. Valine and SAM plus threonine also inhibited HSDH; however, increasing concentrations of KCl and calcium had no inhibitory effect.     

Phosphate closes the solution structure of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) from Mycobacterium tuberculosis

The 5-enolpyruvylshikimate-3-phosphate synthase catalyses the sixth step of the shikimate pathway that is responsible for synthesizing aromatic compounds and is absent in mammals, which makes it a potential target for drugs development against microbial diseases. Here, we report the phosphate binding effects at the structure of the 5-enolpyruvylshikimate-3-phosphate synthase from Mycobacterium tuberculosis. This enzyme is formed by two similar domains that close on each other induced by ligand binding, showing the occurrence of a large conformation change. We have monitored the phosphate binding effects using analytical ultracentrifugation, small angle X-ray scattering and, circular dichroism techniques. The low resolution results showed that the enzyme in the presence of phosphate clearly presented a more compact structure. Thermal-induced unfolding experiments followed by circular dichroism suggested that phosphate rigidified the enzyme. Summarizing, these data suggested that the phosphate itself is able to induce conformational change resulting in the closure movement in the M. tuberculosis 5-enolpyruvylshikimate-3-phosphate synthase.