Acid Incubation Research Articles

Fatty Acid Incubation of Myotubes From Humans With Type 2 Diabetes Leads to Enhanced Release of β-Oxidation Products Because of Impaired Fatty Acid Oxidation

OBJECTIVE—Increased availability of fatty acids is important for accumulation of intracellular lipids and development of insulin resistance in human myotubes. It is unknown whether different types of fatty acids like eicosapentaenoic acid (EPA) or tetradecylthioacetic acid (TTA) influence these processes.RESEARCH DESIGN AND METHODS—We examined fatty acid and glucose metabolism and gene expression in cultured human skeletal muscle cells from control and type 2 diabetic individuals after 4 days of preincubation with EPA or TTA.RESULTS—Type 2 diabetes myotubes exhibited reduced formation of CO2 from palmitic acid (PA), whereas release of β-oxidation products was unchanged at baseline but significantly increased with respect to control myotubes after preincubation with TTA and EPA. Preincubation with TTA enhanced both complete (CO2) and β-oxidation of palmitic acid, whereas EPA increased only β-oxidation significantly. EPA markedly enhanced triacylglycerol (TAG) accumulation in myotubes, more pronounced in type 2 diabetes cells. TAG accumulation and fatty acid oxidation were inversely correlated only after EPA preincubation, and total level of acyl-CoA was reduced. Glucose oxidation (CO2 formation) was enhanced and lactate production decreased after chronic exposure to EPA and TTA, whereas glucose uptake and storage were unchanged. EPA and especially TTA increased the expression of genes involved in fatty acid uptake, activation, accumulation, and oxidation.CONCLUSIONS—Our results suggest that 1) mitochondrial dysfunction in diabetic myotubes is caused by disturbances downstream of fatty acid β-oxidation; 2) EPA promoted accumulation of TAG, enhanced β-oxidation, and increased glucose oxidation; and 3) TTA improved complete palmitic acid oxidation in diabetic myotubes, opposed increased lipid accumulation, and increased glucose oxidation.

The role of acid incubation in rapid immobilization of hydrogen-producing culture in anaerobic upflow column reactors

An approach of acidification was examined on formation of hydrogen-producing granules and biofilms in upflow column-shaped reactors. The reactors were fed with synthetic glucose wastewater and operated at 37 °C and pH 5.5. The acclimated anaerobic culture was inoculated in four reactors designated R1, R2, R3 and R4, with R3 and R4 filled with granular activated carbon as support medium. To unveil the roles of acidification, microbial culture in R2 and R3 was subject to an acid incubation for 24 h by shifting the culture pH from 5.5 to 2.0. The experimental results suggested that the acidification substantially accelerated microbial granulation, but not biofilm formation. Microbial activities were inhibited by the acid incubation for about 78 h, resulting in the retarded formation of biofilms of the acidified culture. Reducing culture pH resulted in improvement in cell surface physicochemical properties favoring microbial adhesion and immobilization. Zeta potential increased from −25.3 mV to 11.9 mV, hydrophobicity in terms of contact angle improved from 31° to 38° and production of extracellular polymers increased from 66 mg/g-VSS to 136 mg/g-VSS. As a result of the formation of granules and biofilms, high hydrogen production rates of 6.98 and 7.49 L/L h were achieved in granule-based and biofilm-based reactors, respectively. It is concluded that acid incubation is an efficient means to initiate the rapid formation of granules by regulating the surface characteristics of microbial culture. The use of support media as starting nuclei may result in rapid formation of biofilms without the acidification.

Effects of trans MUFA from dairy and industrial sources on muscle mitochondrial function and insulin sensitivity

Epidemiological studies suggest that chronic consumption of trans MUFA may alter muscle insulin sensitivity. The major sources of dietary trans MUFA (dairy fat vs. industrially hydrogenated oils) have different isomeric profiles and thus probably different metabolic consequences. These effects may involve alterations in muscle mitochondrial oxidative capacity, which may in turn promote insulin resistance if fatty acid oxidation is reduced. We report that in Wistar rats, an 8 week diet enriched (4% of energy intake) in either dairy, industrial, or control MUFA did not alter insulin and glucose responses to an intraperitoneal glucose tolerance test (1g/kg). In C2C12 myotubes, vaccenic and elaidic acids did not modify insulin sensitivity compared with oleic acid. Furthermore, the ex vivo total, mitochondrial and peroxisomal oxidation rates of [1-(14)C]oleic, vaccenic, and elaidic acids were similar in soleus and tibialis anterior rat muscle. Finally, an 8 week diet enriched in either dairy or industrial trans MUFA did not alter mitochondrial oxidative capacity in these two muscles compared with control MUFA but did induce a specific reduction in soleus mitochondrial ATP and superoxide anion production (P<0.01 vs. control). In conclusion, dietary trans MUFA of dairy or industrial origin have similar effects and do not impair muscle mitochondrial capacity and insulin sensitivity.

Characterization of Cold-Responsive Extracellular Chitinase in Bromegrass Cell Cultures and Its Relationship to Antifreeze Activity

A cold-responsive chitinase gene, BiCHT1, was isolated from bromegrass (Bromus inermis) 'Manchar' suspension cells. BiCHT1 messenger RNA was detected at low levels in nonstressed bromegrass cells, whereas its accumulation was induced by incubation at 10 degrees C and 4 degrees C as detected by northern- and western-blot analyses. BiCHT1 was highly homologous to rye CHT9, known to encode an antifreeze protein. BiCHT1 was overexpressed in Escherichia coli and bromegrass cells using genetic transformation procedures. BiCHT1 products expressed in both systems had chitinase activity, but the expressed proteins did not affect the growth of ice crystals in any conditions tested. Besides cold stress, the expression of the BiCHT1 gene was up-regulated by exposure to 35 degrees C, but not by salt or osmotic stress, abscisic acid, or ethephon. BiCHT1 messenger RNA did not accumulate in response to methyl jasmonate and salicylic acid, but was slightly increased by prolonged culture at 25 degrees C and only transiently by chitin. Antifreeze activity detected in the culture medium was induced at 4 degrees C but only slightly at 10 degrees C. It was also induced by ethephon treatment, but not by abscisic acid, chitin, or prolonged incubation at 25 degrees C. The results of transgenics and expression analyses suggest that the BiCHT1 product is a major protein with chitinase activity secreted in the medium of cold-treated cells and is unlikely to be responsible for the antifreeze activity detected in the culture medium.

Enumeration of Lactobacilli in the Fecal Flora of Infant Using Two Different Modified de-Man Rogosa Sharpe Media under Aerobic and Anaerobic Incubation

Regarding the importance of the presence of intestinal lactobacilli and their population in infants, four different treatments were evaluated for Lactobacillus isolation efficiency via reduction in the growth of other groups of bacteria capable of growing on de-Man Rogosa Sharpe (MRS) medium from fecal samples of 11 Iranian infants. MRS-Vancomycin (1 mg L(-l)) was used as a base medium and application of lactic acid and aerobic incubation of inoculated plates were performed as selective factors. Each fecal sample was cultivated as duplicate on to the base medium with or without lactic acid to reduce the pH to 5.4 +/- 0.2. Half of the plates were incubated aerobically and the rest of them incubated under 10% CO2 concentration. Total count and Lactobacillus count of all samples were recorded according to the age differences of infants. The counts of false positive colonies were recorded with respect to their cell morphology and gram reaction in all treatments. Anaerobic incubation of lactic acid modified MRS-Vancomycin gave the most Lactobacillus percentage coverage, about 93% among the Lactobacillus positive samples. Using this treatment, the median Lactobacillus count yielded 8.29 log10 cfu g(-1) in the younger and 5.70 log10 cfu g(-1) in the elder group. It could be concluded that lactic acid might be a proper pH reducing agent when enumeration of lactobacilli from fecal samples is of interest.

Radio-activation of hTERT promoter in larynx squamous carcinoma cells: An ‘indirected-activator’ strategy in radio-gene-therapy

The usefulness of human telomerase reverse transcriptase (hTERT) gene promoter has been proposed in cancer-targeted gene therapy. However, this promoter may not be strong enough to achieve therapeutic levels of transgene expression. In this study, we tested an 'indirected-activator' strategy that utilizes radiation to increase the activity of the hTERT gene promoter. We demonstrated that hTERT may participate in the process of DNA repair induced by irradiation. We found that Zidovudine (AZT, an hTERT inhibitor) can decrease the telomerase activity in human HEp-2 larynx squamous carcinoma cells and lower the survival fraction of HEp-2 cells exposed to radiation. In HEp-2 cells exposed to 6 Gy-radiation, the hTERT promoter showed 2.9-fold higher activity compared with unirradiated cells. Importantly, an increased expression of enzyme horseradish peroxidase (HRP) controlled by the hTERT promoter was found in the transfected cells after irradiation, which coincided with a higher killing rate for HEp-2 cells after prodrug indole-3-acetic acid (IAA; converted by HRP into a cytotoxin) incubation combined with irradiation or not. Our observations suggest that hTERT promoter-mediated gene therapy could be improved in combination with radiotherapy, which may be due to cellular DNA damage responses.

Production, purification, and characterization of thermostable alkaline protease under solid state fermentation conditions for application in bio-detergent technology

Screening studies were carried out for one hundred, and fifty-three bacterial isolates with respect to their ability to produce protease(s), after growing on some industrial wastes at 55ºC, and pH 9.Two most potent thermophilic bacterial isolates concerning of alkaline thermostable protease(s) production were identified as Bacillus licheniformis B42 and Geobacillus stearothermophilus B78. Alkaline thermostable proteases productivity by the two most potent bacterial isolates was affected by substrate concentrations (solid substrate), carbon source, nitrogen source, mineral ions, vitamins, amino acid supplements, incubation temperature, incubation period, and inoculum size. Maximum both enzymes production by B. licheniformis B42 and G. stearothermophilus B78 were obtained on slaughter house wastes (SHW) concentrations,1.5,2.5 %;carbon sources; galactose, no tested sugar; nitrogen sources; diammonium hydrogen phosphate, ammonium molybdate; chelating agents; ethylene diamine tetra acetic acid (EDTA) (500 ppm), no tested ions; vitamins; thiamine pyridoxine; amino acids; arginine, DL-tyrosine at 55°C for 72 h. when inoculated by 0.5, and 2.5 ml respectively. The protease yield under all optimal conditions was increased many folds from 563.68 to 17825 U/ml (31 fold) by B. licheniformis and from 23.7 to 7096 U/ml (299 fold) by G. stearothermophilus. The purification fold of B. licheniformis B42 alkaline thermostable protease increased to 394.7 after applying Sephadex G200 and 411.9 times after Sephadex G100 column chromatography techniques. Fifteen amino acids were detected in the partially purified protease. Optimum conditions of the partially purified alkaline thermostable protease on Sephadex G200 followed by Sephadex G100 produced by B. licheniformis B42 were: incubation temperature, 50°C; thermostable between 50-70°C; pH,10; and incubation time,36 h exhibited maximal activity. The purified enzymes exhibited good stability towards chlorine. Compatibility of the purified enzyme with various commercial detergents reported that purified protease was unstable in presence of commercial detergents. Calcium chloride was able to restore and enhance the activity of the partially purified enzyme in the presence of commercial detergents. The alkaline serine proteases secreted by both strains are industrially important in respect of their abilities to act in alkaline pH and to show stability in broad pH ranges in addition to their stabilities toward sodium dodecyl sulphate(SDS), and hydrogen peroxide (H2O2), suggesting that it is a potentially candidate as an additive in the detergents formulations. Egyptian Journal of Biotechnology Vol. 25 (1) 2007: pp. 111-129

Optimisation of rhamnolipids produced byPseudomonas aeruginosa 181 using Response Surface Modeling

This work investigated the optimisation of the fermented culture medium for maximisation of rhamnolipids production produced byPseudomonas aeruginosa 181 using Response Surface Modeling (RSM). A two full factorial central composite experimental design was used in the design of experiments and in the analysis of results. This procedure limited the number of actual experiments performed while allowing for possible interactions between the parameters (pH, stirring rate, casamino acid concentration and incubation period) on the production of biosurfactants. Production carried out at larger volumes of one litre using Bioreactor under RSM-optimised conditions yielded 3.61 g l−1 of products after purification by acid precipitation.

Cloning and expression analysis of heat shock cognate 70 gene promoter in tiger shrimp ( Penaeus monodon)

Heat shock cognate 70 (HSC70) functions as a molecular chaperon and plays an important role in protein folding. HSC70 cDNA of tiger shrimp ( Penaeus monodon) was cloned and characterized in our previous study. After shrimps were treated with the 1-hr heat shock, the HSC70 mRNA level in hemocytes increased (∼ 8 fold) using real-time quantitative PCR. An hsc70 clone was obtained from genomic library screening. The gene contains 2 exons separated by a 1557-bp intron. The 5′-flanking region sequence (∼ 1 kb) ahead of the hsc70 gene contains a putative core promoter region and transcription elements including perfect heat shock element (HSE), imperfect HSE, CAAT elements, SP1, NF-κB and GC box. In insect Sf21 cells, the region could drive expression of the enhanced green fluorescent protein (EGFP) and luciferase gene to verify its promoter function. In the luciferase assay system, the effects of serial deletions on the hsc70 promoter were elucidated. Autographa californica multiple nuclear polyhedrosis virus infection (MOI = 0.1) on Sf21 cells significantly increased the hsc70 promoter activity. In addition, the effects of amino acid analogs and arsenic acid incubation with the cells on the hsc70 promoter activity were examined.

Rapid formation of hydrogen‐producing granules in an anaerobic continuous stirred tank reactor induced by acid incubation

A novel approach to rapidly initiate granulation of hydrogen-producing sludge was developed in an anaerobic continuous stirred tank reactor at 37 degrees C. To induce microbial granulation, the acclimated culture was subject to an acid incubation for 24 h by shifting the culture pH from 5.5 to 2.0. The culture was resumed to pH 5.5 after the incubation and the reactor was operated at hydraulic retention times (HRTs) of 12, 6, 2, 1, and 0.5 h in sequence. Microbial aggregation took place immediately with the initiation of acid incubation and granules were developed at 114 h. No granule was observed in the absence of acid incubation in the control test. Changing the culture pH resulted in improvement in surface physicochemical properties of the culture favoring microbial granulation. The zeta potential increased from -11.6 to -3.5 mV, hydrophobicity in terms of contact angle improved from 31 degrees to 43 degrees and extracellular proteins/polysaccharides ratio increased from 0.2 to 0.5-0.8. Formation of granular sludge facilitated biomass retention of up to 32.2 g-VSS/L and enhanced hydrogen production. The hydrogen production rate and hydrogen yield increased with the reduction in HRT at an influent glucose concentration of 10 g/L once steady granular sludge layer was formed, achieving the respective peaks of 3.20 L/L x h and 1.81 mol-H(2)/mol-glucose at 0.5 h HRT. The experimental results suggested that acid incubation was able to initiate the rapid formation of hydrogen-producing granules by regulating the surface characteristics of microbial aggregates in a well-mixed reactor, which enhanced the hydrogen production.

A new multiparameter flow cytometric method for human semen analysis

The objectives of this study were (i) to evaluate whether the combined use of Syto 16 and 7-amino-actinomycin-D (7-AAD) allows the detection of sperm apoptosis and (ii) to describe a new multiparameter flow cytometric method to assess simultaneously sperm concentration (SC), viability and apoptosis as well as leukocyte concentration. Semen samples from 68 patients were evaluated according to World Health Organization (WHO) criteria (normal, n=26; abnormal, n=42). The detection of activated caspases before and after betulinic acid (BA) incubation was carried out in 13 semen samples by flow cytometry using fluorescein-labelled inhibitors of caspases (FLICA). A multiparameter flow cytometric analysis was performed in 55 semen samples. Fluorescent microspheres were used to assess SC. Sperm apoptosis was detected by staining sperm with Syto 16 and 7-AAD. Leukocytes were counted using monoclonal anti-CD45. A significant correlation between the percentage of the spermatozoa with low Syto 16 fluorescence and the percentage of spermatozoa containing activated caspases was found (r=0.68, P=0.0106; n=13). After incubation with BA, an increase of the percentage of apoptotic cells was observed in all samples, using both the Syto 16/7-AAD and the caspase activation methods. There was a good correlation between flow cytometry and optical microscopy for sperm (r=0.98, P < 0.0001) and leukocyte counting (r=0.64, P <0.0001). The percentage of apoptotic sperm was inversely correlated with both SC (r=-0.303, P=0.0246) and morphology (r=-0.384, P=0.0050) but not with motility. The combination of Syto 16/7-AAD provides a sensitive assay to detect sperm apoptosis. The multiparameter flow cytometric method described offers the possibility of a simultaneous, simple, rapid and accurate assessment of several semen parameters.

Identification of alkaline proteins that are differentially expressed in an overgrowth‐mediated growth arrest and cell death of Escherichia coli by proteomic methodologies

The available Escherichia coli genome sequences offer an opportunity to further expand our understanding of this bacterium. In the current study, we present a rapid method for the isolation of bacterial alkaline proteins using acid incubation, purification and protein array by 2-DE, followed by protein identification using MS. Fifty-seven proteins were randomly chosen, in which 55 were identified by a database searching of MS data. The searching results showed that most of these alkaline proteins were involved in special functions within the cell, suggesting that alkaline proteome is an ideal fraction for an understanding of their special functions. Furthermore, alkaline proteomes were compared between the period of majority live bacteria (18-h culture), the period of similar amount of live and dead bacteria (30-h culture) and the period of majority dead bacteria (48-h culture). Six proteins were identified as differentially expressed targets, in which putative transcriptional regulator and superoxide dismutase genes were cloned and expressed for antiserum preparations. The antisera were applied for the confirmation of results obtained from 2-DE. The presented data clearly reveal that alkaline proteome analysis by 2-DE with MS plays an important role in the understanding of protein functions within the cell, and six alkaline proteins are determined as key ones in an overgrowth-mediated growth cycle of E. coli.

Endothelin and nitric oxide mediate adaptation of the cortical collecting duct to metabolic acidosis

Endothelin (ET) and nitric oxide (NO) modulate ion transport in the kidney. In this study, we defined the function of ET receptor subtypes and the NO guanylate cyclase signaling pathway in mediating the adaptation of the rabbit cortical collecting duct (CCD) to metabolic acidosis. CCDs were perfused in vitro and incubated for 3 h at pH 6.8, and bicarbonate transport or cell pH was measured before and after acid incubation. Luminal chloride was reversibly removed to isolate H(+) and HCO(3)(-) secretory fluxes and to raise the pH of beta-intercalated cells. Acid incubation caused reversal of polarity of net HCO(3)(-) transport from secretion to absorption, comprised of a 40% increase in H(+) secretion and a 75% decrease in HCO(3)(-) secretion. The ET(B) receptor antagonist BQ-788, as well as the NO synthase inhibitor, N(G)-nitro-l-arginine methyl ester (l-NAME), attenuated the adaptive decrease in HCO(3)(-) secretion by 40%, but only BQ-788 inhibited the adaptive increase in H(+) secretion. There was no effect of inactive d-NAME or the ET(A) receptor antagonist BQ-123. Both BQ-788 and l-NAME inhibited the acid-induced inactivation (endocytosis) of the apical Cl(-)/HCO(3)(-) exchanger. The guanylate cyclase inhibitor LY-83583 and cGMP-dependent protein kinase inhibitor KT-5823 affected HCO(3)(-) transport similarly to l-NAME. These data indicate that signaling via the ET(B) receptor regulates the adaptation of the CCD to metabolic acidosis and that the NO guanylate cyclase component of ET(B) receptor signaling mediates downregulation of Cl(-)/HCO(3)(-) exchange and HCO(3)(-) secretion.

Point:Counterpoint: Lactic acid accumulation is an advantage/disadvantage during muscle activity

Point:Counterpoint: Lactic acid accumulation is an advantage/disadvantage during muscle activity

Tissue Engineering of Viable Pulmonary Arteries for Surgical Correction of Congenital Heart Defects

Tissue-engineered pulmonary arteries could overcome the drawbacks of homografts or prosthetic conduits used in the repair of many congenital cardiac defects. However, the ideal scaffold material for tissue-engineered conduits is still subject of intensive debate. In this study, we evaluated an acellularized allogeneic matrix scaffold for pulmonary artery tissue engineering with and without in-vitro reseeding with autologous endothelial cells in the pulmonary circulation in a growing sheep model. Ovine pulmonary arteries (n = 10) were acellularized by trypsin/ethylenediamine tetraacetic acid incubation. Autologous endothelial cells were harvested from carotid arteries, and the pulmonary conduits were seeded with endothelial cells. We implanted in-vitro, autologous, reendothelialized (group A, n = 5) and acellularized pulmonary conduits (group B, n = 5) in the pulmonary circulation. The animals were sacrificed 6 months after the operation. Explanted valves were examined histologically and by immunohistochemistry. The conduit diameter increased in both groups (group A, 44% +/- 11%; group B, 87% +/- 18%; p < 0.05). In group A, however, a proportional increase in diameter was present, whereas in group B, a disproportionate increase resulting in aneurysm formation was observed. Histologically, the conduit wall integrity was destroyed in group B and preserved in group A. In group B, the extracellularmatrix degenerated with a reduced amount of collagens and proteoglycanes. Furthermore, no elastic fibers were detectable. In contrast, the extracellularmatrix in group A was close to native ovine tissue. Tissue-engineered pulmonary conduits (autologous endothelial cells and allogeneic matrix scaffolds) functioned well in the pulmonary circulation. They demonstrated an increase in diameter and an extracellular matrix comparable to that of native ovine tissue.

Differential regulation of ATP‐binding cassette transporter A1 (ABCA1) expression by fatty acids in HepG2 and RAW 264.7 macrophages

We investigated whether individual fatty acids affect the expression of ATP binding cassette transporter A1 (ABCA1), a membrane transporter critical for high-density lipoprotein formation by effluxing cellular cholesterol and phospholipids to acceptors. HepG2 cells (human hepatoma cell line) and murine RAW 264.7 macrophages were incubated with a selection of individual fatty acids, including 16:0, 16:1, 18:0, 18:1, 18:2, 18:3 and EPA (100 μM, 2.5:1 of fatty acid to BSA molar ratio), along with a control for 6-12 hrs. After fatty acid incubation, a vehicle or TO901317 (10 μM), a liver X receptor (LXR) agonist, were added to media for additional 18-hr incubation. Realtime PCR analysis was performed to determine mRNA abundance of ABCA1. In the absence of TO901317, all unsaturated fatty acids decreased ABCA1 mRNA levels by 60–90% in RAW 264.7 macrophages, whereas only 18:2 reduced (50%) and EPA increased (75%) ABCA1 mRNA levels in HepG2 cells compared with control treatment. When HepG2 cells were treated with TO901317, repression of ABCA1 mRNA expression by 18:2 disappeared, and all of the polyunsaturated fatty acids (18:2, 18:3, and EPA) induced a 2-3.5-fold increase in ABCA1 expression. In contrast, saturated and monounsaturated fatty acids did not alter the ABCA1 expression compared with control. The data suggest the presence of differential regulation of ABCA1 expression by fatty acids, and activation of LXR pathway antagonizes the inhibitory effect of 18:2 on ABCA1 expression and further increases ABCA1 expression in the presence of polyunsaturated fatty acids in HepG2 cells. (Supported by NSF-EPSCoR grant EPS-0346476)

Infertile Men With Varicocele Show a High Relative Proportion of Sperm Cells With Intense Nuclear Damage Level, Evidenced by the Sperm Chromatin Dispersion Test

The frequency of sperm cells with fragmented DNA was studied in a group of 18 infertile patients with varicocele and compared with those obtained in a group of 51 normozoospermic patients, 103 patients with abnormal standard semen parameters, and 22 fertile men. The spermatozoa were processed to discriminate different levels of DNA fragmentation using the Halosperm kit, an improved Sperm Chromatin Dispersion (SCD) test. In this technique, after an acid incubation and subsequent lysis, those sperm cells without DNA fragmentation show big or medium-sized halos of dispersion of DNA loops from the central nuclear core. Otherwise, those spermatozoa containing fragmented DNA either show a small halo, exhibit no halo with solid staining of the core, or show no halo and irregular or faint stain of the remaining core. The latter, that is, degraded type, corresponds to a much higher level of DNA-nuclear damage. The varicocele patients showed 32.4% +/- 22.3% of spermatozoa with fragmented DNA, significantly different from the group of fertile subjects (12.6% +/- 5.0%). Nevertheless, this was not different from that of normozoospermic patients (31.3% +/- 16.6%) (P = .83) and with abnormal semen parameters (36.6% +/- 15.5%) (P = .31). No significant differences were found between the normozoospermic patients and the patients with abnormal semen parameters. Strikingly, the proportion of the degraded cells in the total of sperm cells with fragmented DNA was 1 out of 4.2 (23.9% +/- 12.9%) in the case of varicocele patients, whereas it was 1 out of 8.2 to 9.7 in the normozoospermic patients (11.1% +/- 9.9%) in the patients with abnormal sperm parameters (12.2% +/- 8.3%) and in the fertile group (10.3% +/- 7.2%). Thus, whereas no differences in the percentage of sperm cells with fragmented DNA were evident with respect to other infertile patients, individuals with varicocele exhibit a higher yield of sperm cells with the greatest nuclear DNA damage level in the population with fragmented DNA. This finding illustrates the value of assessing different patterns of DNA-nuclear damage within each sperm cell and the particular ability of the Halosperm kit to reveal them.

Is there morphological difference between branchiomeric and somitic muscles submitted to alcohol consumption? An experimental study in rats (Rattus norvegicus)

Alcoholism is considered a physical dependence disorder. More than 18 million people are alcoholics in the USA and England and between 1/3 to ½ of them present some kind of physical disorder. In general the literature is focused on alcoholic trunk muscle disorders. These muscles have different embryological origins if compared to the masticatory muscles. The aim of this research was to evaluate the effects of alcohol on the masticatory muscles in order to compare them with the somitic muscles. For this purpose, 15 male Wistar rats weighing around 250g were used. The rats were divided into three groups: Normal control (N), Alcoholic (A) and Isocaloric (I). Slices of the masseter muscle, temporalis muscle and rectus abdominal muscle were harvested and submitted to histochemical reactions (m-ATPase: acid and alkaline pre incubation and NADH-TR). The myofibers were classified in SO, FOG and FG. The results showed atrophy of the fast fibers (FG and FOG) in the masticatory muscles but this atrophy was not statistically significant in this study (p< 0.05). On the other hand, significant atrophy occurred in the rectus abdominal muscle (p<0.05). Based on these data it can concluded that the effect of alcohol on the branchiomeric jaw elevator muscles (masseter and temporalis muscles) is different compared to the effect on somitic muscle (rectus abdominal muscle).

Lack of proinflammatory effects of free fatty acids on human umbilical cord vein endothelial cells and leukocytes

To determine whether the free fatty acids (FFAs), oleic, linoleic, and palmitic acid, found elevated before 20 weeks of pregnancy in those women who later develop preeclampsia, induced changes in expression of the vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), or E-selectin in cultured human umbilical cord vein endothelial cells (HUVEC), and integrin subunit CD11b, L-selectin or intracellular reactive oxygen species (ROS) in leukocytes. The VCAM-1, ICAM-1, and E-selectin expression were measured using ELISA in HUVEC after incubation with 100 micromol of either oleic, linoleic, or palmitic acid for 6 hr and 24 hr. The co-reactivity with lipopolysaccharide (LPS), the amount of VCAM-1 mRNA in the cells, and soluble VCAM-1 in the incubation medium were measured as well. Leukocyte adhesion molecules and ROS were measured after incubation with 750 microm of either of the FFAs in a whole blood model using flow cytometry. No effects of the FFAs tested were found on the HUVEC or leukocyte adhesion molecule expression or intracellular ROS. The only exception to this was palmitic acid incubation, which significantly lowered the VCAM-1 expression in HUVEC after 24-hr incubation and also slowed the decay of VCAM-1 expressed after stimulation with LPS. The lack of significant proinflammatory changes of the FFAs tested might indicate that the elevated plasma levels of FFAs seen in preeclampsia most probably are products of the preeclamptic process rather than a causative factor.

Differential lectin binding patterns in the oviductal ampulla of the horse during oestrus.

We investigated the oligosaccharide sequence of glycoconjugates, mainly sialoglycoconjugates, in the horse oviductal ampulla during oestrus by means of lectin and pre-lectin methods such as the KOH-neuraminidase procedure to remove sialic acid residues and incubation with N-glycosidase F to cleave N-linked glycans. Ciliated cells displayed N-linked oligosaccharides throughout the cytoplasm. The cilia glycocalyx expressed both N- and O-linked (mucin-type) oligosaccharides, both showing a high variety of terminal sequences. In the most non-ciliated cells, the whole cytoplasm contained N-linked oligosaccharides with terminal alphaGal as well as mucin-type glycans with terminal Forssman pentasaccharides. In a few scattered non-ciliated cells, the whole cytoplasm displayed sialylated N-linked oligosaccharides with terminal Neu5Ac-GalNAc and O-linked glycans terminating with neutral and/or alphaGalNAc, Neu5Ac alpha2,6Gal/GalNAc, Neu5AcGal beta1,3GalNAc. Supra-nuclear granules, probably Golgi zones, of non-ciliated cells showed mainly O-linked glycans rich in sialic acid residues. The luminal surface of non-ciliated cells showed N-linked oligosaccharides, containing terminal/internal alphaMan/alphaGlc, betaGlcNAc and terminal alphaGal, as well as mucin-type oligosaccharides terminating with a large variety of either neutral saccharides or sialylated sequences. Apical protrusions containing O-linked oligosaccharides with terminal Forssman pentasaccharide, Neu5Ac-Gal beta1,4GlcNAc, Neu5Ac-GalNAc were seen in non-ciliated cells scattered along the epithelium. These findings show the presence of sialoglycoconjugates in the oviductal ampulla epithelium of the mare and the existence of different lectin binding profiles between ciliated and non-ciliated (secretory) cells, as well as the presence of non-ciliated cell sub-types which might determine functional differences along the ampullary epithelium of mare oviduct.