Effects of cell concentration during cryopreservation on the post-thaw quality of Santa ines ram sperm.

BACKGROUND: Non-surgical artificial insemination techniques for sheep may benefit from larger numbers of sperm in the insemination dose because the ewe cervix is convoluted and often cannot be traversed with an insemination gun resulting in deposition of the sperm at the os cervix. OBJECTIVE: To compare a range of sperm concentrations when cryopreserving semen from Santa Ines rams and determine the effects of this on post-thaw quality. MATERIALS AND METHODS: One ejaculate from each ram (n = 10) was diluted to four sperm concentrations to obtain the following groups: G-400, G-800, G-1200, and G-1600 x 106 sperm/mL. The semen samples were packaged in 0.25 mL straws, cooled to 5 degree C, cryopreserved in liquid nitrogen vapor, thawed in a water bath (40 degree C per 20 s), and were analyzed for computerized kinetics, capacitation and acrosome integrity, and plasma membrane integrity of sperm. RESULTS: The G-400 treatment resulted in samples with the highest linearity and progressive motion (P < 0.05) and had significantly greater plasma membrane integrity, and lower capacitation and acrosome reaction rates compared to G-1600 (P < 0.05). Overall, use of the G-400 treatment resulted in better kinetics, less plasma membrane damage and less early capacitation. However, despite reducing the ejaculate yield and increasing the costs of the semen freezing process, the G-800 and G-1200 treatments make a greater absolute number of sperm with good kinetics, plasma membrane integrity and capacitation status available. CONCLUSION: Ram sperm concentration impacts cryopreservation, and higher concentrations may be advantageous if a single artificial insemination protocol is desirable. doi.org/10.54680/fr22610110812.

Molecular identification and physiological characterization of Zymomonas mobilis strains from fuel-ethanol production plants in north-east Brazil.

Zymomonas mobilis has long attracted attention owing to its capacity to ferment hexose to ethanol. From a taxonomic viewpoint, Z. mobilis is a unique species of the genus Zymomonas, separated into three subspecies, Z. mobilis subsp. mobilis, Z. mobilis subsp. pomaceae and Z. mobilis subsp. francensis on the basis of physiological tests, which are often unreliable owing to the genetic proximity among these species. Currently, the use of molecular techniques is more appropriate for identification of these bacterial subspecies. In this study, the 32 strains of Z. mobilis present in the UFPEDA bacterial collection were characterized using molecular techniques, such as sequencing of the 16S rDNA gene and its theoretical restriction profile, classifying them as members of the subspecies, Z. mobilis subsp. mobilis. In addition, anaerobic cultivations were performed, which showed the biological diversity of the strains in terms of growth, sugar consumption and ethanol production. From these results, it was possible to identify the strain Z-2-80 as a promising bacterium for use in the fermentation process. SIGNIFICANCE AND IMPACT OF THE STUDY: Zymomonas mobilis is a bacterium of great relevance to biotechnology, owing to its capacity to ferment hexose to ethanol. On a molecular basis, 32 isolates were identified as Z. mobilis subsp. mobilis. However, intraspecific diversity was identified when these were grown under strictly anaerobic conditions. The results obtained from this study suggest a strain of Z. mobilis as an alternative for use in the fermentation process.

Isolation of clinically relevant fungal species from solid waste and environment of dental health services.

AIMS: This study was undertaken to detect, identify and determine antifungal susceptibility of yeast strains isolated from dental solid waste and to evaluate airborne fungi in the Brazilian dental health care environment and in the waste storage room. METHODS AND RESULTS: A group of 17 yeast strains were identified by macroscopic and microscopic characteristics, API 20C Aux system and Multiplex PCR. All 104 airborne fungal colonies were identified by macroscopic and microscopic morphology. The CLSI broth microdilution method was utilized as the susceptibility test. Candida parapsilosis was the prevailing yeast species recovered from waste, followed by Rhodotorula glutinis. Three strains of Candida guilliermondii presented minimal inhibitory concentration values considered to be susceptible dose dependent (2 µg ml(-1)) to voriconazole. Of all airborne fungal species, 69% were recovered from the waste storage room and 31% were recovered from the clinical/surgical environment. Most of them were identified as Cladosporium spp. CONCLUSIONS: These findings reinforce the potential risk of waste handling and point out the need for safe management to minimize the spread of these agents to the environment. Filamentous fungi isolation in almost all sampled environments indicates that a periodic monitoring of airborne microbiota in the dental health care service environment is required. SIGNIFICANCE AND IMPACT OF THE STUDY: The survival of yeast strains for 48 h suggests that dental waste should be carefully controlled and monitored.

Effect of MI-D, a new mesoionic compound, on energy-linked functions of rat liver mitochondria.

MI-D (4-phenyl-5-(4-nitro-cinnamoyl)-1,3,4-thiadiazolium-2-phenylami ne chloride), a new mesoionic compound, depressed the phosphorylation efficiency of liver mitochondria as deduced from an accentuated decrease of the respiratory control coefficient and ADP/O ratio. Analysis of segments of the respiratory chain suggested that the MI-D inhibition site is further on than complex I and between complexes II and III. The transmembrane electrical potential (delta psi) was collapsed dependent on MI-D concentration. ATPase activity was dramatically increased by MI-D in intact mitochondria, but inhibited in carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP)-uncoupled mitochondria. These results suggest that MI-D acts as an uncoupler agent, a property closely related to its structural characteristics.

Effect of the Cymbopogon citratus, Maytenus ilicifolia and Baccharis genistelloides extracts against the stannous chloride oxidative damage in Escherichia coli.

Stannous ion has been used in different sectors of human interest, such as in food industry and in health sciences. Much is known about stannous chloride (SnCl(2)) toxicity, although, there is no general agreement regarding its genotoxicity. Cymbopogon citratus, Maytenus ilicifolia and Baccharis genistelloides extracts have been used in popular medicine. We evaluated the influence of these crude extracts on the survival of the Escherichia coli wild type (AB 1157) strain submitted to SnCl(2) treatment. Reactive oxygen species (ROS) can be generated by a Fenton like reaction induced by SnCl(2). E. coli culture was treated simultaneously with SnCl(2) and a specific extract. Our results showed a reduction of the SnCl(2) effect on the survival of the cultures in presence of the crude extracts. The extract of M. ilicifolia showed the highest level of protection action against the SnCl(2) effect in comparison with the other extracts. This protector effect could due to the redox properties of these crude extracts. The compounds in the crude extracts could (i) chelate stannous ions, protecting them against the oxidation and avoiding the generation of ROS, (ii) be a scavenger of the ROS generated by the SnCl(2) oxidation and/or (iii) have oxidant compounds that could oxidise the stannous ions, abolishing or reducing the SnCl(2) effect.

Assessment of the effect of Fucus vesiculosus extract on the labeling of blood constituents with technetium-99m and the histological modifications on the shape of the red blood cells.

Natural products are widely used as food or food additives or medicines for humans. We are trying to develop a model to assess the possible toxic properties of natural products, such as Fucus vesiculosus, utilized in popular medicine. Red blood cells (RBC) labeled with technetium-99m (99mTc) are used in various procedures in nuclear medicine. This labeling procedure depends on a reducing agent, and stannous chloride is used. There is evidence that this labeling may be altered by drugs. We have investigated the possibility that F. vesiculosus extract is capable of altering the labeling of blood elements with 99mTc. Blood was incubated with F. vesiculosus extract and stannous chloride solution and Tc-99m added. Blood was centrifuged and plasma (P) and blood cells (BC) were isolated. Samples of P or BC were also precipitated, centrifuged and insoluble (IF) and soluble (SF) were separated. The percentages of radioativity (%ATI) in BC, IF-P and IF-BC were calculated. Histological preparations of the RBC treated with F. vesiculosus revealed that this extract is capable of promoting important modifications on the shape of the RBC. The%ATI decreased on BC from 93.6+/-2.3 to 29.0+/-2.7, on IF-P from 77.6+/-1.2 to 7.5+/-1.0 and on IF-BC from 80.0+/-3.4 to 12.6+/-4.8. Once the RBC labeling procedure with 99mTc depends on the presence of stannous (+2) ions, the substances present in the F. vesiculosus extract should increase the valence of these ions to stannic (+4). This would decrease the%ATI on blood elements and indicate the presence of oxidant agents in the F. vesiculosus extract.

Effect of an extract of cauliflower (leaf) on the labeling of blood elements with technetium-99m and on the survival of Escherichia coli AB1157 submitted to the treatment with stannous chloride.

The labeling of red blood cells (RBC) with technetium-99m (99mTc) depends on a reducing agent and stannous chloride (SnCl(2)) and is widely utilized. This labeling may also be altered by drugs, and SnCl(2) reduces the survival of Escherichia coli cultures. Cauliflower (Brassica oleracea L. var. botrytis) is used in folk medicine and we evaluated its influence on (i) the labeling of blood elements with 99mTc, and (ii) on the survival of an E. coli strain. Blood was withdrawn from rats that drank the extract of cauliflower (15 days). Blood was incubated with SnCl(2) and with 99mTc, as sodium pertechnetate, centrifuged and plasma (P) and RBC were isolated. Samples of P and RBC were also precipitated, centrifuged and soluble and insoluble fractions isolated. E. coli culture was treated with SnCl(2) in the presence of cauliflower. The extract of cauliflower did not alter the fixation of 99mTc on blood fractions; however, it abolished the lethal effect of SnCl(2) on the E. coli culture. We suggest that the substances present in the extract of cauliflower probably, would have redox property with different mechanisms of action. The oxidant action of the substances of the extract would not be strong enough to oxidise the stannous ions altering the 99mTc-labeling. However, the referred substances could oxidise these ions sufficiently to protect the E. coli culture against the lethal effect of the stannous ion.

Assessment of the effect of Maytenus ilicifolia (espinheira santa) extract on the labeling of red blood cells and plasma proteins with technetium-99m.

We are trying to develop a model to assess properties of products utilized in popular medicine. Maytenus ilicifolia is used in herbal medicine. Red blood cells (RBC) labeled with technetium-99m (99mTc) are employed in nuclear medicine. This labeling procedure depends on a reducing agent and stannous chloride is used. There is evidence that this labeling may be altered by drugs. We have investigated the possibility of M. ilicifolia extract being capable to alter the labeling of blood elements with 99mTc. Blood was incubated with M. ilicifolia extract. Stannous chloride solution and Tc-99m were added. Blood was centrifuged and plasma (P) and blood cells (C) were isolated. Samples of P or C were also precipitated, centrifuged and insoluble (IF) and soluble (SF) were separated. The percentages of radioactivity (%ATI) in C, IF-P and IF-C was calculated. The %ATI decreased on C from 93.6+/-2.3 to 29.0+/-2.7, on IF-P from 77.6+/-1.2 to 7.5+/-1.0 and on IF-C from 80.0+/-3.4 to 12.6+/-4.8. Once in RBC labeling procedure with 99mTc depends on the presence of stannous (+2) ions, the substances of the M. ilicifolia extract could increase the valence these ions to stannic (+4). This fact would decrease the %ATI on blood elements and indicate the presence of oxidant agents in the M. ilicifolia extract.

Effect of extract of medicinal plants on the labeling of blood elements with Technetium-99m and on the morphology of red blood cells: I--a study with Paullinia cupana.

Drugs can alter the labeling and the morphology of red blood cells. As Paullinia cupana is used in popular medicine, we evaluated its influence on the labeling process using technetium-99m (Tc-99m). Blood was incubated with P. cupana, stannous chloride and Tc-99m. Samples were centrifuged and plasma (P) and blood cells (BC) were separated and precipitated with trichloroacetic acid. Soluble (SF) and insoluble fractions (IF) were isolated. The morphology of the blood cells was evaluated under an optical microscope. The results showed a significant (P = 0.05) decrease in the uptake of radioactivity for the RBC (97.93 +/- 0.74 to 36.90 +/- 4.71%), in IF-P and in IF-BC due to P. cupana extract. The study of the morphology of the RBC revealed alterations in the shape of these cells. We suggest that the P. cupana effect could be explained by an inhibition of the stannous and pertechnetate ions or oxidation of the stannous ion or by damages in the plasma membrane.

Omega-3 prevents behavior response and brain oxidative damage in the ketamine model of schizophrenia.

Supplementation with omega-3 has been identified as an adjunctive alternative for the treatment of psychiatric disorders, in order to minimize symptoms. Considering the lack of understanding concerning the pathophysiology of schizophrenia, the present study hypothesized that omega 3 prevents the onset of symptoms similar to schizophrenia in young Wistar rats submitted to ketamine treatment. Moreover, the role of oxidative stress in this model was assessed. Omega-3 (0.8g/kg) or vehicle was given by orogastric gavage once daily. Both treatments were performed during 21days, starting at the 30th day of life in young rats. After 14days of treatment with omega-3 or vehicle, a concomitant treatment with saline or ketamine (25mg/kg ip daily) was started and maintained until the last day of the experiment. We evaluated the pre-pulse inhibition of the startle reflex, activity of antioxidant systems and damage to proteins and lipids. Our results demonstrate that supplementation of omega-3 prevented: decreased inhibition of startle reflex, damage to lipids in the hippocampus and striatum and damage to proteins in the prefrontal cortex. Furthermore, these changes are associated with decreased GPx in brain tissues evaluated. Together, our results suggest the prophylactic role of omega-3 against the outcome of symptoms associated with schizophrenia.

Effect of maternal deprivation on acetylcholinesterase activity and behavioral changes on the ketamine-induced animal model of schizophrenia.

Maternal deprivation has been associated with physiological and developmental changes that may be related to an increased risk for childhood and adult neuropsychiatric diseases. A growing number of studies demonstrated the importance of childhood experiences in the development of psychosis and schizophrenia in adulthood. Therefore, the present study investigated different behavior responses in rats following maternal deprivation and/or ketamine treatment in adulthood. Male rats were subjected to maternal deprivation for 180 min from postnatal day-01 to postnatal day-10. We evaluated locomotor activity, avoidance task and social interaction of adult male rats deprived or not deprived that were administered with saline or acute subanesthetic doses of ketamine (5, 15 and 25 mg/kg, i.p.). Our results show that only ketamine (25 mg/kg, i.p.) treatment in the adult rats lead to hyperlocomotion but not ketamine (5 and 15 mg/kg) and maternal deprivation alone. However, maternally deprived rats treated with ketamine (5 mg/kg) induced hyperlocomotion. Additionally, ketamine (25 mg/kg) and maternal deprivation alone induced cognitive deficit in the avoidance task. Rats deprived of and treated with ketamine (5, 15 and 25 mg/kg) also lead to memory deficit. Moreover, ketamine (25 mg/kg) and maternal deprivation alone increased latency to start social behavior. However, ketamine (5 mg/kg) and maternal deprivation lead to an increase of latency to start social behavior. Biochemistry data showed that all doses of ketamine and ketamine plus maternal deprivation increased the acetylcholinesterase (AChE) activity in the prefrontal cortex, hippocampus and striatum. The major doses of ketamine associated with maternal deprivation induced a major increase of AChE activity. Together, our results suggest that animals subjected to maternal deprivation had an increased risk for schizophrenia-like behavior and cholinergic alteration.

D-glyceraldehyde-3-phosphate dehydrogenase from HeLa cells--1. Purification and properties of the enzyme.

1. D-GPDH from HeLa cells was isolated and purified. 2. Some basic kinetic constants are reported. 3. Sodium dodecyl polyacrylamide gel electrophoresis gave a single band with a molecular weight of approximately 36 K. 4. ATP and NADH inhibit competitively enzyme activity. 5. Comparative catalytic properties of GPDH from normal and tumor cells were effectuated.

D-glyceraldehyde-3-phosphate dehydrogenase from HeLa cells--2. Immunological characterization.

1. An immunological relationship between GPDH from HeLa cells and those from other phylogenetically different sources was carried out. 2. It was found that HeLa cell anti-GPDH antibody presented an immunological cross-reaction specificity with GPDH from HeLa cells, Caiman sp. muscle and human mammary tumor tissue and a partial one with GPDH from Anas sp. muscle.

Occurrence of the Crabtree effect in HeLa cells.

The occurrence of a Crabtree effect in HeLa cells was detected. Some properties of pyruvate kinase (PK) were also evaluated. Hexose phosphate, triose-phosphate and phosphoenolpyruvate (PEP) significantly decreased the oxygen consumption of digitonin-permeabilized HeLa cells, which were oxidizing succinate. The Crabtree effect promoted by PEP was concentration-dependent and was lowered by an increase of ADP concentration, suggesting a participation of PK. The dependence of fructose-1,6-bisphosphate (FDP) by HeLa cell PK was observed. The PK of HeLa cells was inhibited by L-alanine only in the absence of FDP, while in the presence of the metabolite, an increase in the activity was observed. PK was also inhibited in the presence of L-histidine and L-leucine, while L-serine promoted activation. L-Cysteine and L-phenylalanine also inhibited the PK of HeLa cells. This, together with the sigmoidal character in relation to substrate concentration, suggests the presence of the K-type of PK in HeLa cells.

Alterations induced by citrinin in cultured kidney cells.

The cytotoxicity of citrinin was evaluated in an established cell line of baby hamster kidney cells. The primary effect of the mycotoxin was on the adherence of the cells to the culture bottles. Microscopic evaluation of morphological alterations indicated that the cells which were originally elongated and flattened, became swollen and round. Electron microscopic examination showed that citrinin (0.1, 0.5 and 1.0 mM) incubated for 10 hours with cultured cells, promoted drastic alterations of normal mitochondria, with swelling and cell death. Transplasma membrane redox system is inhibited by citrinin (81%). This effect is dependent not only on the toxin concentration, but also on the time of exposure to the cells.

New Perfluorophtalate Complexes of Platinum(II) With Chemotherapeutic Potential.

Two new platinum(II) complexes have been synthesized and their anti-tumour and anti-HIV activities have been evaluated.THE NEW COMPLEXES ARE: (i) cis-tetrafluorophthalate-ammine-morpholine-platinum(II) or MMF3 and (ii) cis-tetrafluorophthalate- ammine-piperidine-platinum(II) or MPF4. They were characterized by elemental analysis, IR spectra and (1)H and (13)C NMR spectra.They were tested against five human ovarian carcinoma cell lines, viz., CH1, CH1cisR, A2780, A2780cisR and SKOV-3. They were less active than cis-platin and showed cross-resistance with cis-platin in the CH1cisR and A2780cisR acquired resistance lines.They were also tested for possible anti-HIV activity using the HIV-I IIIB virus and C8166 cells, but they were inactive compared with AZT.

Methotrexate increases glycogenolysis in the intact rat liver.

The effect of methotrexate, an anti-cancer drug, on carbohydrate metabolism in the isolated perfused rat liver was investigated. Glucose release from endogenous glycogen (glycogenolysis) is strongly activated by methotrexate. The phenomenon shows a well defined concentration dependence: 100% activation occurs at 2.2 X 10(-4) M. Glycolysis and oxygen consumption are only slightly affected.