Alfonso Cárabez-Trejo

Yeast-mycelial dimorphism of haploid and diploid strains of Ustilago maydis

Summary: Control of the pH of liquid synthetic culture media made possible mycelial growth of both diploid and haploid strains of Ustilago maydis. Whereas at neutral pH the fungus grew as a homogeneous population of budding yeast-like cells (sporidia), at acid pH it developed in the mycelial form. Mycelial cells appeared branched and narrower than yeast cells. Cell morphology was affected by the carbon and nitrogen sources. When the culture medium was removed continuously or intermittently, very long, filamentous cells were formed. Colonies of haploid strains developed aerial mycelium (‘fuzz’ morphology) on acid solid medium. Null b, bW, and bE mutants behaved in the same way as haploid wild-type strains. It is suggested that growth at low pH overcomes the control processes governed by heterologous bE and bW loci.

IDENTIFICATION OF THE STRUCTURAL COMPONENT IN THE CYST WALL OF ENTAMOEBA INVADENS

Cyst walls of Entamoeba invadens were isolated and purified. Both whole cysts and purified walls appeared intensely fluorescent when stained with Calcofluor white M2R. Examination of positive replicas of purified cyst walls with the electron microscope revealed the presence of a microfibrillar structure. The main sugars detected in acid hydrolysates from the walls were hexosamines. X-ray diffraction analysis of purified cyst walls demonstrated that the crystalline polymer present was chitin.

LOCATION OF CHITIN IN THE CYST WALL OF ENTAMOEBA INVADENS WITH COLLOIDAL GOLD TRACERS

Chitin was located in the cyst wall of Entamoeba invadens with colloidal gold-linked wheat germ agglutinin. Cysts stained differentially from trophozoites when encysting cultures were treated with the gold tracer; cysts acquired a wine-red coloration while, in general trophozoites remained unstained. Observation of cells with the electron microscope revealed that the tracer particles were bound specifically to the walls of the surface of the cyst when cells were exposed in suspension, and to the cyst wall cross-section, when cells were exposed to the tracer in thin section, indicating that chitin fibers were distributed on the surface as well as throughout the matrix of the cyst wall.

Influence of hardening procedure and soaking solution on cooking quality of common beans

Two common bean (Phaseolus vulgaris) varieties were seeded in the same location, harvested and cleaned. Three hardening procedures were used (soaking in acetate buffer, pH 4.1 at 37°C for 5 h; storage at 37°C, 100% RH for 28 days; and storage at 31–33°C, 76% RH for 120 days) to have seeds in a hard-to-cook (HTC) state. The adverse effects of HTC condition, in terms of cooking time as assessed by a Mattson bean cooker, were practically eliminated by soaking seeds in salt solutions (1% NaCl+0.75% NaHCO3; and 0.75% NaHCO3) instead of only water. Ultrastructural changes of cotyledon cells from fresh, HTC and softened seeds were observed. Results of this study may be used for the development of a technological procedure to utilize properly HTC beans generated by unefficient storage systems.

INFLUENCE OF SOLID SUBSTRATE FERMENTATION ON THE CHEMICAL COMPOSITION OF CHICKPEA

Abstract A basic procedure was developed to produce a fermented product by solid substrate fermentation using Rhyzopus oligosporus and chickpea as substrate. Water activity was kept at 0.92 throughout the process. Fermentation increased total, ‘true’ and soluble proteins, soluble solids and soluble carbohydrates, and decreased fiber content and pH. About 12% of solids were lost during 72 h of fermentation. The content of most fatty acids was enhanced by fermentation, whereas peroxide value and tannins declined. The color of the fermented product was not deteriorated after 72 h of fermentation. Scanning electron microscopy studies of microbial growth on the substrate showed penetration of the fungus hyphae and degradation effects on the chickpea cotyledon cells.

Melatonin attenuates the effects of sub-acute administration of lead on kidneys in rats without altering the lead-induced reduction in nitric oxide

Exposure to lead induces oxidative stress and renal damage. Although most forms of oxidative stress are characterized by simultaneous elevation of nitrogen and oxidative species, lead-induced oxidative stress is unusual in that it is associated with a reduction in nitric oxide (NO) levels in the kidney. The role of NO in kidney injury is controversial; some studies suggest that it is associated with renal injury, whereas others show that it exerts protective effects. Concentration-dependent effects have also been proposed, linking low levels with vasodilatation and high levels with toxicity. The aim of this study was to evaluate the effects of melatonin co-exposure on the lead-induced reduction in renal NO levels. We found that sub-acute intraperitoneal administration of 10 mg/kg/day of lead for 15 days induced toxic levels of lead in the blood and caused renal toxicity (pathological and functional). Under our experimental conditions, lead induced an increase in lipid peroxidation and a decrease in NO. Melatonin co-treatment decreased lead-induced oxidative stress (peroxidation level) and toxic effects on kidneys without altering the lead-induced reduction in renal NO. These results suggest that, in our experimental model, the reduction in renal NO levels by lead exposure is not the only responsible factor for lead-induced kidney damage.

Thinner inhalation effects on oxidative stress and DNA repair in a rat model of abuse.

Humans can come into contact with thinner by occupational exposure or by intentional inhalation abuse. Numerous studies of workers for genotoxic effects of thinner exposure have yielded conflicting results, perhaps because co‐exposure to variable other compounds cannot be avoided in workplace exposure studies. In contrast, there is no data concerning the genotoxic effects of intentional inhalation abuse. The aim of this project was to examine the genotoxic effects of thinner inhalation in an animal model of thinner abuse (rats exposed to 3000 ppm toluene, a high solvent concentration over a very short, 15 min time period, twice a day for 6 weeks). The data presented here provides evidence that thinner inhalation in our experimental conditions is able to induce weight loss, lung abnormalities and oxidative stress. This oxidative stress induces oxidative DNA damage that is not a characteristic feature of genotoxic damage. No significant difference in DNA damage and DNA repair (biomarkers of genotoxicity) in lymphocytes from thinner‐treated and control rats was found. Lead treatment was used as a positive control in these assays. Finally, bone marrow was evaluated as a biomarker of cellular alteration associated with thinner inhalation. The observed absence of hemopoietic and genetic toxicity could be explained in part by the absence of benzene, the only carcinogenic component of thinner; however, benzene is no longer a common component of thinner. In conclusion, thinner did not cause genotoxic effects in an experimental model of intentional abuse despite the fact that thinner inhalation induces oxidative stress. Copyright

A simple solution for antibody signal enhancement in immunofluorescence and triple immunogold assays.

Immunolocalization techniques are standard in biomedical research. Tissue fixation with aldehydes and cell membrane permeabilization with detergents can distort the specific binding of antibodies to their high affinity epitopes. In immunofluorescence protocols, it is desirable to quench the sample’s autofluorescence without reduction of the antibody-dependent signal. Here we show that adding glycine to the blocking buffer and diluting the antibodies in a phosphate saline solution containing glycine, Triton X-100, Tween20 and hydrogen peroxide increase the specific antibody signal in tissue immunofluorescence and immunogold electron microscopy. This defined antibody signal enhancer (ASE) solution gives similar results to the commercially available Pierce Immunostain Enhancer (PIE). Furthermore, prolonged tissue incubation in resin and fixative and application of ASE or PIE are described in an improved protocol for triple immunogold electron microscopy that is used to show co-localization of GABA-A ρ2 and dopamine D2 receptors in GFAP-positive astrocytes in the mouse striatum. The addition of glycine, Triton X-100, Tween20 and hydrogen peroxide during antibody incubation steps is recommended in immunohistochemistry methods.

Subsurface cistern (SSC) proliferation in Purkinje cells of the rat cerebellum in response to acute and chronic exposure to paint thinner: A light and electron microscopy study

Intentional inhalation and occupational exposure are two ways humans are exposed to thinner, a widely employed solvent in industry. Inhalation of thinner induces toxic effects in various organs, with the cerebellum being one of the most affected structures of the CNS. The aim of this work was to describe specific structural alterations of cerebellum Purkinje cells in rats following exposure to thinner for 16 weeks. A histological analysis of the cerebellum of solvent-exposed rats revealed swollen Purkinje cell dendrites surrounded by empty space, and electronic microscopy showed an increase in the number of subsurface cisterns (SSCs) within their dendritic processes. After a period of non-exposure, the number of SSCs decreased without reaching normal levels, suggesting a degree of plasticity. Purkinje cell SSCs, which are derived from smooth endoplasmic reticulum, contain inositol trisphosphate receptors (IP3Rs), ryanodine receptors (RR), and a recently identified characteristic cluster of large conductance calcium-activated potassium (BKCa) channels. We found that SSCs in Purkinje cell dendrites were closely associated with mitochondria, and immunofluorescence microscopy showed higher levels of RR and calbindin receptors (CB), in Purkinje cells of exposed than normal rats. These changes are probably related to behavioral manifestations of cerebellar alterations, such as imbalance and ataxia, consistent with the suggested involvement of increases in SSCs in ataxia in rats and humans. This increase in SSCs, taken together with the localization of RR, IP3R and BKCa proteins in this structure, suggests altered intracellular calcium-buffering processes in the Purkinje cells of thinner-exposed rats.

Oxidative DNA damage induced by thinner inhalation in rats lymphocytes

Abstract Thinner inhalation is known to induce oxidative stress. Some studies have shown that thinner inhalation causes a decrease of antioxidants and formation of oxidation products of proteins and lipids as well as formamidopyrimidine glycoslyase (Fpg)-sensitive DNA sites. Using the comet assay and the repair-specific enzymes formamido pyrimidine glycosylase (Fpg) and endonuclease III (Endo III) to detect oxidized purines and pyrimidines, respectively, we examined the ability of thinner inhalation to induce oxidative DNA damage in rat lymphocytes. Our results show a high correlation between Fpg- and Endo III-sensitive sites. This, together with our previous results that showed a high correlation between Fpg-sensitive sites and two biomarkers of oxidative stress, suggests that these Fpg-sensitive sites correspond to oxidative damage during the first four weeks of thinner inhalation.