Víctor Tsutsumi

Transient receptor potential (TRPC) channels in human sperm: expression, cellular localization and involvement in the regulation of flagellar motility.

Capacitative Ca2+ entry is a process whereby the activation of Ca2+ influx through the plasma membrane is triggered by depletion of intracellular Ca2+ stores. Some transient receptor potential (TRPC) proteins have been proposed as candidates for capacitative Ca2+ channels. Recent evidence indicates that capacitative Ca2+ entry participates in the sperm acrosome reaction (AR), an exocytotic process necessary for fertilization. In addition, several TRPCs have been detected heterogeneously distributed in mouse sperm, suggesting that they may participate in other functions such as motility. Using reverse transcription‐polymerase chain reaction (RT‐PCR) analysis, RNA messengers for TRPC1, 3, 6 and 7 were found in human spermatogenic cells. Confocal indirect immunofluorescence revealed the presence of TRPC1, 3, 4 and 6 differentially localized in the human sperm, and immunogold transmission electron microscopy indicated that TRPC epitopes are mostly associated to the surface of the cells. Because all of them were detected in the flagellum, TRPC channel antagonists were tested in sperm motility using a computer‐assisted assay. Our results provide what is to our knowledge the first evidence that these channels may influence human sperm motility.

Kupffer cells inhibition prevents hepatic lipid peroxidation and damage induced by carbon tetrachloride.

The aim of this work was to determine if the action mechanism of gadolinium on CCl(4)-induced liver damage is by preventing lipid peroxidation (that may be induced by Kupffer cells) and its effects on liver carbohydrate metabolism. Four groups of rats were treated with CCl(4), CCl(4)+GdCl(3), GdCl(3), and vehicles. CCl(4) was given orally (0.4 g 100 g(-1) body wt.) and GdCl(3) (0.20 g 100 g(-1) body wt.) was administered i.p. All the animals were killed 24 h after treatment with CCl(4) or vehicle. Glycogen and lipid peroxidation were measured in liver. Alkaline phosphatase, gamma-glutamyl transpeptidase, alanine amino transferase activities and bilirubins were measured in rat serum. A liver histological analysis was performed. CCl(4) induced significant elevations on enzyme activities and bilirubins; GdCl(3) completely prevented this effect. Liver lipid peroxidation increased 2.5-fold by CCl(4) treatment; this effect was also prevented by GdCl(3). Glycogen stores were depleted by acute intoxication with CCl(4). However, GdCl(3) did not prevent this effect. The present study shows that Kupffer cells may be responsible for liver damage induced by carbon tetrachloride and that lipid peroxidation is produced or stimulated by Kupffer cells, since their inhibition with GdCl(3) prevented both lipid peroxidation and CCl(4)-induced liver injury.

Expression and differential cell distribution of low-threshold Ca2+ channels in mammalian male germ cells and sperm

Numerous sperm functions including the acrosome reaction (AR) are associated with Ca2+ influx through voltage‐gated Ca2+ (CaV) channels. Although the electrophysiological characterization of Ca2+ currents in mature sperm has proven difficult, functional studies have revealed the presence of low‐threshold (CaV3) channels in spermatogenic cells. However, the molecular identity of these proteins remains undefined. Here, we identified by reverse transcription polymerase chain reaction the expression of CaV3.3 mRNA in mouse male germ cells, an isoform not previously described in these cells. Immunoconfocal microscopy revealed the presence of the three CaV3 channel isoforms in mouse spermatogenic cells. In mature mouse sperm only CaV3.1 and CaV3.2 were detected in the head, suggesting its participation in the AR. CaV3.1 and CaV3.3 were found in the principal and the midpiece of the flagella. All CaV3 channels are also present in human sperm, but only to a minor extent in the head. These findings were corroborated by immunogold transmission electron microscopy. Tail localization of CaV3 channels suggested they may participate in motility, however, mibefradil and gossypol concentrations that inhibit CaV3 channels did not significantly affect human sperm motility. Only higher mibefradil doses that can block high‐threshold (HVA) CaV channels caused small but significant motility alterations. Antibodies to HVA channels detected CaV1.3 and CaV2.3 in human sperm flagella.

Curcumin prevents and reverses cirrhosis induced by bile duct obstruction or CCl4 in rats: role of TGF-β modulation and oxidative stress

Curcumin is a phytophenolic compound, which is highly efficacious for treating several inflammatory diseases. The aim of this study was to evaluate the efficacy of curcumin in preventing or reversing liver cirrhosis. A 4‐week bile duct ligation (BDL) rat model was used to test the ability of curcumin (100 mg/kg, p.o., daily) to prevent cirrhosis. To reverse cirrhosis, CCl4 was administered chronically for 3 months, and then it was withdrawn and curcumin administered for 2 months. Alanine aminotransferase, γ‐glutamyl transpeptidase, liver histopathology, bilirubin, glycogen, reduced and oxidized glutathione, and TGF‐β (mRNA and protein) levels were assessed. Curcumin preserved normal values of markers of liver damage in BDL rats. Fibrosis, assessed by measuring hydroxyproline levels and histopathology, increased nearly fivefold after BDL and this effect was partially but significantly prevented by curcumin. BDL increased transforming growth factor‐beta (TGF‐β) levels (mRNA and proteins), while curcumin partially suppressed this mediator of fibrosis. Curcumin also partially reversed the fibrosis induced by CCl4. Curcumin was effective in preventing and reversing cirrhosis, probably by its ability of reducing TGF‐β expression. These data suggest that curcumin might be an effective antifibrotic and fibrolitic drug in the treatment of chronic hepatic diseases.

β-Defensin Gene Expression during the Course of Experimental Tuberculosis Infection

The kinetics of gene expression and the cellular source of murine beta -defensin-3 (mBD3) and murine beta -defensin-4 (mBD4) were determined in mouse models of progressive pulmonary tuberculosis and latent infection induced by high or low infecting doses, respectively. During progressive disease, there was an initial rapid expression of both defensins by respiratory epithelial cells that correlated with temporary control of bacillary proliferation, but expression decreased during the later progressive phase of the disease. In latent infection, both defensins were expressed continuously, but they were suppressed after reactivation of the disease. Thus, mycobacterial infection induces the expression of mBD3 and mBD4, and both might participate in the control of mycobacterial growth.

Entamoeba histolytica: erythrophagocytosis, collagenolysis, and liver abscess production as virulence markers

High rates of erythrophagocytosis and collagenolysis in vitro have been regarded as indicative of virulence in vivo of Entamoeba histolytica trophozoites. In the present study, the erythrophagocytic index and the collagenolytic activity of 3 axenic lines of E. histolytica, strain HM1:IMSS, were measured. The 3 lines shared the same pathogenic zymodeme but showed clear-cut differences in the extent of liver damage induced in hamsters. A direct correlation between collagenolysis in vitro and the size of liver abscesses produced by each line of E. histolytica trophozoites was found. In contrast, the line with the highest erythrophagocytic index produced small amoebic abscesses in hamsters, whereas the line with a relatively low erythrophagocytic index produced the largest liver lesions. It is concluded that the extent of collagenolytic activity is a better marker of virulence of E. histolytica cultured under axenic conditions than is erythrophagocytosis.

α-Tocopherol modulates liver toxicity of the pyrethroid cypermethrin

The objective of the current study was to analyze the hepatotoxic effect caused by cypermethrin (CYP) in rats, and to evaluate the possible protective effect of the antioxidant alpha-tocopherol (alpha-T). Fifty male Wistar rats were given daily i.p. doses of 300 mg/kg per day of CYP during 7 days. Half of them were administered three previous doses of 100 mg/kg per day of alpha-T, followed by seven subsequent oral doses of 40 mg/kg per day of alpha-T. The levels of biochemical indicators and histological liver damage were determined, as well as DCVA in urine. CYP altered the lipid metabolism. Such alterations were inhibited 32% by alpha-T, except for LDL. Alterations in AST were modulated in 29%. In the histology, alpha-T reduced mitochondria damage, and swelling of the endoplasmic reticulum of the liver cells. The results suggest that alpha-T can modify CYP metabolism, changing the lipidic profile and the histological analysis.

Role of neutrophils in innate resistance to Entamoeba histolytica liver infection in mice

In order to define the role of neutrophils in the innate resistance to Entamoeba histolytica liver infection in mice, we examined the pattern of liver lesion induced by direct injection of E. histolytica trophozoites in normal mice and in neutrophil‐depleted mice. A variety of histological lesions were found, the extent of liver damage was considerably higher in the neutrophil‐depleted mice. Livers from neutrophil‐depleted mice displayed areas of liquefactive (lytic) necrosis containing a large number of amoebae and absence of neutrophils or mononuclear cells. By contrast, in the liver of normal mice, neutrophils were seen associated to E. histolytica at early stages of infection. In both mouse groups, areas of TUNEL‐positive dead hepatocytes were observed and a characteristic internucleosomal banding pattern of genomic DNA consistent with apoptosis was detected in DNA harvested from amoebic liver lesions. These data suggest that neutrophils play an important role in the mechanisms of resistance to amoebic liver infection in mice. In addition, our histological analysis suggests that E. histolytica is capable of producing liver damage in the absence of inflammatory cells.

Entamoeba histolytica: immunohistochemical study of hepatic amoebiasis in mouse. Neutrophils and nitric oxide as possible factors of resistance.

Studies in mice have not rendered conclusive data on cell and humoral factors to support the resistance of this rodent to Entamoeba histolytica infection. In Balb/c and C3H/HeJ mice inoculated with live or fixed trophozoites, we studied the evolution of the hepatic lesion, the kinetics of inflammatory cells, and the participation of some humoral factors in the development of the hepatic amoebic lesion. From the first hour, amoebae were surrounded by neutrophils containing inducible nitric oxide synthase (iNOS); macrophages also expressing iNOS appeared lately, whereas NK cells were not part of the inflammatory infiltrates. On the fourth day, neutrophils, macrophages, T and B lymphocytes, plasma cells, and some NK cells limited the lesions and anti-amoeba antibodies appeared when most parasites had been eliminated. Therefore, the resistance of the mice to E. histolytica probably lies in non-specific immune responses, among which the activation of neutrophils and the production of nitric oxide (NO) may be important amoebicide factors.

Ultrastructural study of the midgut and hindgut in eight species of the genus Dendroctonus Erichson (Coleoptera: Scolytidae)

Abstract Chemical communication mediated by pheromones is a crucial aspect in the life cycle of beetles in the genus Dendroctonus. This communication plays an important role in mate location and in the colonization of host conifers. The study of the alimentary canal of these species is of importance not only because this organ is involved in the processes of digestion, detoxification, nutrient absorption, and transport, but also in the production of semiochemical compounds, such as pheromones. To better understand these functions and where they occur, the ultrastructural differences between the anterior and posterior midgut and the hindgut and their different cellular types were characterized. Adult specimens of both sexes from eight species were dissected and the alimentary canal was removed. It was sectioned into three parts: anterior midgut, posterior midgut, and hindgut, and analyzed by transmission electron microscopy. Results show that the epithelial tissue of the midgut possesses ultrastructural characteristics that permit differentiation of the anterior and posterior midgut. There are no ultrastructural differences within sexes of the same species, but differences exist among species. The ultrastructural characteristics of the hindgut do not differ between sexes or among species, but they do differ from those of the midgut.