Liver growth factor induces testicular regeneration in EDS-treated rats and increases protein levels of class B scavenger receptors.

The aim of the present work was to determine the effects of liver growth factor (LGF) on the regeneration process of rat testes after chemical castration induced by ethane dimethanesulfonate (EDS) by analyzing some of the most relevant proteins involved in cholesterol metabolism, such as hormone sensitive lipase (HSL), 3ß-hydroxysteroid dehydrogenase (3ß-HSD), scavenger receptor SR-BI, and other components of the SR family that could contribute to the recovery of steroidogenesis and spermatogenesis in the testis. Sixty male rats were randomized to nontreated (controls) and LGF-treated, EDS-treated, and EDS + LGF-treated groups. Testes were obtained on days 10 (T1), 21 (T2), and 35 (T3) after EDS treatment, embedded in paraffin, and analyzed by immunohistochemistry and Western blot. LGF improved the recovery of the seminiferous epithelia, the appearance of the mature pattern of Leydig cell interstitial distribution, and the expression of mature SR-BI. Moreover, LGF treatment resulted in partial recovery of HSL expression in Leydig cells and spermatogonia. No changes in serum testosterone were observed in control or LGF-treated rats, but in EDS-castrated animals LGF treatment induced a progressive increase in serum testosterone levels and 3ß-HSD expression. Based on the pivotal role of SR-BI in the uptake of cholesteryl esters from HDL, it is suggested that the observed effects of LGF would facilitate the provision of cholesterol for sperm cell growth and Leydig cell recovery.

Acute up-regulation of the rat brain somatostatin receptor-effector system by leptin is related to activation of insulin signaling and may counteract central leptin actions.

Leptin and somatostatin (SRIF) have opposite effects on food seeking and ingestive behaviors, functions partially regulated by the frontoparietal cortex and hippocampus. Although it is known that the acute suppression of food intake mediated by leptin decreases with time, the counter-regulatory mechanisms remain unclear. Our aims were to analyze the effect of acute central leptin infusion on the SRIF receptor-effector system in these areas and the implication of related intracellular signaling mechanisms in this response. We studied 20 adult male Wister rats including controls and those treated intracerebroventricularly with a single dose of 5 µg of leptin and sacrificed 1 or 6h later. Density of SRIF receptors was unchanged at 1h, whereas leptin increased the density of SRIF receptors at 6h, which was correlated with an elevated capacity of SRIF to inhibit forskolin-stimulated adenylyl cyclase activity in both areas. The functional capacity of SRIF receptors was unaltered as cell membrane levels of αi1 and αi2 subunits of G inhibitory proteins were unaffected in both brain areas. The increased density of SRIF receptors was due to enhanced SRIF receptor subtype 2 (sst2) protein levels that correlated with higher mRNA levels for this receptor. These changes in sst2 mRNA levels were concomitant with increased activation of the insulin signaling, c-Jun and cyclic AMP response element-binding protein (CREB); however, activation of signal transducer and activator of transcription 3 was reduced in the cortex and unchanged in the hippocampus and suppressor of cytokine signaling 3 remained unchanged in these areas. In addition, the leptin antagonist L39A/D40A/F41A blocked the leptin-induced changes in SRIF receptors, leptin signaling and CREB activation. In conclusion, increased activation of insulin signaling after leptin infusion is related to acute up-regulation of the SRIF receptor-effector system that may antagonize short-term leptin actions in the rat brain.

Valoración del fenotipo neoangiogénico y linfangiogénico en lesiones preneoplásicas y primarias de cáncer de pulmón / No disponible

No disponible

In vitro and in vivo characterization of neural stem cells.

Neural stem cells are defined as clonogenic cells with self-renewal capacity and the ability to generate all neural lineages (multipotentiality). Cells with these characteristics have been isolated from the embryonic and adult central nervous system. Under specific conditions, these cells can differentiate into neurons, glia, and non-neural cell types, or proliferate in long-term cultures as cell clusters termed "neurospheres". These cultures represent a useful model for neurodevelopmental studies and a potential cell source for cell replacement therapy. Because no specific markers are available to unequivocally identify neural stem cells, their functional characteristics (self-renewal and multipotentiality) provide the main features for their identification. Here, we review the experimental and ultrastructural studies aimed at identifying the morphological characteristics and the antigenic markers of neural stem cells for their in vitro and in vivo identification.

Cerebral postischemic reperfusion-induced demethylation of the protein phosphatase 2A catalytic subunit.

Brain reperfusion after a period of global ischemia induces changes in the phosphorylation state of a great number of proteins. Neuronal responses to ischemia and reperfusion are quite different depending on the brain region, and phosphorylation changes may be implicated in this tissue-specific response. For this reason, we have used both biochemical and immunohistochemical methods to investigate the potential role of PP2A, the most abundant Ser/Thr phosphatase in the brain, in ischemic injury. PP2A activity as measured with phosphorylase a as substrate was slightly inhibited after 30 min ischemia followed by 30 min reperfusion, and this inhibition correlated with an increased S6K1 and ERK1/2 phosphorylation. Using a monoclonal antibody unable to recognize the methylated form of PP2Ac, we demonstrated that the catalytic subunit of PP2A (PP2Ac) was highly methylated in the brain. In addition, the postischemic reperfusion-induced changes in PP2Ac methylation were studied in sections from cerebral cortex, hippocampus and striatum. Regional differences in PP2Ac methylation were observed within control brains, and the postischemic reperfusion caused a generalized demethylation of PP2Ac. Those regions in the control brains containing highest levels of methylated PP2Ac were the most intensively demethylated after reperfusion and corresponded to the regions most vulnerable to ischemic damage.

Immunohistochemical detection of the retinoid acid receptors (RXR-alpha, -beta, -gamma) and Farnesoid X-activated receptor (FXR) in the marbled newt along the annual cycle.

Retinoid acid receptors (RXR-alpha, -beta, -gamma) and Farnesoid X-activated receptor (FXR) expression in the testis of the marbled newt were investigated with special attention to the changes during the annual testicular cycle, using light microscopy immunohistochemistry and Western blot analysis. The annual testicular cycle of the marbled newt (Triturus marmoratus marmoratus) comprises three periods: (a) proliferative period (germ cell proliferation from primordial germ cells to round spermatids, April-June); (b) spermiogenesis period (July-September); and (c) quiescence period (interstitial and follicular cells form the glandular tissue, October-April). In the proliferative period, primordial germ cells and primary spermatogonia immunostained intensely to the three types of RXRs and also to FXR. In the other periods, immunostaining to these antibodies was weak or absent. Secondary spermatogonia stained weakly to the four antibodies in the proliferative period, and only to FXR, also weakly, in the spermiogenesis period. Immunoreactive primary spermatocytes were weakly labeled with the RXR antibodies in the proliferative period. Spermatids and spermatozoa did not stain to any antibody in any period. Follicular cells only immunostained to RXR-gamma and only in the quiescence period when they are forming the glandular tissue, together with the interstitial cells. As follicular cells, interstitial cells only immunostained in the quiescence period; however, they immunoreacted to the three types of RXRs. These findings suggest that in the newt, RXRs and FXR are involved in spermatogenesis control by regulating the proliferation of primordial germ cells and spermatogonia. In addition, RXR-gamma seems to be also involved in the development of the glandular (steroidogenic) tissue.

Androgen receptor (AR), estrogen receptor-alpha (ER-alpha) and estrogen receptor-beta (ER-beta) expression in the testis of the newt, Triturus marmoratus marmoratus during the annual cycle.

Expression of androgen receptor (AR), estrogen receptor alpha (ER-alpha) and estrogen receptor beta (ER-beta) in the testis of the marbled newt (Triturus marmoratus marmoratus) was investigated, with special attention to changes during the annual testicular cycle, using light microscopy immunohistochemistry and Western blot analysis. Primordial germ cells, primary and secondary spermatogonia and spermatocytes showed a positive reaction to the 3 receptor antibodies during the annual reproductive cycle. Follicular cells were positive to AR, ER-alpha and ER-beta during the spermiogenesis and quiescence periods in the glandular tissue. Interstitial cells showed reactivity to AR, ER-alpha and ER-beta in the spermiogenesis and the quiescence periods, and presented no labelling to these receptors in the proliferative period. These findings suggest that, as in mammals, there is an androgen-estrogen regulation of the function and development of the newt testis.

An osmotic-sensitive taurine pool is localized in rat pancreatic islet cells containing glucagon and somatostatin.

Previous reports have dealt with the hypoglycemic properties of taurine and its effects on insulin secretion by adult and fetal isolated islets. We have studied the presence and cellular distribution of taurine in rat islets, the conditions to evoke its release, and its possible modulatory action on insulin secretion. We localized taurine by techniques of double immunolabeling in most glucagon-positive cells and in some somatostatin-positive cells, whereas insulin-positive cells were not labeled with the taurine antibody. Although high-glucose stimulation did not evoke any taurine release, a hyposmotic solution (17% osmolarity reduction) induced a specific phasic release of taurine and GABA (34 and 52% increase on their basal release rate). On the other hand, taurine (10 mmol/l) application slightly reduced the second phase of insulin secretion induced by glucose stimulation. In conclusion, taurine is highly concentrated in glucagon-containing cells of the islet periphery. It is not liberated by glucose stimulation but is strongly released under hyposmotic conditions. All of these data suggest that taurine plays an osmoregulatory role in alpha-cells.

Developmental expression of fibroblast growth factor (FGF) receptors in neural stem cell progeny. Modulation of neuronal and glial lineages by basic FGF treatment.

Neural stem cells (NSCs) are self-renewable, multipotential cells capable of differentiating into the three major neural cell types, but the mechanisms which regulate their development are not fully understood. Both basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) promote the proliferation of NSCs. However, studies on the role of FGFs in the differentiation of EGF-expanded NSCs are still incomplete. We have studied the expression of distinct FGF receptors (FGFRs) in the progeny of EGF-expanded NSCs isolated from E15 rat striatum. In situ hybridization analysis and immunocytochemistry showed a developmentally related expression pattern and a cell lineage-specific distribution of these receptors. FGFR1 and FGFR2 were identified in many early precursors and in the oligodendrocyte lineage. The latter receptor was also present in a subpopulation of astrocytes. FGFR3 was detected in a restricted population of early precursors, in oligodendroglial progenitors, and in neurons and protoplasmic astrocytes of late-term cultures. Basic FGF treatment of the progeny of NSCs increased the proliferative rate of precursors and the number of oligodendrocytes generated, whereas the number of differentiating neurons was significantly reduced. Together these data provide evidence that FGFs modulate the development of EGF-expanded NSCs, and that this is at least partly determined by a cell lineage-specific expression of multiple FGFRs.

Localization of the lipid receptors CD36 and CLA-1/SR-BI in the human gastrointestinal tract: towards the identification of receptors mediating the intestinal absorption of dietary lipids.

The scavenger receptors CLA-1/SR-BI and CD36 interact with native and modified lipoproteins and with some anionic phospholipids. In addition, CD36 binds/transports long-chain free fatty acids. Recent biochemical evidences indicates that the rabbit CLA-1/SR-BI receptor can be detected in enterocytes, and previous studies showed the presence of mRNA for both CLA-1/SR-BI and CD36 in some segments of the intestinal tract. These findings prompted us to study their respective localization and distribution from the human stomach to the colorectal segments, using immunohistochemical methods. Their expression in the colorectal carcinoma-derived cell line Caco-2 was analyzed by Northern blotting. In the human intestinal tract, CLA-1/SR-BI was found in the brush-border membrane of enterocytes from the duodenum to the rectum. However, CD36 was found only in the duodenal and jejunal epithelium, whereas enterocytes from other intestinal segments were not stained. In the duodenum and jejunum, CD36 co-localized with CLA-1/SR-BI in the apical membrane of enterocytes. The gastric epithelium was immunonegative for both glycoproteins. We also found that CLA-1/SR-BI mRNA was expressed in Caco-2 cells and that its expression levels increased concomitantly with their differentiation. In contrast, the CD36 transcript was not found in this colon cell line, in agreement with the absence of this protein in colon epithelium. The specific localization of CLA-1/SR-BI and CD36 along the human gastrointestinal tract and their ability to interact with a large variety of lipids strongly support a physiological role for them in absorption of dietary lipids.

[Putative chromophobe cell renal carcinoma: are they or are they not?]. / Carcinomas renales de células cromófobas putativos: son o no son?

OBJECTIVE: According to the literature, the typical histological findings and simple colloidal iron staining permit the identification of chromophobe cell renal carcinoma, a genetically well-established entity. Our doubts on whether this tumor type can be recognized by conventional methods are presented in this study. METHODS: 130 cases of renal carcinoma were treated from 1977 to 1997. Of these, 12 showed characteristic general histological features compatible with chromophobe cell renal carcinoma and reticulated and intense, diffuse cytoplasmic positivity on colloidal iron staining. These tumors were reviewed for the following: 1) gross appearance, 2) architecture, 3) cytoplasmic characteristics, 4) nuclear characteristics, 5) colloidal iron histochemical staining which is considered fundamental and exclusive, 6) immunohistochemical phenotype. Ultrastructural study of material fixed in paraffin was also performed. RESULTS: One case met all 6 criteria, 3 met 5 of them, 6 cases met 4, and 2 cases met 3 of the criteria. The ultrastructural study was not useful in making the diagnosis due to the poor quality of the material. CONCLUSIONS: If all cases were chromophobe cell renal carcinoma, then this tumor type can be recognized and diagnosed by simple techniques in any pathology laboratory, and its incidence, presence of necrosis, hemorrhage and high nuclear grade would be even greater than currently accepted. Furthermore, it would be expected that the prognosis, by each grade and stage, would not be so different from that of the conventional renal carcinoma, as some large series have already indicated. On the other hand, if our cases or some of them were in fact conventional renal cell carcinoma that closely resembled chromophobe cell renal carcinoma, an exact diagnosis cannot be made without a genetic or ultrastructural study (using adequately fixed material), and most of the published studies would therefore have to be questioned.

Immunohistochemical localization of taurine in the rat ovary, oviduct, and uterus.

The distribution of the amino acid taurine in the female reproductive organs has not been previously analyzed in detail. The aim of this study was to determine taurine localization in the rat ovary, oviduct, and uterus by immunohistochemical methods. Taurine was localized in the ovarian surface epithelium. The granulosa cells and oocytes of primordial follicles were immunonegative. In primary and antral follicles, taurine was found mainly in theca cells and oocytes, whereas the zona pellucida, antrum, and most granulosa cells were unstained. However, taurine immunoreactivity in theca cells and oocytes decreased during follicular atresia. During corpora lutea development, the number of immunopositive theca lutein cells increased as these cells invaded the granulosa-derived region. Therefore, most luteal cells from the mature corpora lutea were stained. In the regressing corpora lutea, however, taurine staining in luteal cells decreased. In the fimbriae, infundibulum, and uterotubal junction, taurine was localized in most epithelial cells. In the ampullar and isthmic segments, taurine was found in the cilia of most ciliated cells and in the apical cytoplasm of some non-ciliated cells. In the uterus, most epithelial cells were immunopositive during diestrus and metestrus, whereas most of them were immunonegative during estrus and proestrus. Moreover, taurine immunoreactivity in the oviduct and uterus decreased with pregnancy. (J Histochem Cytochem 49:1133-1142, 2001)

Taurine levels and localisation in the stratified squamous epithelia.

The content and distribution of the amino acid taurine in squamous epithelia were studied using high-performance liquid chromatography and immunohistochemical methods. Quantitative analysis demonstrated that taurine was highly concentrated in the epidermis (5.49 mumol/g fresh tissue in the hairless skin of the hind footpad of the rat), although the values in the isolated stratum corneum were extremely low (< 0.073 mumol/g in the horny layer of the same skin area). No other analysed amino acid (such as glutamate, glutamine, glycine or alanine) showed this specific pattern of distribution. The immunohistochemical study revealed that in the dog and rat epidermis, taurine was present in the keratinocytes of the granular and upper spinous layers. The basal layer, lower spinous layer and stratum corneum were immunonegative. A similar immunostaining pattern was found in the epithelia of the different organs studied: the mouth, tongue and oesophagus of the dog and rat, the rat forestomach and the rat corneal epithelium. Other cell types, such as sebaceous and muscle cells, were immunolabelled. The existence of a circulating pool of taurine in the epidermis (via taurine release from keratinocytes before they reach the horny layer and its uptake by nearby cells) and its possible roles in these cells are discussed.

[Renal carcinoma of sarcomatoid chromophobe cells]. / Carcinoma renal de células cromófobas sarcomatoide.

OBJECTIVE: Based on the clinicopathological findings of two additional cases of sarcomatoid chromophobe renal cell carcinoma and a review of the literature, we analyzed the prognosis in this and other forms of sarcomatoid carcinoma to determine the differences, if any, and their histopathological basis. METHODS: Of 139 cases of renal cell carcinoma that were surgically resected during the period 1977-1999, two were sarcomatoid chromophobe renal cell carcinoma, accounting for 15% of 13 cases of chromophobe cell carcinoma and 18% of 11 cases of sarcomatoid cell carcinoma in the same series. RESULTS: The first case was a 73-year-old male with a locally advanced, non-metastatic tumor. Palliative resection was performed and the histological analysis showed a predominantly sarcomatoid mass with small epithelial foci with the morphological, histochemical and immunohistochemical characteristics of chromophobe renal cell carcinoma. The patient died 11 months later. The second case was a 70-year-old female who presented with flank and lumbar pain and episodes of gross hematuria. Anatomopathological analysis showed a chromophobe cell carcinoma with sarcomatous foci, stage pT2pN0M0. The patient is disease-free at 46 months' follow-up. Immunohistochemically, in both cases the sarcomatoid component was found to be strongly positive for vimentin and focally for EMA, and negative for actin, desmin and myoblogin; isolated cells were positive for AE1 and AE3 in the second case. The epithelial component was positive for AE3 and EMA, and negative for AE1, vimentine and CD68. CONCLUSIONS: Like our first case, most of the reported cases of sarcomatoid chromophobe renal cell carcinoma show a sarcomatoid mass with foci of carcinoma, and a poor prognosis. In our view, as in all renal cell carcinomas, prognosis depends on tumor grade and stage, which is the highest for sarcomatoid chromophobe renal cell carcinoma. Furthermore, although the epithelial component (chromophobe, chromophilic, clear cells, etc.) may probably have little significance, the influence that the proportion of epithelial and sarcomatoid component might have in these tumors cannot be completely discarded.

Levels, phosphorylation status and cellular localization of translational factor eIF2 in gastrointestinal carcinomas.

The level of expression and the phosphorylation status of the alpha subunit of initiation factor 2 (eIF2alpha) protein have been determined by comparing samples from human stomach, colon and sigma-rectum carcinomas with normal tissue from the same patients. The unphosphorylated and phosphorylated levels of cytoplasmic eIF2alpha, as well as the percentage of phosphorylated factor over the total, were significantly higher in stomach, colon and sigma-rectum tumours compared with normal tissue. The expression of this factor was also studied by using immunocytochemical methods, where redistribution towards the nucleus in tumour cells as compared with normal tissue was observed. Our results support a likely implication of eIF2alpha in gastrointestinal cancer.

Immunocytochemical localization of taurine in different muscle cell types of the dog and rat.

The presence and distribution of the amino acid taurine in different muscle cell types of the dog and rat was examined by immunocytochemical methods. The light microscope study revealed that smooth muscle cells were similarly immunoreactive for taurine, whereas skeletal muscle fibres showed wide differences in taurine immunoreactivity among individual cells. Some skeletal fibres were strongly immunoreactive whereas others did not display immunolabelling. Mononucleated satellite cells, found adjacent to skeletal fibres in a quiescent stage, were also immunostained. Other myoid cells, such as testicular peritubular cells showed a cytoplasmic and a nuclear pool of taurine. By means of electron microscope immunolabelling, the subcellular localization of taurine was studied in vascular and visceral smooth muscle cells. Taurine was present in most subcellular compartments and frequently appeared randomly distributed. Taurine was localized on myofilaments, dense bodies, mitochondria, the plasma membrane and the cell nucleus. Moreover, the labelling density within individual smooth muscle cells was variable and depended on the state of contraction of each single fibre. Contracted cells showed a higher density of gold particles than relaxed cells. Unmyelinated nerve fibres, found adjacent to smooth muscle cells from the muscularis mucosae and the lamina propria of the stomach, were unstained or poorly stained.

Immunohistochemical localization of taurine in the male reproductive organs of the rat.

The amino acid taurine has been implicated in several aspects of reproductive system physiology. However, its localization in these organs has not been previously analyzed. The aim of this study was to characterize its distribution in male rat reproductive organs by immunohistochemical methods. Taurine was localized in the smooth muscle cells of the tissues studied and in the skeletal fibers of the cremaster muscle. In the testis, taurine was found in Leydig cells, vascular endothelial cells, and other interstitial cells. No immunoreactivity was observed in the cells of the seminiferous tubules, either in germ cells at all spermatogenic stages or in Sertoli cells. However, peritubular myoid cells were immunostained. Most epithelial cells of the efferent ducts were immunolabeled, whereas the epithelial cells of the rete testis (extratesticular segments), epididymis (caput, corpus, and cauda regions), and ductus deferens were unstained. However, most epithelial cells from the intratesticular segments of the rete were immunopositive. Some cells identified as intraepithelial macrophages and lymphocytes, apical cells, and narrow cells were intensely immunolabeled. Regional differences in the distribution of these cell types along the ducts studied were also noted. The possible functional roles for taurine in these cells are discussed.

Cytochemical localization of calcium in prefusion myoblasts from the chick embryo myotome.

Myoblast fusion is a Ca2+-dependent process. The aim of this report was to study the localization of Ca2+ in prefusion myoblasts from the brachial somites of chick embryos (51-108 h of incubation), using the potassium pyroantimonate cytochemical method. When observed under a transmission electron microscope, electron-dense precipitates of Ca2+-antimonate were found in the basement membrane of the myotome, which separates the myotome from the adjacent mesenchyma. Within myoblasts, triads and sarcoplasmic reticulum associated with the first newly formed sarcomeres were observed, but a T-tubule network was not found. Moreover, Ca2+-antimonate precipitates were not observed in structures resembling T-tubules or sarcoplasmic reticulum. The results suggest that sarcomerogenesis and sarcoplasmic reticulum development occur simultaneously and that prefusion myoblasts have neither a T-tubule network nor Ca2+ deposits on sarcoplasmic reticulum. Small Ca2+ pools were found in the myoblast nuclei, cytoplasmic vesicles and mitochondrias. Ca2+-antimonate precipitates periodically distributed at the cell periphery, close to the cell membrane, were observed. These precipitates could represent internal Ca2+ stores located in the peripheral couplings and it is proposed that these pools of Ca2+ could be mobilized before fusion, leading to the increase in free intracellular Ca2+ that precedes myoblast fusion.

Intracellular biogenesis of collagen fibrils in 'activated fibroblasts' of tendo Achillis. An ultrastructural study in the New Zealand rabbit.

We have studied the formation of collagen fibrils in 'activated fibroblasts' of tendo Achillis of rabbits. The tendon was in the process of regeneration after experimental partial tenotomy. Samples were taken from the peri-incisional region and analysed by transmission electron microscopy. Ultrastructural examination showed the presence of a 'fine dense granular substance' inside the rough endoplasmic reticulum and procollagen filaments. These come together to form collagen fibrils in the dilated vacuoles of the rough endoplasmic reticulum. The possible intra- and extracellular origin of collagen fibrils is suggested. Within the cell biosynthesis of collagen fibrils take place with the formation of collagen substance which gives rise to procollagen filaments. These make contact in parallel apposition to produce striated 'spindle-shaped bodies' which elongate by the longitudinal attachment of more procollagen filaments and form intracellular nascent collagen fibrils.