The ubiquitin-conjugating yeast enzyme RAD6 and its human homologs hHR6A and hHR6B are implicated in postreplication repair and damage-induced mutagenesis. The yeast protein is also required for sporulation and may modulate chromatin structure via histone ubiquitination. We report the phenotype of the first animal mutant in the ubiquitin pathway: inactivation of the hHR6B-homologous gene in mice causes male infertility. Derailment of spermatogenesis becomes overt during the postmeiotic condensation of chromatin in spermatids. These findings provide a parallel between yeast sporulation and mammalian spermatogenesis and strongly implicate hHR6-dependent ubiquitination in chromatin remodeling. Since heterozygous male mice and even knockout female mice are completely normal and fertile and thus able to transmit the defect, similar hHR6B mutations may cause male infertility in man.
The cellular localization of the human androgen receptor was visualized immunohistochemically using a mouse monoclonal antibody (MAb) F39.4, directed against a fragment of the N-terminal domain of the androgen receptor. The nuclear immunoreactivity of various human tissues with F39.4 was generally consistent with earlier biochemical and autoradiographic data. However, previously suggested androgen receptor expression in thyroid, pancreatic, gastrointestinal, and bladder tissues was not confirmed immunohistochemically. Stratified squamous epithelia of vagina and cervix showed selective immunostaining of the basal cell layer, whereas in the preputial epithelium the intensity of immunoreactivity decreased gradually with maturation. In contrast, glandular epithelia of the sweat glands, male accessory sex organs, and female breast showed nearly exclusive F39.4 staining of the inner cylindric layer. In the testis, Sertoli cells, peritubular myoid cells, and interstitial cells were immunoreactive with MAb F39.4. Expression of the androgen receptor by smooth muscle tissue was largely confined to the male reproductive organs. The specificity and sensitivity of this simple and rapidly performed immunohistochemical technique in the detection of the human androgen receptor at the cellular and subcellular level makes it worthwhile to study tissue androgen receptor expression by immunohistochemistry in physiological and pathological states.
Glycogen storage disease type II (GSDII; Pompe's disease) is an autosomal recessive disease caused by lysosomal α‐glucosidase deficiency. Skeletal muscle weakness is the most conspicuous clinical symptom of patients suffering from GSDII and skeletal muscle also is prominently involved in the knockout mouse model of this disease. Thus far, however, little detailed information has been published on the pathological changes in other mouse tissues. This paper aims to provide these data and gives a record of the clinical course of the mouse model over a 2‐year period. Four‐month‐old affected mice perform worse in a running wheel than their unaffected littermates, but do not yet display other clear signs of disease. The lysosomal glycogen storage, already evident at birth, becomes more severe in time, leading to muscle wasting by 9–10 months of age and then limb girdle weakness and kyphosis. The disease does not markedly shorten the animal's life span despite the serious tissue pathology, which is not limited to heart and skeletal muscle, but is also seen in the smooth muscle of blood vessels and of the respiratory, digestive, and urogenital tracts. In addition, the mice have lysosomal glycogen storage in the liver, kidney, spleen, and salivary gland; in Schwann cells of the peripheral nerves, and in a subset of neurons in the central nervous system. By pathological criteria, the knockout mouse model parallels the human infantile form of GSDII and is attractive for studying the possible reversal of tissue pathology and symptomatology under different therapeutic regimes. Copyright © 1999 John Wiley & Sons, Ltd.
Summary Various colon carcinoma cell lines were tested in different invasion assays, i.e. invasion into Matrigel, into confluent fibroblast layers and into chicken heart tissue. Furthermore, invasive capacity and metastatic potential were determined in nude mice. The colon carcinoma cells used were the human cell lines Caco-2, SW-480, SW-620 and HT-29, and the murine lines . None of the human colon carcinoma cells migrated through porous membranes coated with Matrigel; of the murine lines, only Colon-26 did. When incubated in a mixture of Matrigel and culture medium non-invading cells formed spheroid cultures, whereas invading cells showed a stellate outgrowth. Only the heterogeneously shaped (epithelioid and stellate) cells of SW-480 and SW-620 and the spindle-shaped cells of Colon-26 invaded clearly confluent skin and colon fibroblasts as well as chicken heart tissue. However, when transplanted into the caecum of nude and syngeneic mice, all the lines tested were invasive with the exception of Caco-2 cells. We conclude that the outcome of in vitro tests measuring the invasive capacity of neoplastic cells is largely dependent on the test system used. Invasive capacity in vitro is strongly correlated with cells having a spindle cell shape, vimentin expression and E-cadherin down regulation. In contrast, HT-29 and Colon-38 cells having an epithelioid phenotype were clearly invasive and metastatic in vivo, but not in vitro. © 1999 Cancer Research Campaign Keywords: Matrigel; confluent fibroblast layers; chicken hearts; orthotopic transplantation 934British Journal of Cancer (1999) 81(6), 934-941 © 1999 Cancer Research Campaign Article no. bjoc.1999 Received 2 In vitro testing Matrigel assayThe assay was carried out as described by Albini et al (1987). Perforated polyvinylpyrrolidone-free polycarbonate membranes of 13-mm diameter (Nucleopore, CA, USA) were used with 8-µm pore size, which is sufficiently large to allow the passage of single carcinoma cells.To promote attachment all the membranes were first coated on the lower side in 4-well dishes with 15 µl of a 0.1% solution of fibronectin (Sigma, St Louis, MO, USA) in serum-free medium according to Sieuwerts et al (1997). Subsequently half of the membranes were coated with a layer of Matrigel to measure invasion. An amount of 20 µl containing 40 µg Matrigel was applied per membrane surface obtained by dilution of a stock solution with serum-free medium as recommended (Collaborative Biomedical Products, Becton and Dickinson Labware, Bedford, UK). The concentration used prevents preliminary detachment of the Matrigel from the filters and falls within the range of concentrations used by others (see Discussion). The Matrigel was gelatinized at 37°C for 20 min. The other half of the membranes was left uncoated to measure migration.The membranes were sealed in a sterilized Boyden chemotaxis chamber (Nucleopore Membrane Products, CA, USA) after filling the lower compartment with 220 µl of serum-free NIH-3T3 conditioned medium as attractant. The upper compartment was ...
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