Sexual dimorphism of the extraorbital lacrimal glands in SF-1 knockout mice

Sexual dimorphism (SD) represents all the differences between males and females of the same species. SD of the murine lacrimal gland and the major effect of testosterone on its formation are well documented. Steroidogenic factor-1 (SF-1, NR5a1) is a nuclear receptor essential for the fetal development of steroid hormones producing organs and SF-1 knockout mice (Sf-1 KO) are therefore born without gonads and adrenal glands. The aim of this study was to investigate whether SD in lacrimal glands is present in the absence of exposure to sex hormones during development. Lacrimal glands from adult Sf-1 KO male and female mice without hormonal exposure, and from males that were treated with testosterone propionate (TP) prior to sacrifice, were examined. After sacrifice, glandular tissue was processed using standard histological procedures. Paraffin sections were analysed by stereology and immunostained against the androgen receptor (AR). Our results showed that there were no statistically significant differences in the mean volumes of acini, connective tissue or ductal system between males, females, and males on TP. The same pertains to the mean length of the ducts in all three groups. In the absence of sex hormones, sex chromosomes proved to be insufficient in inducing sexual dimorphism in LG. However, nuclei of the acinar cells in males on TP were positive for AR, whereas in males without TP no expression of AR was detected. Administration of TP induced the expression of AR in the nuclei of acinar cells of males but did not affect the morphology of LG. We conclude that SD in the lacrimal gland is not present in Sf-1 KO mice and this suggests that sex hormones have a major role in the development of SD in the lacrimal gland.     

Chemically synthesized solid phase oligosaccharide probes for carbohydrate-binding receptors. Interactions of the E-, L- and P-selectins with sialyl-Le(x) and O-sulphated forms linked to biotin or to polyacrylamide

There is a growing interest in chemically defined oligosaccharide reagents for identifying proteins that bind carbohydrates and determining the specificities of carbohydrate-binding proteins. Here, we compare three sets of chemically synthesized commercially available oligosaccharide conjugates as immobilized probes, for the binding signals that they elicit with known carbohydrate-binding receptors of the immune system, the E-, P- and L-selectins. The first set of conjugates is of oligosaccharides linked to biotin via a nine-carbon spacer. The second and third sets are multivalent derivatives in which the oligosaccharides are linked, via a three-carbon spacer to poly[N-(2-hydroxyethyl)acrylamide] (PAA) or to biotinylated PAA with an average of 20% substitution of the hydroxyethyl-amide groups by carbohydrate. The conjugates were immobilized on streptavidin-coated microwells if biotinylated, otherwise by drying in uncoated wells. The most robust binding curves, overall, were with the biotinylated PAA derivatives of the ligands immobilized on streptavidin wells. These reagents have permitted a reevaluation of selectin binding signals elicited by sialyl-Lewis(x) (SLe(x)) analogues having sulphate at position 6 of the galactose (6'SuSLe(x)) or of the N-acetylglucosamine (6SuSLe(x)). The results clarify the role of 6SuSLe(x), rather then 6'SuSLe(x), as a ligand for the selectins.     

Progressive loss of lambda prophage recombinogenicity in UV-irradiated Escherichia coli: the role of RecBCD enzyme

RecBCD enzyme is involved in the radiation-induced process known as prophage inactivation. The process leads to the inability of lambda prophage to excise itself from the Escherichia coli chromosome via site-specific recombination. In this work we sought to further characterize the role of RecBCD enzyme in this process. In addition, we examined the ability of irradiated prophage to recombine with the infecting homologous phage. We used several E. coli mutants differentially altered in RecBCD's activities. The results showed that in the mutants carrying either recB2109 or recD1903, which do not exhibit significant nuclease activities, the prophage progressively loses its capacity for both site-specific and general recombination. In the recB268 null mutant, however, prophage recombinogenicity remained fully preserved. We also showed that the prophage unable to recombine retained its ability to complement the mutant infecting phage and that the recombination frequencies in phage x phage crosses were not affected by postirradiation incubation. Our results suggest that the helicase activity of RecBCD is responsible for the progressive loss of prophage recombinogenicity. This loss is most probably a consequence of the unsuccessful RecBCD-dependent recombinational repair of double-stranded breaks in the cell chromosome, during which some structures unsuitable for further recombination reactions may be produced.     

Modification of the Staphylococcus aureus fibronectin binding phenotype by V8 protease.

The amount of cell surface fibronectin (Fn)-binding protein (FnBP) adhesin expressed by Staphylococcus aureus is maximal during exponential growth but disappears rapidly as the culture progresses into stationary phase. To identify factors responsible for the loss of cell surface FnBP, a culture of S. aureus L170, which shows high levels of Fn binding, was supplemented at the time of inoculation with concentrated stationary-phase supernatant from S. aureus L530, a strain which binds Fn poorly. The resulting exponential-phase cells were devoid of FnBP. The factor responsible for this activity was purified from the culture supernatant and identified as V8 protease. When cultured with 375 ng of exogenous V8 protease ml(-1), exponential-phase cells of S. aureus L170 were devoid of cell surface FnBP, and concentrations as low as 23 ng x ml(-1) resulted in reduced amounts of FnBP. Addition of the protease inhibitor alpha2-macroglobulin to the culture medium prevented the growth-phase-dependent loss of cell surface FnBP, whereas growth with exogenous V8 protease resulted in reduced adherence to the solid-phase N-terminal fragment of Fn and to the extracellular matrix synthesized by fetal rabbit lung fibroblasts. Although FnBP was extremely sensitive to V8 protease, exogenous protease did not exert a significant influence on the amount of cell surface protein A. However, a limited number of other high-molecular-weight cell surface proteins were also sensitive to V8 protease. Therefore, both the adhesive phenotype and cell surface protein profile of S. aureus can be modified by V8 protease activity.     

A critical approach to gamma variate fit of radionuclide-angiography pulmonary histograms

Radionuclide-angiography (RNA_ left-to-right intracardiac-shunt quantification algorithms, based on the part-by-part fit technique and the use of a so-called gamma variate model function (GVF), were tested via simulation analysis using data obtained from normal subjects. A good bolus of radioindicator was obtained by administering it directly into the vena subclavia. Normal subjects were defined as those having pulmonary histograms (PH) with no visible distortion caused by a shunt. Pure, non-superimposed data on the downslope of the PH curves, which are lost in presence of a shunt, proved to be appropriate reference values for testing the accuracy of results of standard shunt quantification algorithms. A generalized four-parameter GVF was introduced in order to extend the flexibility of the model function. The use of the three-parametric GVF to reconstruct the downslope of the PH curve out of the upslope data proved to be inadequate. This reveals an evident source of error in algorithms that calculate the shunt contribution by fitting GVF parameters to so-called difference-curve data. It is concluded that the inherent restricted statistical weight of RNA data prevents accurate results being obtained from standard RNA-shunt-assessment algorithms.     

Effects of high doses of testosterone propionate and testosterone enanthate on rat seminiferous tubules — a stereological and cytological study

The effects of exogenous testosterone on various testicular variables has become of increasing significance because of its potential use in male contraception. For this reason, high doses of two testosterone esters [testosterone propionate (TP) and testosterone enanthate (TE)] were used in a study of their influence on the morphology, length and curvature of the seminiferous tubules of the rat testis, and on cytological smears of the seminiferous tubules epithelium. TP was given for 14 days (3 mg/100 g body weight, i. m.) to assess the acute effects of testosterone on the seminiferous tubules. TE was administered for 60 days (in the same manner as TP) to study possible chronic effects on the rat testis. After TP and TE treatment the seminiferous tubule epithelium showed disorganization and desquamation of spermatogenic cells. In the TP-treated testes the tubules lined with Sertoli cells only were observed. The values for the length and curvature of seminiferous tubules of the TP- and TE-treated rats were significantly reduced (p<0.001). All these changes were observed earlier in the TP-treated than in the TE-treated animals. In cytological smears of the testis of the TP- and TE-treated rats an increase of vacuoles and residual bodies in Sertoli cell cytoplasm was noted. In addition, a reduction of spermatogenic cells, particularly sperms, was manifest in the smears after treatment. Large groups of Sertoli cells were seen in the smears from these testes.The study was supported by a Grant for Scientific Research No. 3-01-041 from the Ministry of Science, Technology and Informatics of the Republic of Croatia     

Lamina propria of sex cords in human fetal testis: an immunohistological and stereological study

Testicular peritubular cells are located in the lamina propria of seminiferous tubules. These cells, significantly contributing to the basal membrane of seminiferous epithelium, have been studied in a number of species. However, there is a lack of data on the development of the lamina propria in the human testis. The aim of our survey was to investigate the characteristics of the lamina propria and, in particular, peritubular cells in the fetal human testes by immunohistological and stereological methods. Therefore, testes (14–39 weeks of gestation, n=45) were dissected and fixed in a 4% buffered paraformaldehyde solution. Several pieces of each testis were embedded in paraffin and processed for immunohistochemical and stereological analysis. All investigated testes have shown sex cords in the process of development and differentiation. Morphologically, peritubular cells in the lamina propria can be divided into two types: fibroblast-like (FL) and myoid-like (ML) type (cells which much resemble mature myoid cells). By immunohistochemistry, both FL and ML cells are found to be strongly positive for the intermediate filament desmin, but negative for -smooth actin. While FL cells intensively express Ki-67 demonstrating proliferative activity, ML cells are found to be negative. The basement membrane of sex cords as well as the blood vessels of the interstitium show strong positivity to collagen IV and laminin. Concerning the correlation between the appearance of the investigated antigens with the gestational age, all antigens have been expressed (in the manner described above) already in the 14th week of gestation. The stereological analysis of the number (Nv) and volume (Vv) of peritubular cells indicates a pulsatile development of these cells in the lamina propria of the human fetal testis. While the stereological variables determined for FL cells show a gradual decrease, the same variables determined for ML cells demonstrate a successive increase. It appears that the lamina propria of the fetal human testes shares many of the properties previously discovered in rodents.     

Synchronous primary carcinomas of the ampulla of vater and ascending colon in a patient with multiple flat adenomas

Multiple primary cancers occurring in the same patients have been reported to represent 1.8–3.9% of all cancers. The majority of all patients reported to have had a combination of simultaneous neoplastic changes in the ampulla of Vater and the colon showed familial adenomatous polyposis (FAP) syndrome. Variants of familial adenomatous polyposis coli are: attenuated adenomatous polyposis coli (AAPC, previously also known as flat adenoma syndrome) and multiple adenoma coli. AAPC is characterized clinically by many, but usually fewer than 100, colonic lesions that are characteristically slightly elevated and plaque-like, with a reddish surface and sometimes central depression. Genetically it represents an extremely rare variant of FAP. Another group of individuals, so-called multiple adenoma patients, have a phenotype similar to AAPC, but most have no demonstrable germ-line adenomatous polyposis coli mutation, as do patients with FAP or AAPC. However, there have been only a few reports that discussed concurrent neoplastic changes in the ampulla of Vater and colon in patients with multiple colonic flat adenomas, but without the florid phenotype of classical FAP. We present rare clinical course of a patient with multiple (more than 60) flat adenomas in the proximal colon and two primary cancers: of the ampulla of Vater and of the ascending colon. This patient and his family history did not show polyposis compatible with FAP or hereditary nonpolyposis colorectal cancer (HNPCC) syndrome.