Sertoli and Leydig cell numbers and gonadotropin receptors in rat testis from birth to puberty

Summary In testes of rats from 2 to 60 days of age, we examined the number of Sertoli cells (SC) and Leydig cells (LC) as well as the binding of radioiodinated gonadotropins to frozen sections and homogenates. The number of SC per testis increased only during the first 2 postnatal weeks, whereas that of LC was stable up to days 7–10 and increased thereafter. The uptake of ¹²⁵I-labelled human follicle-stimulating hormone (¹²⁵I-FSH) to frozen sections was confined to sex cords or seminiferous tubules, while that of ¹²⁵I-labelled human choriogonadotropin (¹²⁵I-hCG) matched the distribution of LC in the interstitium. High affinity receptors for FSH and hCG were found in homogenates at all stages studied. The number of FSH receptors per testis increased steadily, whereas that of hCG receptors was low until days 7–10 and rose afterwards. Thus, SC in rat testis appear to proliferate in the presence of fetal LC during the first 2 postnatal weeks and to differentiate concomitantly with the emergence of the adult LC generation after day 10. The complement of FSH receptors in SC remains constant as they proliferate and increases after day 21 as they differentiate. The hCG receptor number is relatively fixed in each LC generation, being higher in adult compared to fetal LC.     

In search of specific cytotoxic T lymphocytes infiltrating or accompanying human ovarian carcinoma

Summary Lymphocytes infiltrating human ovarian carcinoma obtained directly from the tumour mass (tumour-infiltrating lymphocytes, TIL) or from the carcinomatous ascites (tumour-associated lymphocytes, TAL) were expanded in vitro in long-term cultures with interleukin-2 and tested for their specific cytolytic activity. Killing of the autologous tumour was detected only in a proportion of the patients, less frequently in TIL compared to TAL. In fact two out of ten TIL and four out of nine TAL cultures tested showed significant levels of lysis against the autologous tumour. This cytotoxic activity was not restricted to the autologous tumour, as other tumour cell lines, including non-ovarian ones, were lysed as well. The cultures that were not cytotoxic against the autologous tumour were in most cases able to lyse other tumour cell lines of ovarian or other histology. Cloning of TIL from one patient was performed: of 22 clones tested, 4 displayed higher cytotoxicity against the autologous tumour compared to the uncloned population and 3 out of these 4 did not kill an irrelevant carcinoma cell line. In order to stimulate the expansion of putative specific effectors we performed mixed lymphocyte/tumour cultures (MLTC) with autologous or allogeneic tumour cells. No stimulation of cytotoxicity against the autologous tumour was detected after MLTC in nine different TAL populations, using autologous or allogeneic tumours as stimulators. On the contrary, peripheral blood lymphocytes from two patients after MLTC with the autologous tumour showed increased killing of the autologous and decreased killing of an allogeneic target. In conclusion TIL and TAL from ovarian carcinoma expanded in vitro with interleukin-2 usually have non-MHC-restricted cytotoxicity and variable degrees of reactivity against the autologous tumour. A preferential killing for the autologous tumour was not observed even after MLTC. These results do not exclude the existence of tumour-specific cytotoxic T lymphocytes in ovarian carcinoma; nevertheless they suggest that putative specific effectors have very low frequency and that culture techniques for expanding their growth more selectively are still to be optimized.     

Review of prevalence of Simian Virus 40 (SV40) genomic infection in healthy subjects

Because of the carcinogenicity of SV40 in rodents, and its possible distribution through the polio vaccine, many studies have been conducted to determine if there is an association between SV40 genomic infection and different types of cancer; sometimes, these studies included data on the prevalence of genomic infection in healthy subjects as secondary information. We reviewed all the studies that reported the prevalence of SV40 genomic infection in healthy subjects, tested by PCR based methods. The 20 articles considered here included 1103 samples from healthy subjects, with a prevalence of infection ranging from 0 to 25.6%, with high heterogeneity, and no association with the type of sample analyzed (Mantel-Haenszel OR: 0.74; 95% CI: 0.44-1.23). The wide variation in frequency pose problems in terms of study design; in fact, the representativeness of the samples used as controls in the published studies may be very limited. Larger studies on healthy subjects, tested for SV40 genomic infection at various genomic regions, conducted in different geographic areas, are needed.     

A computational model of the LGI1 protein suggests a common binding site for ADAM proteins

Mutations of human leucine-rich glioma inactivated (LGI1) gene encoding the epitempin protein cause autosomal dominant temporal lateral epilepsy (ADTLE), a rare familial partial epileptic syndrome. The LGI1 gene seems to have a role on the transmission of neuronal messages but the exact molecular mechanism remains unclear. In contrast to other genes involved in epileptic disorders, epitempin shows no homology with known ion channel genes but contains two domains, composed of repeated structural units, known to mediate protein-protein interactions.A three dimensional in silico model of the two epitempin domains was built to predict the structure-function relationship and propose a functional model integrating previous experimental findings. Conserved and electrostatic charged regions of the model surface suggest a possible arrangement between the two domains and identifies a possible ADAM protein binding site in the β-propeller domain and another protein binding site in the leucine-rich repeat domain. The functional model indicates that epitempin could mediate the interaction between proteins localized to different synaptic sides in a static way, by forming a dimer, or in a dynamic way, by binding proteins at different times.The model was also used to predict effects of known disease-causing missense mutations. Most of the variants are predicted to alter protein folding while several other map to functional surface regions. In agreement with experimental evidence, this suggests that non-secreted LGI1 mutants could be retained within the cell by quality control mechanisms or by altering interactions required for the secretion process.     

Activity, stability and structural studies of lactate dehydrogenases adapted to extreme thermal environments

Lactate dehydrogenase (LDH) catalyzes the conversion of pyruvate to lactate with concomitant oxidation of NADH during the last step in anaerobic glycolysis. In the present study, we present a comparative biochemical and structural analysis of various LDHs adapted to function over a large temperature range. The enzymes were from Champsocephalus gunnari (an Antarctic fish), Deinococcus radiodurans (a mesophilic bacterium) and Thermus thermophilus (a hyperthermophilic bacterium). The thermodynamic activation parameters of these LDHs indicated that temperature adaptation from hot to cold conditions was due to a decrease in the activation enthalpy and an increase in activation entropy. The crystal structures of these LDHs have been solved. Pairwise comparisons at the structural level, between hyperthermophilic versus mesophilic LDHs and mesophilic versus psychrophilic LDHs, have revealed that temperature adaptation is due to a few amino acid substitutions that are localized in critical regions of the enzyme. These substitutions, each having accumulating effects, play a role in either the conformational stability or the local flexibility or in both. Going from hot- to cold-adapted LDHs, the various substitutions have decreased the number of ion pairs, reduced the size of ionic networks, created unfavorable interactions involving charged residues and induced strong local disorder. The analysis of the LDHs adapted to extreme temperatures shed light on how evolutionary processes shift the subtle balance between overall stability and flexibility of an enzyme.     

'Click' synthesis of a triazole-based inhibitor of Met functions in cancer cells

The use of Cu(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition permitted the synthesis of a new compound that is able to inhibit the HGF-induced scattering of MDCK (epithelial cells) and in vitro tumorigenesis of H1437 (non-small-cell lung cancer) and GTL-16 (human gastric carcinoma). In agreement with biochemical and biological results, docking studies within the ATP binding site of Met suggested for the new synthesized compound a binding mode similar to that of the active compound Triflorcas previously reported.     

Binding of BiP to an assembly-defective protein in plant cells

The binding protein (BiP) has been implicated as a mediator of protein folding and assembly in the endoplasmic reticulum of mammalian cells and has often been found in stable association with structurally defective proteins. To acquire information on the activity of BiP in plant cells, we have expressed in tobacco protoplasts the wild type form and an assembly-defective form of bean phaseolin. Phaseolin (PHSL) is a soluble, trimeric, storage glycoprotein co-translationally inserted into the lumen of the endoplasmic reticulum and then transported along the secretory pathway to the protein storage vacuoles. We have previously shown that a PHSL mutant in which the last 59 amino acids have been deleted (Δ363PHSL) is unable to form trimers and is retained in a pre-Golgi compartment when synthesized in Xenopus oocytes. When transiently expressed in tobacco leaf protoplasts, wild-type PHSL is correctly glycosylated and assembles efficiently and rapidly into trimers. Δ363PHSL is also correctly glycosylated but does not trimerize. Tobacco BiP and Δ363PHSL are co-immunoselected using either anti-PHSL or anti-BiP antibodies. Under the same conditions, co-immunoselection of BiP with wild-type PHSL is not detectable. The BiP bound to Δ363PHSL can be released by treatment of the complex with ATP, indicating that the binding is related to the proposed function of BiP in protein folding and assembly in the endoplasmic reticulum. These data indicate that BiP stably binds structurally defective proteins in plant cells.     

Historical trends in the epidemiology of candidaemia: analysis of an 11-year period in a tertiary care hospital in Brazil

Candida species are an important cause of bloodstream infections (BSI). To evaluate the epidemiological, clinical and microbiological aspects of two cohorts {1994-1999 [period 1 (P1) ]; 2000-2004 [period 2 (P2) ]} of candidaemic patients, we performed a retrospective analysis from a laboratory-based survey. A total of 388 candidaemias were identified, with an incidence of 0.20/1,000 patient-days and a significant increase in P2 vs. P1 (0.25 vs. 0.15, p = 0.04). Cancer and prior antibiotic use were frequent and Candida albicans was the most prevalent species found (42.4%). Resistance to fluconazole was found in 2.47% of the strains. No differences were observed in the species distribution of Candida during the study periods. In the P2 cohort, there were higher prevalence of elderly individuals, cardiac, pulmonary and liver diseases, renal failure, central venous catheters and antibiotic therapy. In P1, there were higher prevalence of neurological diseases and chemotherapy. The crude mortality was 55.4%. In conclusion, our incidence rates remained high. Furthermore, the distribution pattern of Candida species and the fluconazole resistance profile remained unchanged. Moreover, we found a clear trend of higher prevalence of candidaemia among the elderly and among patients with comorbidities. Finally, it is necessary to discuss strategies for the prevention and control of Candida BSI in Brazil.     

Plasma glutamine decreases immediately after surgery and is related to incisiveness

Glutamine (gln) is the most abundant free amino acid in the blood. It is involved in important metabolic and biochemical processes, like cell proliferation and oxidative stress. Previous studies have demonstrated that gln concentration in human plasma decreases in several conditions such as sepsis, ischemia-reperfusion, trauma, major surgery and burn. The aim of the present work was to compare the acute effects of different types of surgical interventions and of anesthetization on blood gln concentration. Plasma samples from 88 subjects (30 males and 58 females) were collected before and after major or minor surgery and the gln concentration was analyzed with high-performance liquid chromatography. The results showed that plasma gln concentration after surgery was lower than pre-surgery values and that in major surgery the decrease of gln was higher than in minor surgery. No significant effect was shown for sex or type of anesthesia. These results demonstrate the importance of a gln supplementation before a surgical intervention and show that the amount of gln supplementation should also be adjusted based on the type of surgery.