Human Sertoli cells in vitro: morphological features and androgen-binding protein secretion.

Sertoli cells play a pivotal role in the regulation of spermatogenesis as they provide the anatomical basis of the blood-testis barrier. In the present paper we report some results of our studies on the ultrastructural features, the responsiveness to FSH, and the ability to secrete androgen-binding protein (ABP) of human Sertoli cells in vitro. The nucleus showed the characteristic foldings of the nuclear membrane, scattered chromatin, and a fibrillar nucleolus. In the cytoplasm Charcot-Boettcher crystals were present and active phagocytic activity was documented by the presence of vacuoles containing lipids and cellular debris. Human Sertoli cells in culture responded to FSH with a maximal rise in cAMP that was approx. 3-fold. This response to FSH is comparable to that reported for the adult rat but lower than that of the immature rat, and suggests that human as well as rat Sertoli cells could have a reduced response to FSH since sexual maturation was achieved. As no evidence has been reported on ABP secretion by human Sertoli cells in culture we evaluated the concentration of this protein in the Sertoli cell spent media. Human Sertoli cells in culture produced ABP and the response to FSH was dose-related. The Kd value of human ABP (hABP) was approx. 7.5 nM, being slightly higher than that of the rat ABP and an order of magnitude different from that of sex hormone-binding globulin (SHBG) present in human plasma. We also measured the association and dissociation rates of dihydrotestosterone-hABP complexes and the Kd/Ka ratio was very close to the value of Kd of the Scatchard analysis. The differences between hABP and SHBG may open the way to the selective measurement of ABP in many conditions of male infertility.     

Serotonin Inhibition of the NMDA Receptor/Nitric Oxide/Cyclic GMP Pathway in Rat Cerebellum: Involvement of 5-Hydroxytryptamine2C Receptors

Abstract: Previous studies have shown that 5-hydroxytryptamine (5-HT) can potently inhibit glutamatergic transmission in rat cerebellum through the activation of multiple 5-HT receptors. The aim of this study was to subclassify the 5-HT₂ receptor mediating inhibition of the cyclic GMP response elicited by N -methyl- d -aspartate in adult rat cerebellar slices. Seven receptor antagonists, endowed with relative selectivities for the 5-HT_2A, 5-HT_2B, and 5-HT_2C subtypes, differentially affected the inhibition by (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane of the cyclic GMP response, suggesting that the receptor involved belongs to the 5-HT_2C subtype.     

A Comparison Between Different Methods for the Determination of Reduced and Oxidized Glutathione in Mammalian Tissues

In this study, three rapid assay techniques for the determination of glutathione, one enzymatic, one flu-orometric and one newly patented colorimetric method, were compared by measuring reduced (GSH) and oxidized (GSSG) glutathione in guinea-pig heart and liver. The HPLC technique was used as a standard, since it is considered the most reliable assay method. In heart, all methods measured the same levels of GSH (about 1 µmole/g wet tissue), whereas in liver the fluorometric assay gave GSH levels about half as high as those measured by the other methods (about 3 vs. 7 µmoles/g wet tissue). Conversely, the fluorometric assay grossly overestimated GSSG concentration (by 5 to 8 times) in both heart and liver. These results confirm previous doubts about the use of the fluorometric technique for GSSC determination in mammalian tissues and also raise some questions about its use for the measurement of GSH in liver. In this tissue, the GSH concentration determined by the fluorometric method was shown to be inversely correlated with the size of the sample, suggesting the presence of some quenching material.     

Gastric Juice Polymerase Chain Reaction: An Alternative to Histology in the Diagnosis of Helicobacter pylori Infection

Background. Infection from Helicobacter pylori plays a role in several gastroduodenal diseases. The recent availability of molecular techniques, particularly the polymerase chain reaction (PCR), allows us to detect small amounts of this bacterium. The aims of this study were to compare PCR and histological findings and to ascertain the clinical usefulness of H. pylori PCR identification in different biological samples.
Materials and Methods. We studied 94 consecutive patients. Saliva, gastric juice, and four antral and four body biopsies were obtained from each patients. H. pylori was evaluated histologically in two antral and two body biopsies (Giemsa or Warthin-Starry stain). After extraction, DNA was submitted for PCR amplification using the two primers HPU1 and HPU2, which amplified a 411-bp product from the urease gene A.
Results. Forty-nine patients were H. pylori -positive at histological workup. The sensitivity of PCR was 92% for gastric juice, 73% for antral biopsies, 61% for body biopsies, and 13% for saliva. Of the 45 H. pylori -negative patients at histological assessment, 7 (16%) had positive findings on PCR, mainly when gastric juice was examined.
Conclusions. These results indicate that PCR is as sensitive as histological assessment. We suggest that PCR H. pylori detection in gastric juice is a sensitive method for diagnosing this infection.     

Geometry of guanidinium groups in arginines

The restraints in common usage today have been obtained based on small molecule X‐ray crystal structures available 25 years ago and recent reports have shown that the values of bond lengths and valence angles can be, in fact, significantly different from those stored in libraries, for example for the peptide bond or the histidine ring geometry. We showed that almost 50% of outliers found in protein validation reports released in the Protein Data Bank on 23 March 2016 come from geometry of guanidine groups in arginines. Therefore, structures of small molecules and atomic resolution protein crystal structures have been used to derive new target values for the geometry of this group. The most significant difference was found for NE‐CZ‐NH1 and NE‐CZ‐NH2 angles, showing that the guanidinium group is not symmetric. The NE‐CZ‐NH1 angle is larger, 121.5(10)?, than NE‐CZ‐NH2, 119.2(10)?, due to the repulsive interaction between NH1 and CD1 atom.     

Pharmacological induction of ferritin prevents osteoblastic transformation of smooth muscle cells

Vascular calcification is a frequent complication of atherosclerosis, diabetes and chronic kidney disease. In the latter group of patients, calcification is commonly seen in tunica media where smooth muscle cells (SMC) undergo osteoblastic transformation. Risk factors such as elevated phosphorus levels and vitamin D₃ analogues have been identified. In the light of earlier observations by our group and others, we sought to inhibit SMC calcification via induction of ferritin. Human aortic SMC were cultured using β‐glycerophosphate with activated vitamin D₃, or inorganic phosphate with calcium, and induction of alkaline phosphatase (ALP) and osteocalcin as well as accumulation of calcium were used to monitor osteoblastic transformation. In addition, to examine the role of vitamin D₃ analogues, plasma samples from patients on haemodialysis who had received calcitriol or paricalcitol were tested for their tendency to induce calcification of SMC. Addition of exogenous ferritin mitigates the transformation of SMC into osteoblast‐like cells. Importantly, pharmacological induction of heavy chain ferritin by 3H‐1,2‐Dithiole‐3‐thione was able to inhibit the SMC transition into osteoblast‐like cells and calcification of extracellular matrix. Plasma samples collected from patients after the administration of activated vitamin D₃ caused significantly increased ALP activity in SMC compared to the samples drawn prior to activated vitamin D₃ and here, again induction of ferritin diminished the osteoblastic transformation. Our data suggests that pharmacological induction of ferritin prevents osteoblastic transformation of SMC. Hence, utilization of such agents that will cause enhanced ferritin synthesis may have important clinical applications in prevention of vascular calcification.     

The role of auxin in hairy root induction

Summary We have investigated the relative role of auxin and of Agrobacterium rhizogenes T-DNA in the induction of hairy roots. By infecting carrot discs with suitably constructed bacterial strains containing different T-DNA complements, we have shown that both auxin and the presence of T-DNA in the carrot cells are required for root growth on the discs. Auxin added alone or in combination with cytokinin is not sufficient to induce rooting on uninfected discs. Also cells transformed by T-DNA containing only auxin synthetic genes very rarely differentiate into roots. On the other hand auxin is necessary for hairy root induction since A. rhizogenes devoid of T-DNA-borne auxin genes is not capable of eliciting symptoms in the absence of hormone. Auxin is not required for either T-DNA transfer or T-DNA expression in the transformed host. Cells infected in the absence of auxin, which do not respond by rooting, do contain T-DNA whose expression is shown by the synthesis of hairy root opines; subsequent addition of auxin to these quiescent transformed cells results in root development. A model for hairy root induction where the action of T-DNA is envisaged as conferring auxin responsiveness to the transformed cells is discussed.     

Cytotoxicity, DNA fragmentation and sister-chromatid exchange in Chinese hamster ovary cells exposed to the lipid peroxidation product 4-hydroxynonenal and homologous aldehydes

The cytotoxic and genotoxic activities of 4-hydroxypentenal (HPE), 4-hydroxyhexenal (HHE), 4-hydroxyoctenal (HOE), 4-hydroxynonenal (HNE) and 4-hydroxyundecenal (HUE) were investigated in Chinese hamster ovary (CHO) cells. All five 4-hydroxyalkenals reduced plating efficiency in a concentration (ranging from 7 to 170 microM) lower than that producing a parallel reduction of trypan blue-excluding cells, but with both methods the increase in molarity needed to obtain a lethal effect was constantly rather small. With all five 4-hydroxyalkenals a significant amount of DNA fragmentation, as revealed either by the alkaline elution assay or by alkaline denaturation followed by chromatographic partition of single- and double-stranded DNA, was detected only after cell exposure to a cytotoxic concentration. HPE, HHE and HOE induced a clear-cut increase of sister-chromatid exchange (SCE) frequency, while that displayed by cells treated with HNE and HUE was minimal, even if dose-dependent and statistically significant. Since 4-hydroxyalkenals have been shown to originate from biomembrane lipids peroxidation, these findings should be taken into consideration in the assessment of the genotoxic role of lipoperoxidation in humans.     

Induction of sperm abnormalities in mice by norfloxacin.

Norfloxacin was tested in the mouse sperm morphology test. Data obtained suggest that norfloxacin may have 2 different effects on sperm development: a stimulating effect on spermatogenesis and a possible mutagenic effect that results in an increase in sperm abnormalities. The first effect might be caused by a hormonal action. A dose-response relationship was not observed in sperm morphology changes. Consequently norfloxacin cannot with certainty be judged a positive inducer of abnormal sperm, but further studies are essential to clarify the obtained results.