Intracellular distribution of pregnenolone metabolites in testis of macaque (Macaca fascicularis)

The metabolism of pregnenolone in subcellular fractions of the testes of the macaque (Macaca fascicularis) has been studied using capillary gas chromatography to characterize and quantify the metabolites, after their conversion into the O-methyloxime and/or trimethylsilyl ether derivatives. The microsomal incubations yielded the greatest quantities of metabolites, with lesser amounts in the mitochondrial fraction. The cytosolic fraction contained no significant quantity of metabolites after incubation, except for 5alpha-androst-16-en-3 beta-ol. This, and other odorous androst-16-enes, found in the microsomal fraction, are of particular interest in the context of animal communication because of their possible pheromonal role. Pregnenolone was converted into androst-5-ene-3 beta,17 beta-diol, androst-4-ene-3,17-dione and testosterone, suggesting that both classical pathways for testosterone synthesis were operating. Testosterone was further converted into 5 alpha-reduced androstanediols, especially in the microsomal fraction.     

Aspects of the demetalation and remetalation of ceruloplasmin

This study dealt with the demetalation and remetalation chemistry of the copper-containing protein ceruloplasmin. For the enzyme from human plasma, dialysis against cyanide at 4°C readily removed copper. Although the apoprotein took up copper(I) at the same temperature, the characteristic blue color of the native protein did not return to any significant extent. However, excellent reconstitution occurred when we added copper(I) at room temperature. With porcine ceruloplasmin, it was more practical to carry out the copper removal step at room temperature, but the reconstitution went smoothly at 4°C. With either source of ceruloplasmin, the binding of the six essential copper ions was generally a highly cooperative process, but the results were different when we combined the apoprotein with Hg(II). After the protein took up 2 mercury ions, it would take up only 1–2 more metal ions even after exposure to a large excess of copper(I). In order to accomodate the various experimental results, we have proposed that a reversible conformational change must occur during the demetalation and remetalation processes. During the remetalation process, it is therefore important that metal uptake occurs in the proper sequence.     

Facile Synthesis of Benzimidazoles via Oxidative Cyclization of Acyclic Monoterpene Aldehyde with Diamines: Studies on Antimicrobial and in Vivo Evaluation of Zebrafish

Citral ( 1a ), a bioactive component of Cymbopogon citratus (lemongrass) could be isolated and semi-synthetic analogs synthesized with improved therapeutic properties. Herein we first report describes citral ( 1a ) as a primary material for the synthesis of benzimidazole derivatives between various o-phenylenediamines ( 2a – l ) in the presence of Diisopropylethylamine (DIPEA) as a commercially available environmentally benign base, ethanol as a green solvent and the yield of all benzimidazole derivatives ( 3a – l ) was between 68–76 %; The semi-synthetically prepared benzimidazole derivatives ( 3a – l ) were assessed for their anti-bacterial and anti-fungal properties. The benzimidazole compounds ( 3a – b , and 3g – j ) exhibit good anti-microbial activity. In addition, in silico study was carried out to determine the specific binding affinity of the diamine halogen substituted benzimidazole derivatives to the specific target proteins. In silico analysis revealed a high correlation between docking results and experimental results. Finally, benzimidazole demonstrated significant antibacterial and antifungal activity. Zebrafish embryos were subjected to In vivo toxicological test found that all of the benzimidazole compounds ( 3a – l ) were non-toxic and had low embryotoxicity after 96 h, with an LC₅₀ of 36.425 μg, which could facilitate the design of novel antimicrobial agents using a cost-effective method.     

Linoleic acid-induced endothelial cell injury: Role of membrane-bound enzyme activities and lipid oxidation

High plasma levels of linoleic acid (18:2) may injure endothelial cells, resulting in decreased barrier function of the vascular endothelium. The effects of linoleic acid on endothelial barrier function (transendothelial movement of albumin), membrane-bound enzyme activities, and possible autooxidation of linoleic acid under experimental conditions were studied. The exposure of endothelial monolayers to 18:2 for 24 hr at 60, 90, and 120 μM. fatty acid concentrations caused a significant increase in transendothelial movement of albumin, with maximum albumin transfer at 90 μM. Fatty acid treatment resulted in the increased appearance of cytosolic lipid droplets. Activities of the membrane-bound enzymes, angiotensin-converting enzyme (ACE), and Ca²⁺-ATPase increased steadily with increasing time of cell exposure to 90 μM 18:2, reaching significance at 24 hr. Treatment of endothelial cultures with up to 120 μM 18:2 did not cause cytotoxicity, as evidenced by a nonsignificant change in cellular release of [₃H]-adenine. Incubation of 18:2-supplemented serum-containing culture media with 1000 μM 18:2 at 37°C for up to 48 hr did not result in formation of autooxidation products. These results suggest that 18:2 itself, and not its oxidation products, plays a major role in disrupting endothelial barrier function.     

Renal brush border membrane bound intrinsic factor

A highly active receptor for intrinsic factor (IF)-cobalamin (Cbl) complex has been detected and reported in mammalian kidney earlier (Seetharam, B., et al. (1988) J. Biol. Chem. 263, 4443-4449). The physiological role of this receptor in normal Cbl homeostasis is not known. In addition to binding of exogenously added IF-[57Co]Cbl, the renal apical membranes contain endogenous IF or IF-Cbl. Washing with pH 5/EDTA buffer enhanced the binding of exogenously added IF-[57Co]Cbl to renal apical but not basolateral membranes. The pH 5/EDTA extract from renal apical membranes bound [57Co]Cbl. The complex also bound to rat ileal brush border membrane and promoted ileal transport of [57Co]Cbl. On immunoblots using monospecific antiserum to IF a 62 kDa protein was identified in renal and intestinal apical membranes, serum and in tissue extracts of unperfused rat liver, kidney and heart. The 62 kDa band was eliminated from the renal apical membranes following pH 5/EDTA wash. Rat urine demonstrated unsaturated [57Co]Cbl binding (0.2 to 0.4 pmol/day) of which only 30-40% was immunoprecipitated with anti IF and could be identified on immunoblots. The identification of IF in rat renal apical membranes (160-200 ng/mg protein) and secretion of only traces of IF in urine suggest that the renal IF-Cbl receptor may play a role in sequestering IF/IF-Cbl and prevent urinary loss of Cbl.     

The effect of polyamines on tyrosine hydroxylase and L-Aromatic amino acid decarboxylase

The polyamines stimulated tyrosine hydroxylase in whole homogenates of bovine caudate nuclei approximately 2 fold. TheV _max forl-tyrosine increased by 2.3 fold while theK _m s forl-tyrosine and for the cofactor (DMPH₄) were unchanged.l-Aromatic amino acid decarboxylase from whole rat brain homogenate was stimulated by about 40% in the presence of polyamines. These findings suggest that increased polyamine levels associated with increased cellular synthetic activity can modify the synthesis of neurotransmitters.     

Mechanism of interaction of myelin basic protein and S-100 protein: metal binding and fluorescence studies.

Abstract— It has been reported that myelin basic protein (MBP) forms a specific complex with S-100 protein in the presence of either Ca²⁺ or Mn²⁺, as detected by Immunoelectrophoresis. We have now studied the binding of Ca²⁺ and Mn²⁺ to these two proteins. We find that MBP binds 1 mol of Mn²⁺/mol of protein, and this binding produces an increment in its fluorescence, indicating a conformational change. Ca²⁺ does not bind to MBP nor does it affect the fluorescence of MBP. S-100 protein, as has been reported, binds about 10 mol of Ca²⁺/mol and this binding produces a conformational change. S-100 protein also has 25 binding sites for Mn²⁺, but this binding does not alter fluorescence and does not appear to affect conformation. Competitive binding experiments demonstrate that the binding sites of S-100 protein for Ca²⁺ and Mn²⁺ are independent. The alteration of electrophoretic migration in gels of S-100 protein produced by Ca²⁺ and of MBP produced by Mn²⁺ are in accord with the observations based on fluorescence. Mn²⁺ does not affect the electrophoretic mobility of S-100. These results indicate that the formation of the complex between MBP and S-100 protein in the presence of either Ca²⁺ or Mn²⁺ is due to the conformational change induced by these ions in S-100 protein, MBP, or both.     

Low oxygen tension potentiates proliferation and stemness but not multilineage differentiation of caprine male germline stem cells

The milieu of male germline stem cells (mGSCs) is characterized as a low-oxygen (O₂) environment, whereas, their in-vitro expansion is typically performed under normoxia (20–21% O₂). The comparative information about the effects of low and normal O₂ levels on the growth and differentiation of caprine mGSCs (cmGSCs) is lacking. Thus, we aimed to investigate the functional and multilineage differentiation characteristics of enriched cmGSCs, when grown under hypoxia and normoxia. After enrichment of cmGSCs through multiple methods (differential platting and Percoll-density gradient centrifugation), the growth characteristics of cells [population-doubling time (PDT), viability, proliferation, and senescence], and expression of key-markers of adhesion (β-integrin and E-Cadherin) and stemness (OCT-4, THY-1 and UCHL-1) were evaluated under hypoxia (5% O₂) and normoxia (21% O₂). Furthermore, the extent of multilineage differentiation (neurogenic, adipogenic, and chondrogenic differentiation) under different culture conditions was assessed. The survival, viability, and proliferation were significantly (p?<?0.05) improved, thus, yielding a significantly (p?<?0.05) higher number of viable cells with larger colonies under hypoxia. Furthermore, the expression of stemness and adhesion markers were distinctly upregulated under lowered O₂ conditions. Conversely, the differentiated regions and expression of differentiation-specific genes [C/EBPα (adipogenic), nestin and β-tubulin (neurogenic), and COL2A1 (chondrogenic)] were significantly (p?<?0.05) reduced under hypoxia. Overall, the results demonstrate that culturing cmGSCs under hypoxia augments the growth characteristics and stemness but not the multilineage differentiation of cmGSCs, as compared with normoxia. These data are important to develop robust methodologies for ex-vivo expansion and lineage-committed differentiation of cmGSCs for clinical applications.