GC-1 mRHBDD1 knockdown spermatogonia cells lose their spermatogenic capacity in mouse seminiferous tubules

Background  

Apoptosis is important for regulating spermatogenesis. The protein mRHBDD1 (mouse homolog of human RHBDD1)/rRHBDD1 (rat homolog of human RHBDD1) is highly expressed in the testis and is involved in apoptosis of spermatogonia. GC-1, a spermatogonia cell line, has the capacity to differentiate into spermatids within the seminiferous tubules. We constructed mRHBDD1 knockdown GC-1 cells and evaluated their capacity to differentiate into spermatids in mouse seminiferous tubules.     

Transport of liposomal and albumin loaded curcumin to living cells: an absorption and fluorescence spectroscopic study

Curcumin, a lipid soluble antioxidant, exhibits solvent and medium sensitive absorption and fluorescence properties. Using such changes, the average binding constants of curcumin to phosphatidylcholine (PC) liposomes and human serum albumin (HSA) were estimated to be 2.5 x 10(4) M(-1) and 6.1 x 10(4) M(-1) respectively. From the studies on temperature dependent fluorescence anisotropy of liposomal curcumin and its fluorescence quenching by acrylamide and iodide, it was concluded that curcumin is located in the gel phase of the liposomes. Similarly from the studies on quenching of tryptophan fluorescence in HSA by curcumin, it was found to be in the same domain as that of tryptophan. Both liposomal and HSA vehicles were examined for the transfer of curcumin to spleen lymphocyte cells, EL4 lymphoma cell line and compared with aqueous DMSO vehicles. From these studies it was found that liposomal vehicle is capable of loading more curcumin in to cells than HSA or aqueous-DMSO, and lymphoma cells show preferential uptake of curcumin to lymphocytes. The fluorescence of curcumin in EL4 lymphoma cells was found to be significantly higher as compared to the lymphocytes. The present study demonstrates a simple and quantitative method of estimation of curcumin delivered to cells by different vehicles using absorption and fluorescence spectroscopy.     

Cloning and expression analysis of two distinct HIF-alpha isoforms – gcHIF-1alpha and gcHIF-4alpha – from the hypoxia-tolerant grass carp,Ctenopharyngodon idellus

Background  

Hypoxia-inducible factors (HIFs) are involved in adaptive and survival responses to hypoxic stress in mammals. In fish, very little is known about the functions of HIFs.     

Unusual complexation of Cu(I) by pyrimidine/pyridine-pyrazole derived ligands exploiting the molecular function of 2-mercapto-4,6-dimethylpyrimidine - Syntheses, crystal structures and electrochemistry

Reaction of 2 equiv. amount of copper(II) chloride dihydrate with 2 equiv. of methyl-5-methyl-1-(4,6-dimethyl-2-pyrimidyl)pyrazole-3-carboxylate (DpymPzC) in presence of 1 equiv. of 2-mercapto-4,6-dimethylpyrimidine (DpymtH) at pH ∼ 6 afforded the tricoordinated copper(I) complex [Cu(DpymPzC)Cl] (1). The same reaction with copper(II) perchlorate hexahydrate, as the metal salt under the same equivalent ratio at pH ∼ 6 formed the tetracoordinated copper(I) complex [Cu(DpymPzC)₂]ClO₄ (2). In both the cases, the role of DpymtH is nothing but only to reduce the copper(II) salt in situ finally forming the copper(I) complex. On the other hand, the direct reaction between the copper(I) thiocyanate and DpymPzC in 2:2 equiv. ratio produced a tricoordinated copper(I) complex [Cu(DpymPzC)SCN] (3). In a similar reaction of 2 equiv. amount of copper(II) chloride dihydrate with 2 equiv. amount of ethyl-5-methyl-1-(2-pyridyl)pyrazole-3-carboxylate (PyPzC) in presence of 1 equiv. of DpymtH at pH ∼ 6, an intense red coloured microcrystalline compound (4) was obtained. In contrast, 1 equiv. of PyPzC and 2 equiv. of DpymtH on reaction with 1 equiv. of copper(II) chloride dihydrate at pH ∼ 6 produced a novel tetranuclear mixed coordinated [Cu₄(DpymtH)₄Cl₄] complex (5). Here DpymtH plays dual role - a reducing agent for the copper(II) salt followed by a chelating ligand towards copper(I) so formed in situ. Among the above species, 1, 2 and 5 are crystallographically characterized. In 1, the central copper atom is in distorted triangular planar geometry with N₂Cl chromophore whereas in 2, the same is in distorted tetrahedral geometry with N₄ chromophore. Notably, the extent of distortion from the ideal geometry is more in 2. In 5, which is in chair conformation, out of four copper atoms, two being in S₂Cl chromophore are tricoordinated and the remaining two are tetracoordinated with NS₂Cl chromophore. The metal centers are bridged through DpymtH in its ‘thione’ form. Interestingly, the chelation (in part) results in formation of the highly stable four-membered two chelate rings around the two tetracoordinated copper atoms in 5. The two copper centers along the long arm of the chair are separated through a distance of 5.190 Å while those in the short arm are at a length of 3.629 Å. The electronic, IR spectra and electrochemistry of the complexes 1, 2 and 5 have also been investigated.     

In vitro plant regeneration from cotyledonary nodes of Withania somnifera (L.) Dunal and assessment of clonal fidelity using RAPD and ISSR markers

An efficient large-scale clonal propagation protocol has been described for Withania somnifera (L.) Dunal, a valuable medicinal plant, using cotyledonary nodes derived from axenic seedlings. Murashige and Skoog’s (Physiol Plant 15:473–497, 1962) (MS) medium supplemented with 1.0 mg l^?1 N ⁶-benzyladenine (BA) was found to be optimum for production of multiple shoots (100 % shoot proliferation frequency and 16.93 shoots per explant). Successive shoot cultures were established by repeatedly sub-culturing the original cotyledonary node on a fresh medium after each harvest of newly formed shoots. Multiple shoot proliferation was also achieved from nodal segments derived from in vitro raised shoots on MS medium augmented with 1.0 mg l^?1 BA. Regenerated shoots were best rooted (95.2 %, 38.7 roots per shoot) in half-strength MS medium supplemented with 1.0 mg l^?1 indole-3-butyric acid. The plantlets were successfully acclimated and established in soil. Random amplified polymorphic DNA and inter-simple sequence repeats analysis revealed a homogeneous amplification profile for all micropropagated plants analyzed validating the genetic fidelity of the in vitro regenerated plants.     

Inhibitory property of the Piper betel phenolics against photosensitization-induced biological damages

The Piper betel phenolics, allylpyrocatechol (APC) and chavibetol (CHV), were found to protect photosensitization-mediated lipid peroxidation (LPO) of rat liver mitochondria effectively, APC being significantly more potent. The better activity of APC vis-à-vis CHV could be attributed to its higher reactivity with (1)O(2), as revealed from the rate constant values of (1)O(2) quenching by the respective phenolics. APC also prevented the detrimental effects of the Type II photosensitization-induced toxicity to mouse fibroblast L929 cells. The results suggest that APC may play an important role in protecting biological systems against damage, by eliminating (1)O(2) generated from certain endogenous photosensitizers.     

Cellular La protein shields nonsegmented negative-strand RNA viral leader RNA from RIG-I and enhances virus growth by diverse mechanisms

The La antigen (SS-B) associates with a wide variety of cellular and viral RNAs to affect gene expression in multiple systems. We show that La is the major cellular protein found to be associated with the abundant 44-nucleotide viral leader RNA (leRNA) early after infection with respiratory syncytial virus (RSV), a nonsegmented negative-strand RNA virus. Consistent with this, La redistributes from the nucleus to the cytoplasm in RSV-infected cells. Upon RNA interference knockdown of La, leRNA is redirected to associate with the RNA-binding protein RIG-I, a known activator of interferon (IFN) gene expression, and this is accompanied by the early induction of IFN mRNA. These results suggest that La shields leRNA from RIG-I, abrogating the early viral activation of type I IFN. We mapped the leRNA binding function to RNA recognition motif 1 of La and showed that while wild-type La greatly enhanced RSV growth, a La mutant defective in RSV leRNA binding also did not support RSV growth. Comparative studies of RSV and Sendai virus and the use of IFN-negative Vero cells indicated that La supports the growth of nonsegmented negative-strand RNA viruses by both IFN suppression and a potentially novel IFN-independent mechanism.