Oral toxicity of Madhuca butyracea Macb. saponins to albino rats

Saponins, isolated from M. butyracea, were assessed for their acute and subacute oral toxicity in albino rats. Acute doses of saponins caused mortalities and LD50 and LD90 values were 330 and 430 mg/kg body wt respectively. Severe diarrhoea, restlessness and histopathological changes were observed in liver and kidney. Diets containing saponins at 0,250,500 and 1000 ppm for 14 weeks did not affect food intake, growth or organ weights, but induced mild histological changes in liver and kidney and altered the serum levels of alkaline phosphatase, blood urea nitrogen, cholesterol and proteins, particularly in female rats.     

4-Hydroxynonenal Induces G2/M Phase Cell Cycle Arrest by Activation of the Ataxia Telangiectasia Mutated and Rad3-related Protein (ATR)/Checkpoint Kinase 1 (Chk1) Signaling Pathway

4-Hydroxynonenal (HNE) has been widely implicated in the mechanisms of oxidant-induced toxicity, but the detrimental effects of HNE associated with DNA damage or cell cycle arrest have not been thoroughly studied. Here we demonstrate for the first time that HNE caused G₂/M cell cycle arrest of hepatocellular carcinoma HepG2 (p53 wild type) and Hep3B (p53 null) cells that was accompanied with decreased expression of CDK1 and cyclin B1 and activation of p21 in a p53-independent manner. HNE treatment suppressed the Cdc25C level, which led to inactivation of CDK1. HNE-induced phosphorylation of Cdc25C at Ser-216 resulted in its translocation from nucleus to cytoplasm, thereby facilitating its degradation via the ubiquitin-mediated proteasomal pathway. This phosphorylation of Cdc25C was regulated by activation of the ataxia telangiectasia and Rad3-related protein (ATR)/checkpoint kinase 1 (Chk1) pathway. The role of HNE in the DNA double strand break was strongly suggested by a remarkable increase in comet tail formation and H2A.X phosphorylation in HNE-treated cells in vitro. This was supported by increased in vivo phosphorylation of H2A.X in mGsta4 null mice that have impaired HNE metabolism and increased HNE levels in tissues. HNE-mediated ATR/Chk1 signaling was inhibited by ATR kinase inhibitor (caffeine). Additionally, most of the signaling effects of HNE on cell cycle arrest were attenuated in hGSTA4 transfected cells, thereby indicating the involvement of HNE in these events. A novel role of GSTA4-4 in the maintenance of genomic integrity is also suggested.     

Regulation of nucleo-cytoplasmic shuttling of human annexin A2—a proposed mechanism

Studies have long been focused on the functions of annexin A2 in the cytoplasm. However, the involvement of annexin A2 in DNA replication as a part of primer recognition protein complex and the presence of nuclear export signal (NES) suggest that annexin A2 is also functional in the nucleus, and its localization in the nucleus is under regulation by interaction with other nuclear factors through its N-terminus. During the study of the mechanism of annexin A2 sequestering in the nucleus and the regulation of its export from the nucleus, in this study, we show that endogenous annexin A2 is present in both the cytoplasm and the nucleus in HeLa, PC-3 and DU-145 cells. While exogenously expressed annexin A2 is excluded from nuclei of annexin A2-null LNCaP cells in a CRM1 (Chromosome Maintenance Region 1) mediated nuclear export, endogenous annexin A2 in HeLa, PC-3 and DU-145 cell lines does not undergo the CRM1 mediated nuclear export. While investigating the mechanism of the nuclear retention of annexin A2, we found that an anti-annexin A2 antibody that recognizes the C-terminus of annexin A2 (D1/274.5) cannot recognize nuclear annexin A2, suggesting that the domain recognized by this antibody may be masked in the nuclei. In order to find out the role of annexin A2 C-terminus in the nuclear retention of annexin A2, we transiently transfected green fluorescence protein (GFP)-fused N-terminal 29 amino acids of annexin A2 to LNCaP, PC-3 and DU-145 cells, and determined that the C-terminus is not required for the nuclear retention of annexin A2. Based on the finding described above, we propose a model for nuclear retention of annexin A2 where the regulation sites reside in the N-terminus and are adjacent to the NES, and upon modification, the NES is exposed and annexin A2 is exported from the nucleus. Electronic Supplementary Material The online version of this article (doi) contains supplementary material, which is available to authorized users.     

Scale up, optimization and stability analysis of Curcumin C3 complex-loaded nanoparticles for cancer therapy

ABSTRACT: BACKGROUND: Nanoparticle based delivery of anticancer drugs have been widely investigated. However, a very important process for Research & Development in any pharmaceutical industry is scaling nanoparticle formulation techniques so as to produce large batches for preclinical and clinical trials. This process is not only critical but also difficult as it involves various formulation parameters to be modulated all in the same process. METHODS: In our present study, we formulated curcumin loaded poly (lactic acid-co-glycolic acid) nanoparticles (PLGA-CURC). This improved the bioavailability of curcumin, a potent natural anticancer drug, making it suitable for cancer therapy. Post formulation, we optimized our process by Reponse Surface Methodology (RSM) using Central Composite Design (CCD) and scaled up the formulation process in four stages with final scale-up process yielding 5 g of curcumin loaded nanoparticles within the laboratory setup. The nanoparticles formed after scale-up process were characterized for particle size, drug loading and encapsulation efficiency, surface morphology, in vitro release kinetics and pharmacokinetics. Stability analysis and gamma sterilization were also carried out. RESULTS: Results revealed that that process scale-up is being mastered for elaboration to 5 g level. The mean nanoparticle size of the scaled up batch was found to be 158.5 [PLUS-MINUS SIGN] 9.8 nm and the drug loading was determined to be 10.32 [PLUS-MINUS SIGN] 1.4 %. The in vitro release study illustrated a slow sustained release corresponding to 75 % drug over a period of 10 days. The pharmacokinetic profile of PLGA-CURC in rats following i.v. administration showed two compartmental model with the area under the curve (AUC0-[INFINITY]) being 6.139 mg/L h. Gamma sterilization showed no significant change in the particle size or drug loading of the nanoparticles. Stability analysis revealed long term physiochemical stability of the PLGA-CURC formulation. CONCLUSIONS: A successful effort towards formulating, optimizing and scaling up PLGA-CURC by using Solid-Oil/Water emulsion technique was demonstrated. The process used CCD-RSM for optimization and further scaled up to produce 5 g of PLGA-CURC with almost similar physicochemical characteristics as that of the primary formulated batch.     

Tissue angiotensin I-converting enzyme activity in spontaneously hypertensive hamsters.

The purpose of this study was to measure angiotensin I-converting activity in heart, kidney, lung and cheek pouch tissue homogenates of spontaneously hypertensive and normotensive hamsters. We also determined inhibitor sensitivity and the effects of chloride anion concentration on kidney angiotensin I-converting activity in these animals. We found no significant differences in angiotensin I-converting activity between hypertensive and normotensive hamsters in all tissues tested. Inhibitor sensitivity of kidney angiotensin I-converting activity with captopril and lisonopril was similar in both groups. Finally, kidney angiotensin I-converting activity increased significantly in both groups as chloride anion concentration in the assay buffer increased. Substituting chloride anion for citrate abrogated the increase in angiotensin I-converting enzyme activity.     

Impact of Race/Ethnicity on the Relationship Between Visceral Fat and Inflammatory Biomarkers

The purpose of this study was to determine whether racial/ethnic differences exist in the relationship between visceral adipose tissue (VAT) and selected inflammatory biomarkers. Subjects included 136 African‐American, 133 Hispanic, and 100 white men and women, aged ≥45. Waist circumference and BMI were measured using standard methods. Total VAT, and VAT and subcutaneous adipose tissue (SAT) at the L4L5 spinal level were measured using computed tomography. Interleukin‐6 (IL‐6), C‐reactive protein (CRP), and fibrinogen were measured from fasting blood samples. Results revealed that waist circumference and BMI were similar among groups but African Americans had significantly lower L4L5 VAT compared with Hispanics and whites. Despite lower VAT, African‐American men had similar concentrations of inflammatory biomarkers. On the other hand, African‐American women had higher CRP and IL‐6 than white women, and higher fibrinogen than both Hispanic and white women. After controlling for L4L5 VAT, L4L5 SAT, and age, African‐American women had higher concentrations of IL‐6 and fibrinogen. Stratified analyses for CRP indicated that L4L5 SAT was associated with CRP in African‐American and white women after controlling for L4L5 VAT and age, but that the reverse was not true. These data indicate that African Americans had lower VAT but similar or higher concentrations of inflammatory biomarkers. African‐American women consistently displayed greater inflammation compared with whites, even after controlling for VAT or SAT.     

Selection of template initiation sites and the lengths of RNA primers synthesized by DNA primase are strongly affected by its organization in a multiprotein DNA polymerase alpha complex.

Synthesis of (p)ppRNA-DNA chains by purified HeLa cell DNA primase-DNA polymerase alpha (pol alpha-primase) was compared with those synthesized by a multiprotein form of DNA polymerase alpha (pol alpha 2) using unique single-stranded DNA templates containing the origin of replication for simian virus 40 (SV40) DNA. The nucleotide locations of 33 initiation sites were identified by mapping G*pppN-RNA-DNA chains and identifying their 5'-terminal ribonucleotide. Pol alpha 2 strongly preferred initiation sites that began with ATP rather than GTP, thus frequently preferring different initiation sites than pol alpha-primase, depending on the template examined. The initiation sites selected in vitro, however, did not correspond to the sites used during SV40 DNA replication in vivo. Pol alpha 2 had the greatest effect on RNA primer size, typically synthesizing primers 1-5 nucleotides long, while pol alpha-primase synthesized primers 6-8 nucleotides long. These differences were observed even at individual initiation sites. Thus, the multiprotein form of DNA primase-DNA polymerase alpha affects selection of initiation sites, the frequency at which the sites are chosen, and length of RNA primers.     

Resolution and purification of free primase activity from the DNA primase-polymerase alpha complex of HeLa cells.

DNA primase activity has been resolved from a purified DNA primase-polymerase alpha complex of HeLa cells by hydrophobic affinity chromatography on phenylSepharose followed by chromatography on hexylagarose. This procedure provides a good yield (55%) of DNA primase that is free from polymerase alpha. The free DNA primase activity was purified to near homogeneity and its properties characterized. Sodium dodecyl sulfate polyacrylamide gel electrophoretic analysis of the purified free DNA primase showed a major protein staining band of Mr 70,000. The native enzyme in velocity sedimentation has an S20'W of 5. DNA primase synthesizes RNA oligomers with single-stranded M-13 DNA, poly(dT) and poly(dC) templates that are elongated by the DNA polymerase alpha in a manner that has already been described for several purified eukaryotic DNA primase-polymerase alpha complexes. The purified free DNA primase activity is resistant to neutralizing anti-human DNA polymerase alpha antibodies, BuPdGTP and aphidicolin that specifically inhibit the free DNA polymerase alpha and also DNA polymerase alpha complexed with the primase. The free primase activity is more sensitive to monovalent salt concentrations and is more labile than polymerase alpha. Taken together these results indicate that the DNA primase and polymerase alpha activities of the DNA primase-polymerase alpha complex reside on separate polypeptides that associate tightly through hydrophobic interactions.     

The protein-tyrosine kinase substrate, calpactin I heavy chain (p36), is part of the primer recognition protein complex that interacts with DNA polymerase alpha

Primer recognition proteins (PRP) stimulate the activity of DNA polymerase alpha on DNA substrates with long single-stranded template containing few primers. Purified PRP from HeLa cells and human placenta are composed of two subunits of 36,000 (PRP 1) and 41,000 (PRP 2) daltons. By amino acid sequence homology, we have identified PRP 2 as the glycolytic enzyme 3-phosphoglycerate kinase. Here we present data that establishes PRP 1 to be the protein-tyrosine kinase substrate, calpactin I heavy chain. Amino acid sequence analysis of six tryptic peptides of PRP 1 followed by homology search in a protein sequence data base revealed 100% identity of all six peptides with the deduced amino acid sequence of human calpactin I heavy chain. The activities of PRP and calpactin I coelute on gel filtration columns, and a high correlation of PRP and calpactin I activities was seen at different stages of purification. A rabbit polyclonal anti-chicken calpactin I antibody was shown to cross-react with PRP 1 polypeptide at various stages of PRP purification, and the homogeneous preparation of PRP exhibits 3-phosphoglycerate kinase (PRP 2) and calpactin I (PRP 1) activities. PRP activity is neutralized by a mouse monoclonal anti-calpactin II antibody although having no effect on the polymerase alpha activity itself. Calpactin II has a 50% amino acid sequence homology with calpactin I. However, PRP 1 is not calpactin II as shown by lack of cross-reaction to a monoclonal anti-calpactin II antibody on Western blots. Calpactin I and 3-phosphoglycerate kinase, purified independently, cannot be efficiently reconstituted into the PRP complex, indicating that their association in the PRP complex involves specific protein-protein interactions that remain to be elucidated. The biochemical and immunological data presented here revealing the identity of PRP 1 as calpactin I provide evidence for one physiological role of calpactin I in the cell.     

Mammalian alpha-polymerase: cloning of partial complementary DNA and immunobinding of catalytic subunit in crude homogenate protein blots

A new polyclonal antibody against the alpha-polymerase catalytic polypeptide was prepared by using homogeneous HeLa cell alpha-polymerase. The antibody neutralized alpha-polymerase activity and was strong and specific for the alpha-polymerase catalytic polypeptide (Mr 183,000) in Western blot analysis of crude extracts of HeLa cells. The antibody was used to screen a cDNA library of newborn rat brain poly(A+) RNA in lambda gt11. A positive phage was identified and plaque purified. This phage, designated lambda pol alpha 1.2, also was found to be positive with an antibody against Drosophila alpha-polymerase. The insert in lambda pol alpha 1.2 (1183 base pairs) contained a poly(A) sequence at the 3' terminus and a short in-phase open reading frame at the 5' terminus. A synthetic oligopeptide (eight amino acids) corresponding to the open reading frame was used to raise antiserum in rabbits. Antibody affinity purified from this serum was found to be immunoreactive against purified alpha-polymerase by enzyme-linked immunosorbent assay and was capable of immunoprecipitating alpha-polymerase. This indicated the lambda pol alpha 1.2 insert encoded an alpha-polymerase epitope and suggested that the cDNA corresponded to an alpha-polymerase mRNA. This was confirmed in hybrid selection experiments using pUC9 containing the cDNA insert and poly(A+) RNA from newborn rat brain; the insert hybridized to mRNA capable of encoding alpha-polymerase catalytic polypeptides. Northern blot analysis of rat brain poly(A+) RNA revealed that this mRNA is approximately 5.4 kilobases.