Differential epithelium DNA damage response to ATM and DNA-PK pathway inhibition in human prostate tissue culture

The ability of cells to respond and repair DNA damage is fundamental for the maintenance of genomic integrity. Ex vivo culturing of surgery-derived human tissues has provided a significant advancement to assess DNA damage response (DDR) in the context of normal cytoarchitecture in a non-proliferating tissue. Here, we assess the dependency of prostate epithelium DDR on ATM and DNA-PKcs, the major kinases responsible for damage detection and repair by nonhomologous end-joining (NHEJ), respectively. DNA damage was caused by ionizing radiation (IR) and cytotoxic drugs, cultured tissues were treated with ATM and DNA-PK inhibitors, and DDR was assessed by phosphorylation of ATM and its targets H2AX and KAP1, a heterochromatin binding protein. Phosphorylation of H2AX and KAP1 was fast, transient and fully dependent on ATM, but these responses were moderate in luminal cells. In contrast, DNA-PKcs was phosphorylated in both luminal and basal cells, suggesting that DNA-PK-dependent repair was also activated in the luminal cells despite the diminished H2AX and KAP1 responses. These results indicate that prostate epithelial cell types have constitutively dissimilar responses to DNA damage. We correlate the altered damage response to the differential chromatin state of the cells. These findings are relevant in understanding how the epithelium senses and responds to DNA damage.Key words: DNA damage, prostate, γH2AX, ATM, DNA-PK     

Biotransformation of withanolides by cell suspension cultures of Withania somnifera (Dunal)

The biotransformation potential of cell suspension cultures generated from Withania somnifera leaf was investigated, using withanolides, i.e. withanolide A, withaferin A, and withanone as precursor substrates. Interestingly, the cell suspension cultures showed inter-conversion of withanolides, as well converted to some unknown compounds, released to the culture media. The bio-catalyzed withanolide was detected and quantified by TLC and HPLC, respectively. There is noticeable conversion of withanolide A to withanone, and vice versa though at a lower level. The type of reaction of this biotransformation appears to be substitution of 20-OH group to 17-OH in withanolide A. In this paper, we present for the first time the possibility of biotransformation by inter-conversion of withanolides of pharmacological importance through cell suspension culture of W. somnifera. The possible role of putative cytochrome P₄₅₀ hydroxylases is implicated in the conversion.     

Antibacterial property of the antipsychotic agent prochlorperazine, and its synergism with methdilazine

The antipsychotic drug prochlorperazine was screened in vitro for possible antimicrobial property against 157 strains of bacteria, belonging to gram positive and gram negative genera. The minimum inhibitory concentration (MIC) of prochlorperazine was determined by agar dilution method, which ranged from 25 to 200 microg/ml with respect to most of the strains. Based on such findings, a further study was undertaken to determine whether the efficacy of this drug could be enhanced in the presence of an antihistaminic agent methdilazine, reported to have remarkable antimicrobial action. Four bacterial strains, sensitive to prochlorperazine as well as to three antibacterial chemotherapeutics, viz., methdilazine, fluphenazine and thioridazine were chosen. Disc diffusion tests with prochlorperazine and methdilazine revealed marked synergism between the combination, compared to their individual effects. The synergism was found to be statistically significant (p<0.01). To assess the degree of synergism, the checkerboard analysis was performed. The FIC index of this combination turned out to be 0.37, which confirmed synergism. Therefore, this synergistic drug combination might open a new therapeutic approach to combat drug-resistance in bacterial infections.     

An unusual presentation of a malignant jejunal tumor and a different management strategy

BACKGROUND: Malignant small bowel tumors are very rare and leiomyosarcoma accounts for less than 15% of the cases. Management of these tumors is challenging in view of nonspecific symptoms, unusual presentation and high incidence of metastasis. In this case report, an unusual presentation of jejunal sarcoma and management of liver metastasis with radiofrequency ablation (RFA) is discussed. CASE PRESENTATION: A 45-year-old male presented with anemia and features of small bowel obstruction. Operative findings revealed a mass lesion in jejunum with intussusception of proximal loop. Resection of bowel mass was performed. Histopathological findings were suggestive of leiomyosarcoma. After 3-years of follow-up, the patient developed recurrence in infracolic omentum and a liver metastasis. The omental mass was resected and liver lesion was managed with radiofrequency ablation. CONCLUSION: Jejunal leiomyosarcoma is a rare variety of malignant small bowel tumor and a clinical presentation with intussusception is unusual. We suggest that an aggressive management approach using a combination of surgery and a newer technique like RFA can be attempted in patients with limited metastatic spread to liver to prolong the long-term survival in a subset of patients.     

Crosslinked enzyme crystals of glucoamylase as a potent catalyst for biotransformations

Glucoamylase (E.C: 3.2.1.3, alpha-(1-->4)-glucan glucohydrolase) mainly hydrolyzes starch and has been extensively used in the starch, glucose (dextrose), and fermentation industries. Immobilized glucoamylase has an inherent disadvantage of lower conversion rates and low thermostability of less than 55 degrees C when used in continuous operations. We have developed crosslinked enzyme crystals (CLEC) of glucoamylase that overcome the above disadvantages, possess good thermal stability and retain 98.6% of their original activity at 70 degrees C for 1h, 77% activity at 80 degrees C for 1h, and 51.4% activity at 90 degrees C for 0.5h. CLEC glucoamylase has a specific activity of 0.0687 IU/mg and a yield of 50.7% of the original activity of the enzyme under optimum conditions with starch as the substrate. The crystals obtained are rhombohedral in shape having a size approximately 10-100 microm, a density of 1.8926 g/cm(3) and a surface area of 0.7867 m(2)/g. The pH optimum of the glucoamylase crystals was sharp at pH 4.5, unlike the soluble enzyme. The kinetic constants V(max) and K(m) exhibited a 10-fold increase as a consequence of crystallization and crosslinking. The continuous production of glucose from 10% soluble starch and 10% maltodextrin (12.5 DE) by a packed-bed reactor at 60 degrees C had a productivity of 110.58 g/L/h at a residence time of 7.6 min and 714.1g/L/h at a residence time of 3.4 min, respectively. The CLEC glucoamylase had a half-life of 10h with 4% starch substrate at 60 degrees C.     

Effect of simultaneous exposure to lead and cadmium on gonadotropin binding and steroidogenesis on granulosa cells: an in vitro study

Effects of lead (Pb) and cadmium (Cd) both alone or in combination on the binding of LH and FSH on isolated granulosa cells were studied. Granulosa cells isolated from proestrous rats were incubated (in vitro) with lead acetate and/or cadmium acetate (0.03 microM of Pb or Cd) for 1 hr. LH binding was dropped to 84% in Pb treated cells, 72.5% in Cd treated cells and 74.8% in combined metal treated cells compared to control. FSH binding dropped to 85.5% in Pb treated cells, 71.16% in Cd treated cells and 72.5% in combined metal treated cells compared to control. Activity of 17beta Hydroxy Steroid Dehydrogenase (17betaHSDH), a key steroidogenic enzyme was reduced by 52% in Cd and 37% in combined metal exposed cells whereas Pb exposed cells showed 31% reduction in the enzyme activity. Pretreatment with SH groups protectants (glutathione [GSH], dithiothretol [DTT]) and zinc caused an ameriolation in enzyme activity whereas Zn pretreatment showed an increase in gonadotropin binding in metal exposed cells. These results suggest that both Pb and Cd can cause a reduction in LH and FSH binding, which significantly alters steroid production in vitro and exerts a direct influence on granulosa cell function.     

Translesion synthesis past equine estrogen-derived 2'-deoxycytidine DNA adducts by human DNA polymerases eta and kappa

Estrogen replacement therapy (ERT), composed of equilenin, is associated with increased risk of breast, ovarian, and endometrial cancers. Several diastereoisomers of unique dC and dA DNA adducts were derived from 4-hydroxyequilenin (4-OHEN), a metabolite of equilenin, and have been detected in women receiving ERT. To explore the miscoding property of 4-OHEN-dC adduct, site-specifically modified oligodeoxynucleotides (Pk-1, Pk-2, Pk-3, and Pk-4) containing a single diastereoisomer of 4-OHEN-dC were prepared by a postsynthetic method. Among them, major 4-OHEN-dC-modified oligodeoxynucleotides (Pk-3 and Pk-4) were used to prepare the templates for primer extension reactions catalyzed by DNA polymerase (pol) alpha, pol eta, and pol kappa. Primer extension was retarded one base prior to the lesion and opposite the lesion; stronger blockage was observed with pol alpha, while with human pol eta or pol kappa, a fraction of the primers was extended past the lesion. Steady-state kinetic studies showed that both pol kappa and pol eta inserted dCMP and dAMP opposite the 4-OHEN-dC and extended past the lesion. Never or less-frequently, dGMP, the correct base, was inserted opposite the lesion. The relative bypass frequency past the 4-OHEN-dC lesion with pol eta was at least 3 orders of magnitude higher than that for pol kappa, as observed for primer extension reactions. The bypass frequency past the dA.4-OHEN-dC adduct in Pk-4 was 2 orders of magnitude more efficient than that past the adduct in Pk-3. Thus, 4-OHEN-dC is a highly miscoding lesion capable of generating C --> T transitions and C --> G transversions. The miscoding frequency and specificity of 4-OHEN-dC were strikingly influenced by the adduct stereochemistry and DNA polymerase used.     

Acceleration of neutrophil precursors' maturation and immunostimulation of CD3+, CD4+ lymphocytes by stanozolol in mice

The abuse of anabolic-androgenic steroids (AAS) for improved physical performance is associated with many deleterious effects. The present study aims to evaluate the short-term effect of an AAS compound stanozolol, on lipoprotein profile, granulopoiesis and immune response in adult female mice. The mice were assigned to five experimental groups and different doses of stanozolol (low - 0.05 mg, medium - 0.5 mg, high - 5 mg and highest dose - 7.5 mg/kg bwt or only vehicle respectively) were administered s.c. for 15 days. A decrease in high density lipoprotein cholesterol (HDL-c) as well as total cholesterol (TC) in all the stanozolol treated groups and an increase in low density lipoprotein (LDL-c) in high and the highest dose treated groups indicate that stanozolol alters serum lipoprotein profile. A significant increase in the percentage of myelocytes, metamyelocytes and neutrophils in all the treated mice unveils the stimulation of granulopoiesis through the acceleration of neutrophil precursors' maturation in the bone marrow of mice. The stimulation of erythropoiesis was also noted in all the treated groups. The flow cytometry analysis of lymphocyte subpopulations (CD3(+) and CD4(+)) revealed immunoenhancing response of stanozolol at optimum physiological dose, however, it is immunosuppressive at supraphysiologic level. We conclude that stanozolol accelerates granulopoiesis and stimulates immune response (at physiologic level only), though it alters the lipoprotein profile in mice.