Flow cytometric analysis of DNA indices, expression of p53 and multidrug resistance genes in multiple myeloma patients

OBJECTIVE: To perform a quantitative analysis of DNA ploidy, S-phase fraction % (SPF%), p53 and multidrug resistance (MDR) gene expression as independent prognostic parameters and to compare these parameters with stage of the disease in multiple myeloma (MM) patients. STUDY DESIGN: Peripheral blood bone marrow samples were analyzed for DNA ploidy and SPF% using a FACScan flow cytometer (Becton Dickinson). Detection of p53 and MDR gene expression was done using immunocytochemistry. RESULTS: Aneuploidy was found in 5/48 (10.42%) total myeloma patients, all of whom revealed hyperdiploidy. High SPF% was noted in 18/37 (48.65%) newly diagnosed MM patients and 5/11 (45.45%) follow-up cases of myeloma. p53 Gene product was noted in 8/48 (16.66%) myeloma patients, 6 newly diagnosed and 2 on follow-up. MDR gene expression was detected in 4/27 (10.81%) newly diagnosed patients and in 1/11 (9.09%)follow-up patients. CONCLUSION: All myeloma patients with aneuploidy revealed hyperdiploidy. The majority of cases with high SPF% were at advanced stages, indicating the prognostic significance of SPF%. Although, there was no statistical significance of DNA ploidy, SPF%, p53 and MDR gene product expression, they are important prognostic parameters. Our results can provide baseline data for comparison with future studies since these parameters have not been reported earlier from the Indian subcontinent.     

Behavioural and psychological symptoms of dementia in primary care: a survey of general practitioners in Ireland

Background Management of neuropsychiatric symptoms is a challenging task in primary care. Aims To assess self-reported confidence and knowledge of general practitioners (GPs) regarding the identification and management of behavioural and psychological symptoms of dementia (BPSD).Methods A self-designed two-page paper questionnaire was sent to a random sample of 160 GPs practising in north Dublin. They were asked to evaluate their confidence and knowledge on several aspects of diagnosis and management of BPSD.Results Completed questionnaires were returned from 109 GPs (response rate = 68%), of which 106 were usable. In general, GPs were somewhat critical of their self-reported skills in diagnosing (76.4%) and managing (77.4%) BPSD, as well as in discriminating BPSD from other behavioural disturbances (71.7%). Many of them (67.9%) also encountered difficulty accessing specialist services. There was no correlation between demographic characteristics of GPs or patient caseload with respect to their responses to questionnaire items. Although many GPs (92.5%) highly valued the important role of non-pharmacological interventions in BPSD, none of them reported recommending these in their daily practice.Conclusions Despite the fact that GPs have a wealth of knowledge about BPSD, they are largely critical of their knowledge and management skills of these symptoms. Efforts should be focused on supporting GPs by means of educational interventions that consider all aspects of dementia, but additionally highlight the more challenging neuropsychiatric components of the illness. Health services need to be structured in a way that promotes collaboration between GPs and mental health professionals for a seamless delivery of care.     

Desferrithiocin analogue iron chelators: iron clearing efficiency,tissue distribution,and renal toxicity

The current solution to iron-mediated damage in transfusional iron overload disorders is decorporation of excess unmanaged metal, chelation therapy. The clinical development of the tridentate chelator deferitrin (1, Table 1) was halted due to nephrotoxicity. It was then shown by replacing the 4′-(HO) of 1 with a 3,6,9-trioxadecyloxy group, the nephrotoxicity could be ameliorated. Further structure–activity relationship studies have established that the length and the position of the polyether backbone controlled: (1) the ligand’s iron clearing efficiency (ICE), (2) chelator tissue distribution, (3) biliary ferrokinetics, and (4) tissue iron reduction. The current investigation compares the ICE and tissue distribution of a series of (S)-4,5-dihydro-2-[2-hydroxy-4-(polyether)phenyl]-4-methyl-4-thiazolecarboxylic acids (Table 1, 3–5) and the (S)-4,5-dihydro-2-[2-hydroxy-3-(polyether)phenyl]-4-methyl-4-thiazolecarboxylic acids (Table 1, 8–10). The three most effective polyether analogues, in terms of performance ratio (PR), defined as mean ICE_primate/ICE_rodent, are 3 (PR 1.1), 8, (PR 1.5), and 9, now in human trials, (PR 2.2). At the onset of the clinical trial on 9, no data were available for ligand 3 or 8. This is unfortunate, as 3 has many advantages over 9, e.g., the ICE of 3 in rats is 2.5-fold greater than that of 9 and analogue 3 achieves very high levels in the liver, pancreas, and heart, the organs most affected by iron overload. Finally, the impact of 3 on the urinary excretion of kidney injury molecule-1 (Kim-1), an early diagnostic biomarker for monitoring acute kidney toxicity, has been carried out in rats; no evidence of nephrotoxicity was found. Overall, the results suggest that 3 would be a far superior clinical candidate to 9.     

Sequence-related amplified polymorphism (SRAP) molecular marker system and its applications in crop improvement

Plant biotechnology has great potential for improving target traits in crops. This can be achieved by the production of transgenic crops and marker-assisted breeding. Marker-assisted breeding has gained momentum in recent times since it does not need biosafety regulations. Several kind of molecular markers are available for use in crop breeding, such as restriction fragment length polymorphism, microsatellites, sequence characterized amplified region, sequence-tagged site, inter-simple sequence repeat amplification, amplified fragment length polymorphism and single nucleotide polymorphism. Sequence-related amplified polymorphism is a novel molecular marker system which is based on open reading frames (ORFs) developed from genome sequence data of Arabidopsis. It provides a unique combination of forward and reverse primers which can be selected arbitrarily giving a large number of primer combinations. Since this is an ORF-based marker system, it targets functional genes and has potential for their application in crop breeding. This marker system was first used and demonstrated by Li and Quiros in Brassica oleracea in 2001, and since then there have been several reports in different plant species ranging from field crops to tree species for assessing genetic diversity, mapping and tagging of genes, hybrid identification and sex determination. This review provides an overview of SRAP markers and their applications in crop improvement.     

Analysis of P-Loop and its Flanking Region Subsequence of Diverse NTPases Reveals Evolutionary Selected Residues

The P-loop NTPases are involved in diverse cellular functions. Members of the P-loop NTPase superfamily are characterized by presence of a highly conserved sequence pattern GxxxxGKS/T, known as Walker A motif. This motif adopts an archetypal P-loop conformation which allows accommodation of the triphosphate moiety of a bound nucleotide. Despite the presence of Walker A as a common sequence motif, P-loop NTPases exhibit extreme sequence divergence which hampers their phylogenetic or evolutionary classification. Here, we show that P-loop and its flanking region subsequence (termed as “extended-WalkerA motif”) contain distinct signatures that can be utilized to classify NTPase domain of functionally diverse proteins. We find a clearly classified group of diverse NTPases of Conserved Domain Database such as G-proteins, Ylqf, RecA like, DExDc, AAA, CPT, NK, ABC transporter and NifH proteins.     

Metformin Induces Apoptosis and Cell Cycle Arrest Mediated by Oxidative Stress,AMPK and FOXO3a in MCF-7 Breast Cancer Cells

Recent studies have demonstrated that the anti-diabetic drug, metformin, can exhibit direct antitumoral effects, or can indirectly decrease tumor proliferation by improving insulin sensitivity. Despite these recent advances, the underlying molecular mechanisms involved in decreasing tumor formation are not well understood. In this study, we examined the antiproliferative role and mechanism of action of metformin in MCF-7 cancer cells treated with 10 mM of metformin for 24, 48, and 72 hours. Using BrdU and the MTT assay, it was found that metformin demonstrated an antiproliferative effect in MCF-7 cells that occurred in a time- and concentration- dependent manner. Flow cytometry was used to analyze markers of cell cycle, apoptosis, necrosis and oxidative stress. Exposure to metformin induced cell cycle arrest in G₀-G₁ phase and increased cell apoptosis and necrosis, which were associated with increased oxidative stress. Gene and protein expression were determined in MCF-7 cells by real time RT-PCR and western blotting, respectively. In MCF-7 cells metformin decreased the activation of IRβ, Akt and ERK1/2, increased p-AMPK, FOXO3a, p27, Bax and cleaved caspase-3, and decreased phosphorylation of p70S6K and Bcl-2 protein expression. Co-treatment with metformin and H₂O₂ increased oxidative stress which was associated with reduced cell number. In the presence of metformin, treating with SOD and catalase improved cell viability. Treatment with metformin resulted in an increase in p-p38 MAPK, catalase, MnSOD and Cu/Zn SOD protein expression. These results show that metformin has an antiproliferative effect associated with cell cycle arrest and apoptosis, which is mediated by oxidative stress, as well as AMPK and FOXO3a activation. Our study further reinforces the potential benefit of metformin in cancer treatment and provides novel mechanistic insight into its antiproliferative role.     

Intragraft gene expression profile associated with the induction of tolerance by allochimeric MHC I in the rat heart transplantation model

The MHC class I allochimeric protein containing donor‐type epitopes on recipient‐type heavy chains induces indefinite survival of heterotopic cardiac allografts in rats. We analyzed gene expression profile of heart allograft tissue. Mutated peptide [α1h1/u]‐RT1.Aa that contains donor‐type (Wistar Furth, WF; RT1u) immunogenic epitopes displayed on recipient‐type (ACI, RT1a) was delivered into ACI recipients of WF hearts at the time of transplantation in addition to a 3 days course of oral cyclosporine. Microarray analysis was performed using Affymetrix Rat 230 2.0 Microarray. Allochimeric molecule treatment caused upregulation of genes involved in structural integrity of heart muscle, downregulation of IL‐1β a key modulator of the immune response, and downregulation of partitioning defective six homolog gamma PAR6, which is involved in T cell polarity, motility, and ability to scan dendritic cells (DC). These indicate that the immunosuppressive function of allochimeric molecule and/or the establishment of allograft tolerance depend on the induction of genes responsible for the heart tissue integrity, the suppression of cytokine pathway(s), and possibly the impairment of T cells mobility and their DC scanning ability. These novel findings may have important clinical implications for inhibition of chronic rejection in transplant recipients. genesis 48:8–19, 2010. © 2009 Wiley‐Liss, Inc.     

Influence of explant types,non-embryogenic synseed and reduced oxygen environment on in vitro conservation of Bacopa monnieri (L.) Wettst

In Vitro Cellular & Developmental Biology - Plant - The effect of using encapsulated shoot tips (ST) and nodal segment (NS) explants, in combination with mineral oil (MO) overlay, for in vitro...     

L-Asparaginase: A Promising Chemotherapeutic Agent

ABSTRACT

This article comprises detailed information about L-asparaginase, encompassing topics such as microbial and plant sources of L-asparaginase, treatment with L-asparaginase, mechanism of action of L-asparaginase, production, purification, properties, expression and characteristics of l-asparaginase along with information about studies on the structure of L-asparaginase. Although L-asparaginase has been reviewed by , our effort has been to include recent and updated information about the enzyme covering new aspects such as structural modification and immobilization of L-asparaginase, recombinant L-asparaginase, resistance to L-asparaginase, methods of assay of L-asparagine and L-asparaginase activity using the biosensor approach, L-asparaginase activity in soil and the factors affecting it. Also, side-effects of L-asparaginase treatment in acute lymphoblastic leukemia (ALL) have been discussed in the current review. L-asparaginase has been and is still one of the most widely studied therapeutic enzymes by researchers and scientists worldwide.