Differentiation of mitotic melanoma cells from G2 cells and their isolation by use of 5-bromo-2'-deoxyuridine and propidium iodide

This report describes a method by which mitotic cells were isolated from nonsynchronized Cloudman melanoma cells that had been pulse labeled with 5-bromo-2'-deoxyuridine (BrdUrd) and double-stained with a fluoresceinated monoclonal antibody to BrdUrd and with propidium iodide (PI). In initial experiments, melanoma cells were first pulse labeled with BrdUrd, treated with prostaglandin E1 (PGE1 10 micrograms/m1) or vehicle (0.1% ethanol) for up to 24 hours, then stained with anti-BrdUrd and PI. PGE1-treated cells monitored at 3-hour intervals were observed to migrate from S phase to G2 phase, then, enigmatically, back into the late S phase region of the distribution. In other experiments, cells treated with PGE1 were pulse labeled with BrdUrd at the end of the treatment period and harvested. In these experiments, there was a small, discrete subpopulation of cells within the late S phase region of the DNA distribution that was negative for anti-BrdUrd. This subpopulation of cells was sorted and examined by light microscopy. We observed that 95% of these BrdUrd-negative "S phase" cells were mitotic cells. Since mitotic cells and G2 cells have equivalent amounts of DNA, the reduced red fluorescence exhibited by these cells may be due to a greater sensitivity to denaturation, which has been described for DNA of mitotic cells, and would account for the phenomenon of cells appearing to move "backwards" in the cell cycle. This report indicates that although the BrdUrd/PI method can further define the cell cycle into four compartments, it can also lead to over-estimation of S phase cells in kinetic studies because of contaminating mitotic cells.     

Gaseous ozone: A potent pest management strategy to control Callosobruchus maculatus (Coleoptera: Bruchidae) infesting green gram

Extending the storage life of legumes by protecting it from the Callosobruchus maculatus infestation is a major concern for the producers, processors and exporters. Legume processing industry requires “greener” alternatives to the conventional fumigants. Gaseous ozone has a great potential as an insect management strategy that is suited for this niche. Nevertheless, the efficacy of ozone against C. maculatus is yet unknown. A laboratory study was conducted to test the insecticidal effect of ozone in controlling the infestation of C. maculatus in green gram. We have determined the concentration of ozone exposure time–mortality relationship for all the stages of C. maculatus that were exposed to 500–1,500 ppmv ozone. The percentages of mortality for different stages of C. maculatus increased with the increase in ozone concentration and exposure time. It was documented that adult stage is least tolerant to ozone (500 ppmv for 274.40 min exposure required to kill 90%), whereas the most tolerant stage is pupa (500 ppmv for 1816.54 min is required to kill 90%). The results indicate that gaseous ozone is the attractive alternative to the synthetic fumigants.     

birgHPC: creating instant computing clusters for bioinformatics and molecular dynamics

birgHPC, a bootable Linux Live CD has been developed to create high-performance clusters for bioinformatics and molecular dynamics studies using any Local Area Network (LAN)-networked computers. birgHPC features automated hardware and slots detection as well as provides a simple job submission interface. The latest versions of GROMACS, NAMD, mpiBLAST and ClustalW-MPI can be run in parallel by simply booting the birgHPC CD or flash drive from the head node, which immediately positions the rest of the PCs on the network as computing nodes. Thus, a temporary, affordable, scalable and high-performance computing environment can be built by non-computing-based researchers using low-cost commodity hardware. AVAILABILITY: The birgHPC Live CD and relevant user guide are available for free at http://birg1.fbb.utm.my/birghpc.     

Proinflammatory cytokines (TNF-alpha and IL-6) in Egyptian patients with SLE: its correlation with disease activity

BACKGROUND: Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by a wide variety of autoantibodies, some of which are pathogenic. In recent years it has become more evident that the polyclonal B cell activation in SLE is T-cell dependent. The stimulation of the autoantibody producing B cells is likely mediated by the TH2 subtype of T cells producing IL-4, IL-5, IL-6 and IL- 10, whereas the TH1 subtype secreting IL-2 and IFN-gamma predominates in cell-mediated immune response. Tumor Necrosis Factor (TNF-alpha) is both a proinflammatory and an immunoregulatory cytokine. TNF-alpha has differential effects on B cells, on T cells and on dendritic cells as well as on the process of programmed cell death. Understanding how the immune system integrates the pleiotropic properties of TNF-alpha is a challenge, particularly so in diseases like SLE. Meanwhile the role of IL-6 in the pathogenesis of SLE is controversial. Objective: To investigate whether serum levels of TNF-alpha and IL-6 is higher in Egyptian patients with SLE than healthy control volunteers and its correlation with the clinical activity in patients with different activity scores as measured by Systemic Lupus Erythmatosus Disease Activity Index (SLEADI). Methods: Sixty individuals (40 patients with Systemic lupus Erythmatosus and 20 healthy control volunteers) were the subject of this study, they were subjected to thorough clinical examination, laboratory investigations, their clinical disease activity was scored according to SLEDAI, and serum sampling was obtained for TNF-alpha and IL-6 levels assay. Renal biopsy was carried out and examined by light microscopy by a pathologist blinded with the clinical activity. Results: The mean level of TNF-alpha was (766.95+/-357.82Pg/ml) for patients with active disease while it was (314.01+/-100.87Pg/ml) for those with inactive disease and (172.7+/-39.19Pg/ml) for the healthy control group. The difference was statistically significant (P=.002). The mean level of IL-6 was (135.4+/-54.23Pg/ml) for patients with active disease while it was (47.33+/-18.61Pg/ml) for those with inactive disease and (21.15+/-10.99Pg/ml) for the healthy control group. The difference was statistically significant (P=.002). A significant correlations between TNF-alpha and IL-6 serum levels and the SLEDAI score was observed (r=.743 and .772, respectively). Conclusion: Serum TNF-alpha and IL-6 are sensitive markers of SLE disease activity. They may be useful independent markers for prediction of SLE disease activity and to differentiate normal subjects from those having SLE. Possible therapeutic implications in the treatment of SLE in the future deserve wide scale trials.     

Diversity of pigmentation in cultured human melanocytes is due to differences in the type as well as quantity of melanin

Cultured human melanocytes differ tremendously in visual pigmentation, and recapitulate the pigmentary phenotype of the donor's skin. This diversity arises from variation in type as well as quantity of melanin produced. Here, we measured contents of eumelanin (EM) and pheomelanin (PM) in 60 primary human melanocyte cultures (51 neonatal and nine adults), and correlated some of these values with the respective activity and protein levels of tyrosinase, and the melanocortin-1 receptor (MC1R) genotype. Melanocytes were classified into four phenotypes (L, L+, D, D+) as depicted by visual pigmentation using light microscopy, and by the pigmentary phenotype of the donor's skin. There were large differences in total melanin (TM) and EM, which increased progressively for L, L+, D and D+ melanocytes. TM content, the sum of EM and PM, showed a good correlation with TM measured spectrophotometrically, and with the activity and protein levels of tyrosinase. Log EM/PM ratio did not correlate with MC1R genotype. We conclude that: (i) EM consistently correlates with the visual phenotype; (ii) lighter melanocytes tend to be more pheomelanic in composition than darker melanocytes; (iii) in adult melanocyte cultures, EM correlates with the ethnic background of the donors (African-American > Indian > Caucasian); and (iv) MC1R loss-of-function mutations do not necessarily alter the phenotype of cultured melanocytes.     

Melanin content and MC1R function independently affect UVR-induced DNA damage in cultured human melanocytes

Malignant transformation of melanocytes leads to melanoma, the most fatal form of skin cancer. Ultraviolet radiation (UVR)-induced DNA photoproducts play an important role in melanomagenesis. Cutaneous melanin content represents a major photoprotective mechanism against UVR-induced DNA damage, and generally correlates inversely with the risk of skin cancer, including melanoma. Melanoma risk is also determined by susceptibility genes, one of which is the melanocortin 1 receptor (MC1R) gene. Certain MC1R alleles are strongly associated with melanoma. We hereby present experimental evidence for the role of two melanoma risk factors, constitutive pigmentation, as assessed by total melanin, eumelanin and pheomelanin contents, and MC1R genotype and function, in determining the induction and repair of DNA photoproducts in cultured human melanocytes after irradiation with increasing doses of UVR. We found that total melanin and eumelanin contents (MC and EC) correlated inversely with the extent of UVR-induced growth arrest, apoptosis and induction of cyclobutane pyrimidine dimers (CPD), but not with hydrogen peroxide release in melanocytes expressing functional MC1R. In comparison, melanocytes with loss-of-function MC1R, regardless of their MC or EC, sustained more UVR-induced apoptosis and CPD, and exhibited reduced CPD repair. Therefore, MC, mainly EC, and MC1R function are independent determinants of UVR-induced DNA damage in melanocytes.     

An S188V Mutation Alters Substrate Specificity of Non-Stereospecific α-Haloalkanoic Acid Dehalogenase E (DehE)

The non-stereospecific α-haloalkanoic acid dehalogenase E (DehE) degrades many halogenated compounds but is ineffective against β-halogenated compounds such as 3-chloropropionic acid (3CP). Using molecular dynamics (MD) simulations and site-directed mutagenesis we show here that introducing the mutation S188V into DehE improves substrate specificity towards 3CP. MD simulations showed that residues W34, F37, and S188 of DehE were crucial for substrate binding. DehE showed strong binding ability for D-2-chloropropionic acid (D-2CP) and L-2-chloropropionic acid (L-2CP) but less affinity for 3CP. This reduced affinity was attributed to weak hydrogen bonding between 3CP and residue S188, as the carboxylate of 3CP forms rapidly interconverting hydrogen bonds with the backbone amide and side chain hydroxyl group of S188. By replacing S188 with a valine residue, we reduced the inter-molecular distance and stabilised bonding of the carboxylate of 3CP to hydrogens of the substrate-binding residues. Therefore, the S188V can act on 3CP, although its affinity is less strong than for D-2CP and L-2CP as assessed by K_m. This successful alteration of DehE substrate specificity may promote the application of protein engineering strategies to other dehalogenases, thereby generating valuable tools for future bioremediation technologies.     

Semi-rigid tripeptide agonists of melanocortin receptors

A series of 30 RCO–HfR–NH₂ derivatives show preference for the mouse MC1R vs MC3-5Rs. trans-4-HOC₆H₄CHCHCO–HfR–NH₂ (13) [EC₅₀ (nM): MC1R 83, MC3R 20500, MC4R 18130 and MC5R 935; ratio 1:246:217:11] is 11 times more potent than the lead compound LK-394 Ph(CH₂)₃CO–HfR–NH₂ (2) and only 11 times less potent than the native tridecapeptide α-MSH at mMC1R. Differences in conformations of 2 and 13 are discussed.