Co-expressed recombinant human Translin-Trax complex binds DNA

Trax, expressed alone aggregates into insoluble complexes, whereas upon co-expression with Translin becomes readily soluble and forms a stable heteromeric complex ( approximately 430 kDa) containing both proteins at nearly equimolar ratio. Based on the subunit molecular weights, estimated by MALDI-TOF-MS, the purified complex appears to comprise of either an octameric Translin plus a hexameric Trax (calculated MW 420 kDa) or a heptamer each of Trax and Translin (calculated MW 425 kDa) or a hexameric Translin plus an octameric Trax (calculated MW 431 kDa). The complex binds single-stranded/double-stranded DNA. ssDNA gel-shifted complex shows both proteins at nearly equimolar ratio, suggesting that Translin "chaperones" Trax and forms heteromeric complex that is DNA binding competent.     

Executive function is less sensitive to estradiol than spatial memory: performance on an analog of the card sorting test in ovariectomized aged rhesus monkeys

Functions supported by the frontal lobes are particularly sensitive to the detrimental effects of aging. Recent studies on postmenopausal women find that estrogen replacement therapy benefits performance on tasks dependent on the frontal lobes. To determine whether estrogen has a similar influence in a rhesus monkey model of menopause, we tested five aged, long-term ovariectomized rhesus monkeys in a modified version of the Wisconsin Card Sort test which had been adapted to the nonhuman primate. In this test, monkeys had to select 3-D objects based either on color (blue, red, yellow) or shape (block, tube, cup) and had to be able to switch their response as a function of reinforcement contingencies. The monkeys were treated with placebo and ethinyl estradiol (EE2, 450 ng/kg/day) in alternation with each successive test. Contrary to our hypothesis, estradiol treatment did not affect performance. Because previous studies in the same monkeys [Neurobiol. Aging 23 (2002) 589] had shown that EE2 improves performance on a spatial memory task dependent on the hippocampus, but not on another task dependent upon the frontal lobes (the delayed response), we conclude that executive processes may be less sensitive to the effects of estradiol than hippocampal-dependent tasks.     

Cancer preventive potential of Momordica charantia L. against benzo(a)pyrene induced fore-stomach tumourigenesis in murine model system

Bitter melon ( Momordica charantia Linnaeus) fruit extract was tested against 3,4 benzo(a)pyrene [B(a)P] induced forestomach papillomagenesis in Swiss albino mice. Extract of M. charantia in two concentrations, 2.5 and 5% of standard mice feed was used for the short-term and long-term studies. A significant decrease in tumour burden was observed in short and long-term treatment. Also, total tumour incidence reduced to 83.33% with 2.5% dose and 90.90% with 5% dose in short term treatment, while in long-term treatment tumor incidence decreased to 76.92% with 2.5% dose and 69.23% with 5% dose of M. charantia. The possible mechanism involved in the cancer chemoprevention has also been discussed.     

In vitro (CTG)*(CAG) expansions and deletions by human cell extracts

The mechanism of disease-associated (CTG)*(CAG) expansion may involve DNA replication slippage, replication direction, Okazaki fragment processing, recombination, or repair. A length-dependent bias for expansions is observed in humans affected by a trinucleotide repeat-associated disease. We developed an assay to test the effect of replication direction on (CTG)*(CAG) instabilities incurred during in vitro (SV40) DNA replication mediated by human cell extracts. This system recapitulates the bias for expansions observed in humans. Replication by HeLa cell extracts generated expansions and deletions that depended upon repeat tract length and the direction of replication. Templates with 79 repeats yielded predominantly expansions (CAG as lagging strand template) or predominantly deletions (CTG as lagging strand template). Templates containing 17 repeats were stable. Thus, replication direction determined the type of mutation. These results provide new insights into the orientation of replication effect upon repeat stability. This system will be useful in determining the contribution of specific human proteins to (CTG)*(CAG) expansions.     

Genetic variation in IRF4 expression modulates growth characteristics,tyrosinase expression and interferon‐gamma response in melanocytic cells

A SNP within intron4 of the interferon regulatory factor4 (IRF4) gene, rs12203592*C/T, has been independently associated with pigmentation and age‐specific effects on naevus count in European‐derived populations. We have characterized the cis‐regulatory activity of this intronic region and using human foreskin‐derived melanoblast strains, we have explored the correlation between IRF4 rs12203592 homozygous C/C and T/T genotypes with TYR enzyme activity, supporting its association with pigmentation traits. Further, higher IRF4 protein levels directed by the rs12203592*C allele were associated with increased basal proliferation but decreased cell viability following UVR, an etiological factor in melanoma development. Since UVR, and accompanying IFNγ‐mediated inflammatory response, is associated with melanomagenesis, we evaluated its effects in the context of IRF4 status. Manipulation of IRF4 levels followed by IFNγ treatment revealed a subset of chemokines and immuno‐evasive molecules that are sensitive to IRF4 expression level and genotype including CTLA4 and PD‐L1.     

Circulating tumor cell-derived organoids: Current challenges and promises in medical research and precision medicine

Traditional 2D cell cultures do not accurately recapitulate tumor heterogeneity, and insufficient human cell lines are available. Patient-derived xenograft (PDX) models more closely mimic clinical tumor heterogeneity, but are not useful for high-throughput drug screening. Recently, patient-derived organoid cultures have emerged as a novel technique to fill this critical need. Organoids maintain tumor tissue heterogeneity and drug-resistance responses, and thus are useful for high-throughput drug screening. Among various biological tissues used to produce organoid cultures, circulating tumor cells (CTCs) are promising, due to relative ease of ascertainment. CTC-derived organoids could help to acquire relevant genetic and epigenetic information about tumors in real time, and screen and test promising drugs. This could reduce the need for tissue biopsies, which are painful and may be difficult depending on the tumor location. In this review, we have focused on advances in CTC isolation and organoid culture methods, and their potential applications in disease modeling and precision medicine.     

In vitro biosynthesis of the Pseudomonas aeruginosa quorum-sensing signal molecule N-butanoyl-L-homoserine lactone

In Pseudomonas aeruginosa , synthesis of the quorum-sensing signal molecules N -butanoyl- L -homoserine lactone (BHL) and N -hexanoyl- L -homoserine lactone (HHL) requires the LuxI homologue RhlI(VsmI). By using thin-layer chromatography in conjunction with high-performance liquid chromatography (HPLC) and mass spectrometry, we show that purified RhlI can catalyse the biosynthesis of BHL and HHL using either S -adenosylmethionine (SAM) or homoserine lactone (HSL) but not homoserine as the source of the homoserine lactone moiety. As we were unable to detect homoserine lactone in cytoplasmic extracts of Escherichia coli , we conclude that SAM is the natural substrate for RhlI-directed N -acylhomoserine lactone (AHL) biosynthesis. The N -acyl chain of BHL and HHL can be supplied by the appropriately charged coenzyme A derivative (either n -butanoyl-CoA or n -hexanoyl-CoA). The specificity of RhlI for charged CoA derivatives is demonstrated as RhlI was unable to generate AHLs detectable in our bioassays from acetyl-CoA, malonyl-CoA, n -octanoyl-CoA, n -decanoyl-CoA, DL-β-hydroxybutanoyl-CoA or crotonoyl-CoA. RhlI was also unable to use N -acetyl- S -3-oxobutanoylcysteamine, a chemical mimic for 3-oxobutanoyl-CoA. Furthermore, the RhlI-catalysed synthesis of BHL and HHL was most efficiently driven when NADPH was included in the reaction mixture.     

Effects of oral contraceptive pills on drug metabolizing enzymes and acid soluble sulfhydryl level in mouse liver.

The present paper reports the modulatory influence of two widely used combined oral contraceptive pills "OVRAL" and "NORACYCLINE" on hepatic phase I and II drug metabolizing enzymes and acid soluble sulfhydryl group of the mouse. Three different doses of the pills were used in this study i.e. D1 (1/2000th of a pill), D2 (1/200th of a pill) and D3 (1/20th of a pill). The sulfhydryl group increased significantly with the D1 and D2 dose of Ovral and the D2 dose of Noracycline. Dose D2 of both pills decreased cyt.P450 and cyt.b5 contents. D3 of Noracycline, however increased both the cytochrome levels. Dose D3 of Ovral and all three doses of Noracycline reduced the glutathione S-transferase activity.     

Cell density-regulated recovery of starved biofilm populations of ammonia-oxidizing bacteria.

The speed of recovery of cell suspensions and biofilm populations of the ammonia oxidizer Nitrosomonas europaea, following starvation was determined. Stationary-phase cells, washed and resuspended in ammoniumfree inorganic medium, were starved for periods of up to 42 days, after which the medium was supplemented with ammonium and subsequent growth was monitored by measuring nitrite concentration changes. Cultures exhibited a lag phase prior to exponential nitrite production, which increased from 8.72 h (no starvation) to 153 h after starvation for 42 days. Biofilm populations of N. europaea colonizing sand or soil particles in continuous-flow, fixed column reactors were starved by continuous supply of ammonium-free medium. Following resupply of ammonium, starved biofilms exhibited no lag phase prior to nitrite production, even after starvation for 43.2 days, although there was evidence of cell loss during starvation. Biofilm formation will therefore provide a significant ecological advantage for ammonia oxidizers in natural environments in which the substrate supply is intermittent. Cell density-dependent phenomena in a number of gram-negative bacteria are mediated by N-acyl homoserine lactones (AHL), including N-(3-oxohexanoyl)-L-homoserine lactone (OHHL). Addition of both ammonium and OHHL to cell suspensions starved for 28 days decreased the lag phase in a concentration-dependent manner from 53.4 h to a minimum of 10.8 h. AHL production by N. europaea was detected by using a luxR-luxAB AHL reporter system. The results suggest that rapid recovery of high-density biofilm populations may be due to production and accumulation of OHHL to levels not possible in relatively low-density cell suspensions.