Discovery of novel inhibitors targeting the Mycobacterium tuberculosis O-acetylserine sulfhydrylase (CysK1) using virtual high-throughput screening

Cysteine biosynthesis in Mycobacterium tuberculosis (MTB) is crucial for this pathogen to combat oxidative stress and for long term survival in the host. Hence inhibition of this pathway is attractive for developing novel drugs against tuberculosis. In the present study, the crystal structure of the mycobacterial enzyme O-acetylserine sulfhydrylase CysK1 bound to an oligopeptide inhibitor was used as a framework for virtual screening of the BITS-Pilani in-house database to identify new scaffolds as CysK1 inhibitors. Thirty compounds were synthesized and evaluated in vitro for their ability to inhibit CysK1, activity against M. tuberculosis and cytotoxicity as steps towards the derivation of structure–activity relationships (SAR) and lead optimization. Compound 8-nitro-4-(2-(trifluoromethyl)phenyl)-4,4a-dihydro-2H-pyrimido[5,4-e]thiazolo[3,2-a]pyrimidine-2,5(3H)-dione (4n) emerged as the most promising lead with an IC₅₀ of 17.7 μM for purified CysK1 and MIC of 7.6 μM for M. tuberculosis, with little or no cytotoxicity (>50 μM).     

Diabetes,metformin use,and colorectal cancer survival in postmenopausal women

Background: Observational studies have associated metformin use with lower colorectal cancer (CRC) incidence but few studies have examined metformin's influence on CRC survival. We examined the relationships among metformin use, diabetes, and survival in postmenopausal women with CRC in the Women's Health Initiative (WHI) clinical trials and observational study. Methods: 2066 postmenopausal women with CRC were followed for a median of 4.1 years, with 589 deaths after CRC diagnosis from all causes and 414 deaths directly attributed to CRC. CRC-specific survival was compared among women with diabetes with metformin use (n = 84); women with diabetes with no metformin use (n = 128); and women without diabetes (n = 1854). Cox proportional hazard models were used to estimate associations among metformin use, diabetes and survival after CRC. Strategies to adjust for potential confounders included: multivariate adjustment with known predictors of colorectal cancer survival and construction of a propensity score for the likelihood of receiving metformin, with model stratification by propensity score quintile. Results: After adjusting for age and stage, CRC specific survival in women with diabetes with metformin use was not significantly different compared to that in women with diabetes with no metformin use (HR 0.75; 95% CI 0.40–1.38, p = 0.67) and to women without diabetes (HR 1.00; 95% CI 0.61–1.66, p = 0.99). Following propensity score adjustment, the HR for CRC-specific survival in women with diabetes with metformin use compared to non-users was 0.78 (95% CI 0.38–1.55, p = 0.47) and for overall survival was 0.86 (95% CI 0.49–1.52; p = 0.60). Conclusions: In postmenopausal women with CRC and DM, no statistically significant difference was seen in CRC specific survival in those who used metformin compared to non-users. Analyses in larger populations of colorectal cancer patients are warranted.     

Knockdown of a laccase in Populus deltoides confers altered cell wall chemistry and increased sugar release

Plant laccases are thought to function in the oxidation of monolignols which leads to higher order lignin formation. Only a hand‐full of laccases in plants have been functionally evaluated, and as such little is known about the breadth of their impact on cell wall chemistry or structure. Here, we describe a previously uncharacterized laccase from Populus, encoded by locus Potri.008G064000, whose reduced expression resulted in transgenic Populus trees with changes in syringyl/guaiacyl ratios as well as altered sugar release phenotypes. These phenotypes are consistent with plant biomass exhibiting reduced recalcitrance. Interestingly, the transgene effect on recalcitrance is dependent on a mild pretreatment prior to chemical extraction of sugars. Metabolite profiling suggests the transgene modulates phenolics that are associated with the cell wall structure. We propose that this particular laccase has a range of functions related to oxidation of phenolics and conjugation of flavonoids that interact with lignin in the cell wall.     

Immunogenicity of plant‐produced porcine circovirus‐like particles in mice

Porcine circovirus type 2 (PCV‐2) is the main causative agent associated with a group of diseases collectively known as porcine circovirus‐associated disease (PCAD). There is a significant economic strain on the global swine industry due to PCAD and the production of commercial PCV‐2 vaccines is expensive. Plant expression systems are increasingly regarded as a viable technology to produce recombinant proteins for use as pharmaceutical agents and vaccines. However, successful production and purification of PCV‐2 capsid protein (CP) from plants is an essential first step towards the goal of a plant‐produced PCV‐2 vaccine candidate. In this study, the PCV‐2 CP was transiently expressed in Nicotiana benthamiana plants via agroinfiltration and PCV‐2 CP was successfully purified using sucrose gradient ultracentrifugation. The CP self‐assembled into virus‐like particles (VLPs) resembling native virions and up to 6.5 mg of VLPs could be purified from 1 kg of leaf wet weight. Mice immunized with the plant‐produced PCV‐2 VLPs elicited specific antibody responses to PCV‐2 CP. This is the first report describing the expression of PCV‐2 CP in plants, the confirmation of its assembly into VLPs and the demonstration of their use to elicit a strong immune response in a mammalian model.     

Hsp90-mediated inhibition of FKBP38 regulates apoptosis in neuroblastoma cells

The FK506-binding protein 38 (FKBP38) is a pro-apoptotic regulator of Bcl-2 in neuroblastoma cells. Hsp90 inhibits the pro-apoptotic FKBP38/CaM/Ca(2+) complex and thus prevents interactions between FKBP38 and Bcl-2. Here we show that Hsp90 increases cell survival rates of neuroblastoma cells after apoptosis induction. Depletion of FKBP38 by short interference RNA significantly decreased the anti-apoptotic effect of Hsp90 expression. In addition, the influence of high cellular Hsp90 levels was only observed in post-stimulation apoptosis that is sensitive to selective FKBP38 active site inhibition. Similar anti-apoptotic effects in neuroblastoma cells were observed after stimulation of endogenous Hsp90 expression. Hence, the inhibition of FKBP38 by Hsp90 participates in programmed cell death control of neuroblastoma cells.     

UBE1L2, a novel E1 enzyme specific for ubiquitin

UBE1 is known as the human ubiquitin-activating enzyme (E1), which activates ubiquitin in an ATP-dependent manner. Here, we identified a novel human ubiquitin-activating enzyme referred to as UBE1L2, which also shows specificity for ubiquitin. The UBE1L2 sequence displays a 40% identity to UBE1 and also contains an ATP-binding domain and an active site cysteine conserved among E1 family proteins. UBE1L2 forms a covalent link with ubiquitin in vitro and in vivo, which is sensitive to reducing conditions. In an in vitro polyubiquitylation assay, recombinant UBE1L2 could activate ubiquitin and transfer it onto the ubiquitin-conjugating enzyme UbcH5b. Ubiquitin activated by UBE1L2 could be used for ubiquitylation of p53 by MDM2 and supported the autoubiquitylation of the E3 ubiquitin ligases HectH9 and E6-AP. The UBE1L2 mRNA is most abundantly expressed in the testis, suggesting an organ-specific regulation of ubiquitin activation.     

Characterization and the broad cross‐species applicability of 20 anonymous nuclear loci isolated from the Taiwan Hwamei (Garrulax taewanus)

We isolated 20 anonymous nuclear loci (8556 bp in total) from the Taiwan Hwamei (Garrulax taewanus), an endemic songbird of Taiwan. A panel of nine to 15 individuals with unknown relationship was used to characterize polymorphism of these loci. We identified 46 single nucleotide polymorphic sites (SNPs) in 15 polymorphic loci. Frequency of SNPs was one per every 186 bp in average. Nucleotide diversity, θ, ranged from 0.00054 to 0.00371 per locus. We also tested cross‐species applicability of these loci on 17 species from eight different passerine families. All 20 loci could be successfully amplified (ranged from one to 16 species, mean = 7.9 species).     

Evaluation of risk profiles by subcutaneous adipose tissue topography in obese juveniles

Objective: To compare subcutaneous adipose tissue topography (SAT‐top) in obese juveniles with age‐matched normal‐weight controls. Research Methods and Procedures: The optical device LIPOMETER (European Patent EP 0516251) enables the non‐invasive, rapid, safe, and precise measurement of the thickness of subcutaneous adipose tissue. Fifteen defined body sites (1 = neck to 15 = calf) characterize the individual SAT‐top like an individual fingerprint. SAT‐top of 1351 juveniles (obese: 42 boys, 59 girls, normal weight: 680 boys, 570 girls) from 7 to 19 years of age were measured. For visual comparison, the 15‐dimensional SAT‐top information was condensed by factor analysis into a two‐dimensional factor plot. Results: Both female and male obese juveniles had markedly increased adipose tissue layers at 7 = upper abdomen, 8 = lower abdomen, 5 = front chest, and 6 = lateral chest. The pubertal changes of body shape and fat distribution of the normal‐weight boys and girls (boys show thinner adipose tissue layers on their legs, whereas girls had thicker adipose tissue layers at the extremities) were not seen in the obese group. Independently of age and sex, all of the obese juveniles showed a similar, more android body fat distribution with increased trunk fat. Discussion: SAT‐top of the obese juveniles is similar to that of patients with type 2 diabetes, polycystic ovary syndrome, and coronary heart disease. Patients with these metabolic disorders and obese juveniles are located in the factor plot in the same area. This body shape may indicate a risk profile for developing polycystic ovary syndrome (women), type 2 diabetes, and early atherosclerosis (both sexes).     

The rapid mode of calcium uptake into heart mitochondria (RaM): comparison to RaM in liver mitochondria

A mechanism of Ca(2+) uptake, capable of sequestering significant amounts of Ca(2+) from cytosolic Ca(2+) pulses, has previously been identified in liver mitochondria. This mechanism, the Rapid Mode of Ca(2+) uptake (RaM), was shown to sequester Ca(2+) very rapidly at the beginning of each pulse in a sequence [Sparagna et al. (1995) J. Biol. Chem. 270, 27510-27515]. The existence and properties of RaM in heart mitochondria, however, are unknown and are the basis for this study. We show that RaM functions in heart mitochondria with some of the characteristics of RaM in liver, but its activation and inhibition are quite different. It is feasible that these differences represent different physiological adaptations in these two tissues. In both tissues, RaM is highly conductive at the beginning of a Ca(2+) pulse, but is inhibited by the rising [Ca(2+)] of the pulse itself. In heart mitochondria, the time required at low [Ca(2+)] to reestablish high Ca(2+) conductivity via RaM i.e. the 'resetting time' of RaM is much longer than in liver. RaM in liver mitochondria is strongly activated by spermine, activated by ATP or GTP and unaffected by ADP and AMP. In heart, RaM is activated much less strongly by spermine and unaffected by ATP or GTP. RaM in heart is strongly inhibited by AMP and has a biphasic response to ADP; it is activated at low concentrations and inhibited at high concentrations. Finally, an hypothesis consistent with the data and characteristics of liver and heart is presented to explain how RaM may function to control the rate of oxidative phosphorylation in each tissue. Under this hypothesis, RaM functions to create a brief, high free Ca(2+) concentration inside mitochondria which may activate intramitochondrial metabolic reactions with relatively small amounts of Ca(2+) uptake. This hypothesis is consistent with the view that intramitochondrial [Ca(2+)] may be used to control the rate of ADP phosphorylation in such a way as to minimize the probability of activating the Ca(2+)-induced mitochondrial membrane permeability transition (MPT).