Analysis of slow-binding enzyme inhibitors at elevated enzyme concentrations

The improvement in the characterization of slow-binding inhibitors achieved by performing experiments at elevated enzyme concentrations is presented. In particular, the characterization of slow-binding inhibitors conforming to a two-step mode of inhibition with a steady-state dissociation constant that is much lower than the initial dissociation constant with enzyme is discussed. For these systems, inhibition is rapid and low steady-state product concentrations are produced at saturating inhibitor concentrations. By working at elevated enzyme concentrations, improved signal-to-noise ratios are achieved and data may be collected at saturating inhibitor levels. Numerical simulations confirmed that improved parameter estimates are obtained and useful data to discern the mechanism of slow-binding inhibition are produced by working at elevated enzyme concentrations. The saturation kinetics that were unobservable in two previous studies of an enzyme inhibitor system were measured by performing experiments at an elevated enzyme concentration. These results indicate that consideration of the quality of the data acquired using a particular assay is an important factor when selecting the enzyme concentration at which to perform experiments used to characterize the class of enzyme inhibitors examined herein.     

The relative biological effectiveness of densely ionizing heavy-ion radiation for inducing ocular cataracts in wild type versus mice heterozygous for the ATM gene

The accelerated appearance of ocular cataracts at younger ages has been recorded in both astronauts and airline pilots, and is usually attributed to high-energy heavy ions in galactic cosmic ray radiation. We have previously shown that high-LET 1-GeV/nucleon ⁵⁶Fe ions are significantly more effective than X-rays in producing cataracts in mice. We have also shown that mice haploinsufficient for ATM develop cataracts earlier than wild-type animals, when exposed to either low-LET X-rays or high-LET ⁵⁶Fe ions. In this paper we derive quantitative estimates for the relative biological effectiveness (RBE) of high energy ⁵⁶Fe ions compared with X-rays, both for wild type and for mice haploinsufficient for ATM. There is a clear trend toward higher RBE’s in haploinsufficient animals, both for low- and high-grade cataracts. Haploinsufficiency for ATM results in an enhanced sensitivity to X-rays compared with the wild type, and this enhancement appears even larger after exposure to high-LET heavy ions.Dedicated to the memory of Professor Basil V. Worgul, who passed away in January 2006, much missed by all his colleagues.     

Macrotubule-dependent protoplast volume regulation in plasmolysed root-tip cells of Triticum turgidum: involvement of phospholipase D

The probable involvement of phospholipase D (PLD)/phosphatidic acid (PA) signalling in the hyperosmotic stress response of Triticum turgidum root cells was investigated by examining the effects of butanol-1, butanol-2, phosphatidylbutanol (PtdBut), N-acylethanolamine (NAE) and PA on the hyperosmotic response, the organization of the tubulin cytoskeleton and the accumulation of a phosphorylated p38-like mitogen-activated protein (MAP) kinase (phospho-p46) in plasmolysed root cells. The effects of all the treatments were assessed by differential interference contrast (DIC) microscopy of living cells, tubulin immunofluorescence, conventional transmission electron microscopy (TEM), tubulin immunogold localization, protoplast volume measurements and western blot analysis. Butanol-1 and NAE compromised the viability of plasmolysed cells, induced a marked reduction in the plasmolysed protoplast volume, and inhibited hyperosmotically induced tubulin macrotubule formation and the accumulation of phospho-p46. Exogenous PA reinforced the hyperosmotic response of T. turgidum root cells and positively affected tubulin macrotubule formation. Additionally, PA reduced the effects of butanol-1 in plasmolysed cells. Taken together, the data suggest that PLD-mediated PA synthesis occurs upstream of the accumulation of phospho-p46 to regulate hyperosmotically induced macrotubule formation in plasmolysed T. turgidum root cells.     

beta4 integrin and epidermal growth factor coordinately regulate electric field-mediated directional migration via Rac1

Endogenous DC electric fields (EF) are present during embryogenesis and are generated in vivo upon wounding, providing guidance cues for directional cell migration (galvanotaxis) required in these processes. To understand the role of beta (beta)4 integrin in directional migration, the migratory paths of either primary human keratinocytes (NHK), beta4 integrin-null human keratinocytes (beta4-), or those in which beta4 integrin was reexpressed (beta4+), were tracked during exposure to EFs of physiological magnitude (100 mV/mm). Although the expression of beta4 integrin had no effect on the rate of cell movement, it was essential for directional (cathodal) migration in the absence of epidermal growth factor (EGF). The addition of EGF potentiated the directional response, suggesting that at least two distinct but synergistic signaling pathways coordinate galvanotaxis. Expression of either a ligand binding-defective beta4 (beta4+AD) or beta4 with a truncated cytoplasmic tail (beta4+CT) resulted in loss of directionality in the absence of EGF, whereas inhibition of Rac1 blinded the cells to the EF even in the presence of EGF. In summary, both the beta4 integrin ligand-binding and cytoplasmic domains together with EGF were required for the synergistic activation of a Rac-dependent signaling pathway that was essential for keratinocyte directional migration in response to a galvanotactic stimulus.     

Berberine protects against high fat diet-induced dysfunction in muscle mitochondria by inducing SIRT1-dependent mitochondrial biogenesis

Berberine (BBR) has recently been shown to improve insulin sensitivity in rodent models of insulin resistance. Although this effect was explained partly through an observed activation of AMP-activated protein kinase (AMPK), the upstream and downstream mediators of this phenotype were not explored. Here, we show that BBR supplementation reverts mitochondrial dysfunction induced by High Fat Diet (HFD) and hyperglycemia in skeletal muscle, in part due to an increase in mitochondrial biogenesis. Furthermore, we observe that the prevention of mitochondrial dysfunction by BBR, the increase in mitochondrial biogenesis, as well as BBR-induced AMPK activation, are blocked in cells in which SIRT1 has been knocked-down. Taken together, these data reveal an important role for SIRT1 and mitochondrial biogenesis in the preventive effects of BBR on diet-induced insulin resistance.     

Berberine protects against high fat diet-induced dysfunction in muscle mitochondria by inducing SIRT1-dependent mitochondrial biogenesis

Berberine (BBR) has recently been shown to improve insulin sensitivity in rodent models of insulin resistance. Although this effect was explained partly through an observed activation of AMP-activated protein kinase (AMPK), the upstream and downstream mediators of this phenotype were not explored. Here, we show that BBR supplementation reverts mitochondrial dysfunction induced by High Fat Diet (HFD) and hyperglycemia in skeletal muscle, in part due to an increase in mitochondrial biogenesis. Furthermore, we observe that the prevention of mitochondrial dysfunction by BBR, the increase in mitochondrial biogenesis, as well as BBR-induced AMPK activation, are blocked in cells in which SIRT1 has been knocked-down. Taken together, these data reveal an important role for SIRT1 and mitochondrial biogenesis in the preventive effects of BBR on diet-induced insulin resistance.     

Nafamostat mesylate, a serine protease inhibitor, demonstrates novel antimicrobial properties and effectiveness in Chlamydia-induced arthritis

Introduction

Hydroxychloroquine (HCQ) is a common disease modifying therapy for the treatment of rheumatoid arthritis (RA). Prior research suggests that HCQ may reduce the risk of diabetes mellitus in patients with RA. To investigate the mechanism of this effect, we examined the effect of HCQ on insulin resistance, insulin sensitivity, and pancreatic ??-cell secretion of insulin in non-diabetic, obese subjects.

Methods

We recruited 13 obese, non-diabetic subjects without systemic inflammatory conditions for an open-label longitudinal study of HCQ 6.5 mg per kilogram per day for six weeks. Subjects underwent an oral glucose tolerance test at three time points: 0 weeks (pre-treatment with HCQ), 6 weeks (at the end of the HCQ treatment), and 12 weeks (6 weeks post HCQ-treatment). The Matsuda Insulin Sensitivity Index (ISI), HOMA-IR, and HOMA-B were compared across time-points.

Results

The mean age of the cohort was 49 years, 77% females and median body mass index was 36.1 kg/m². After 6 weeks of HCQ therapy, ISI increased from a median (interquartile range) of 4.5 (2.3-7.8) to 8.9 (3.7-11.4) with a p-value of 0.040, and HOMA-IR decreased from a median of 2.1 (1.6-5.4) to 1.8 (1.02-2.1) with a p-value of 0.09. All these variables returned toward baseline at week 12.

Conclusion

HCQ use for 6 weeks in non diabetic obese subjects was associated with a significant increase in ISI and trends toward reduced insulin resistance and insulin secretion. These data suggest that HCQ, a common medication used to treat RA, possesses beneficial effects upon insulin sensitization. Further study of the insulin sensitizing effects of HCQ in patients with RA is warranted.