Proteolytic cleavage of pyridoxal kinase into two structural domains

Chymotryptic digestion of sheep brain pyridoxal kinase, a dimer of identical subunits each of 40 kDa, yields 2 fragments of 24 and 16 kDa with concomitant loss of catalytic activity. These fragments were separated by chromatographic techniques and analyzed for interaction with the ATP analogue, trinitrophenyl-ATP, using fluorescence spectroscopy. The absorption and fluorescence properties of trinitrophenyl-ATP bound to the fragment of 24 kDa (emission maximum, 540 nm, emission anisotropy at 460 nm, 0.30, and fluorescence lifetime, gamma = lns) are indistinguishable from those of the ATP analogue bound to the native enzyme. The fragment of 16 kDa does not bind trinitrophenyl-ATP. The results are consistent with the hypothesis that monomeric pyridoxal kinase is folded into 2 domains connected by a single polypeptide chain sensitive to proteolytic cleavage.     

DHTP is an allosteric inhibitor of the kinesin-13 family of microtubule depolymerases

The kinesin-13 family of microtubule depolymerases is a major regulator of microtubule dynamics. RNA interference-induced knockdown studies have highlighted their importance in many cell division processes including spindle assembly and chromosome segregation. Since microtubule turnovers and most mitotic events are relatively rapid (in minutes or seconds), developing tools that offer faster control over protein functions is therefore essential to more effectively interrogate kinesin-13 activities in living cells. Here, we report the identification and characterization of a selective allosteric kinesin-13 inhibitor, DHTP. Using high resolution microscopy, we show that DHTP is cell permeable and can modulate microtubule dynamics in cells.     

RBM3 regulates temperature sensitive miR-142–5p and miR-143 (thermomiRs), which target immune genes and control fever

Fever is commonly used to diagnose disease and is consistently associated with increased mortality in critically ill patients. However, the molecular controls of elevated body temperature are poorly understood. We discovered that the expression of RNA-binding motif protein 3 (RBM3), known to respond to cold stress and to modulate microRNA (miRNA) expression, was reduced in 30 patients with fever, and in THP-1-derived macrophages maintained at a fever-like temperature (40°C). Notably, RBM3 expression is reduced during fever whether or not infection is demonstrable. Reduced RBM3 expression resulted in increased expression of RBM3-targeted temperature-sensitive miRNAs, we termed thermomiRs. ThermomiRs such as miR-142–5p and miR-143 in turn target endogenous pyrogens including IL-6, IL6ST, TLR2, PGE2 and TNF to complete a negative feedback mechanism, which may be crucial to prevent pathological hyperthermia. Using normal PBMCs that were exogenously exposed to fever-like temperature (40°C), we further demonstrate the trend by which decreased levels of RBM3 were associated with increased levels of miR-142–5p and miR-143 and vice versa over a 24 h time course. Collectively, our results indicate the existence of a negative feedback loop that regulates fever via reduced RBM3 levels and increased expression of miR-142–5p and miR-143.     

Kinetic determinism of lysozyme folding at high temperatures

Reduced, disordered hen egg lysozyme rapidly regains enzymic activity in a nonenzymic system previously reported from this laboratory. When such regenerations are carried out at high temperatures (where the native enzyme is unstable), native enzyme is formed as a transient intermediate. Thermal inactivation does not depend on irreversible changes in the protein, as shown by experiments wherein thermally inactivated regenerated lysozyme spontaneously reactivates at 37°. These findings are inconsistent with thermodynamic determinism of protein structure.     

The antilipemic effectiveness of pentaerythritol tetranicotinate on hyperlipidemic patients with or without diabetes mellitus--a double-blind, randomized and two-period change-over experiment

Pentaerythritol tetranicotinate (Perycit), at an oral dosage of 750 mg daily, was given to 12 patients with idiopathic hyperlipidemia and to 12 patients with hyperlipidemia superimposed with diabetes mellitus (DM). With 2 months off-drug period as the baseline, each patient then received 3 months of placebo and 3 months of Perycit. The sequence of treatment was randomized and balanced in frequency. Blood glycosylated hemoglobin (Hb A1) and fasting plasma glucose (FPG) were used as indices of diabetic control. Serum triglyceride (TG), total cholesterol (TC), high density lipoprotein cholesterol (HDL-C), and TC/HDL-C ratio were measured and calculated in order to compare the antilipemic effectiveness of Perycit with that of placebo. The non-parametric Wilcoxon test was used for the statistical analysis. The results showed that in the idiopathic group, Perycit significantly lowered the serum level of TG and the ratio of TC/HDL-C, and elevated the serum level of HDL-C. In the diabetic group, although there was a similar improvement in diabetic control in both periods of placebo and Perycit treatments, there was no change in the serum levels of TG and HDL-C. There was a slight increase of the serum levels of TC in the periods of Perycit treatment, whereas a small increase of HDL-C resulted in a mild decrease of the TC/HDL-C ratio. There was mild and transient facial flushing during the Perycit treatment in 6 out of 12 diabetic patients. Otherwise, there was no side effects in either group. Pooling the two groups' data together, Perycit increased the serum levels of HDL-C and decreased the TC/HDL-C ratio. It is concluded that Perycit has antilipemic effects in patients with idiopathic hyperlipidemia, and may be helpful in reducing the atherogenic risks in these patients. In patients with hyperlipidemia superimposed with DM, although the serum lipids composition was not significantly changed after Perycit, the atherogenic risks might also be reduced as demonstrated by the decrease of the TC/HDL-C ratio.     

Ammonia or Ammonium Ion as Substrate for Oxidation by Nitrosomonas europaea Cells and Extracts

The effect of pH on the K(m) values for ammonia was studied in its oxidation by Nitrosomonas cells and cell-free extracts. The K(m) values decreased markedly with increasing pH, suggesting (NH(3)) rather than (NH(4) (+)) as the actual substrate for oxidation.     

Stress-induced phenocopy of C. elegans defines functional steps of sensory organ differentiation

The differentiation of male specific sensory rays in the nematode Caenorhabditis elegans is a complex process regulated by multiple genetic components. A novel approach with heat shock treatment was employed to show that multistep regulation is involved in this process. Intervention in this stepwise regulation resulted in phenocopy of specific gene mutations. The results suggest that differential gene function acting at a precise time frame is necessary to guide the normal differentiation of sensory rays.