Onset-potency relationship of nondepolarizing muscle relaxants: a reexamination using simulations

Nondepolarizing muscle relaxants (MRs) display an inverse onset-potency relationship, that is, less potent MRs display a more rapid onset. We have conducted the current investigation to estimate the impact of variable pharmacokinetic or pharmacodynamic properties of the MRs on potency and onset time, and on the onset-potency relationship. Using a model of neuromuscular transmission, we changed either the affinity of MRs for the postsynaptic receptors or the pharmacokinetic properties of the MRs. The elimination rate constant, k(10), which defines the systemic clearance, was assigned one of 9 values and the transport rate constant, k(12), one of 5 values. The transport rate constant into the effect compartment was constant (k(e1) = 0.2 min(-1)). Only one parameter was altered at a time. With constant pharmacokinetics, a 100-fold decrease in affinity caused a proportional decrease in potency, but little change (0.02 min) in onset time. With constant affinity, increasing the clearance from 1 to 250 mL x kg(-1) x min(-1) shortened the onset time from 7.2 to 0.7 min and decreased the potency 12-fold. In a double logarithmic plot, the onset-potency relationship was linear. Lesser affinities produce a nearly parallel rightward shift of the regression lines. The inverse onset-potency relationship may be explained by the pharmacokinetic factors producing changes in both the potency and onset times.     

Discovery and optimization of orally active cyclohexane-based prolylcarboxypeptidase (PrCP) inhibitors

The synthesis, SAR, binding affinities and pharmacokinetic profiles are described for a series of cyclohexane-based prolylcarboxypeptidase (PrCP) inhibitors discovered by high throughput screening. Compounds show high levels of ex vivo target engagement in mouse plasma 20 h post oral dose.     

The Influence of Environmental Factors on the Respiration of Plant-Parasitic Nematodes

Respiration of selected nematode species was measured relative to CO₂ level, temperature, osmotic pressure, humidity, glucose utilization and high ionic concentrations of sodium and potassium.In general, respiration was stimulated most by the dominant environmental factors at levels near those expected in the nematode''s "natural" habitat. Soil-inhabiting nematodes utilized O₂, most rapidly with high (1-2%) CO₂ whereas a foliar nematode (Aphelenchoides ritzemabosi) did so with 0.03% CO₂, the concentration typically found in air. Temperature optima for respiration corresponded closely to those for other activities. Ditylenchus dipsaci and Pratylenchus penetrans adults and Anguina tritici and A. agrostis second-stage larvae respired within the range of osmotic pressures from 0 to 44.8 arm and respiration of their drought-resistant stages was stimulated by increasing osmotic pressure which accompanies the onset of drought. Rehydration of A. tritici and A. agrostis larvae with RH as low as 5% stimulated measurable respiration. Glucose utilization from liquid medium by A. tritici larvae or A. ritzembosi was not detectable. Supplemental Na⁺ stimulated respiration of Anguina tritici, K⁺ did not.     

Inhibition of lindane-induced toxicity using alpha-lipoic acid and vitamin E in the brain of Mus musculus

In the present investigation, we have used adenosine triphosphatase (ATPase) activity as biochemical test of toxic action of lindane that was explained by lipid peroxidation model. Study was also undertaken to ascertain the potential protective role of alpha-lipoic acid (ALA) and vitamin E on the same parameters. Highly acute dose of lindane, i.e., 40 mg/kg bw for 18 h exposure, was used for creating lesions in brain. Lipid peroxidation was measured in terms of glutathione peroxidase and thio barbituric acid-reacting substances (TBARS). Various brain regions under investigation were cerebellum and pons-medulla oblongata. Healthy, male, Swiss mice (7–8 weeks old) were allocated into four groups. First group was control, second group was treated with lindane, third group was treated purely with antioxidants, and fourth group received both antioxidants and lindane treatment. Results revealed the significant difference (at 1% and 5% in all groups) in all studied parameters from control. Increased TBARS level in second group suggests that lindane enhances the production of free radicals in studied brain regions. Antioxidants under test are efficient remedy for neurotoxicity caused by lindane. We conclude that lindane manifests toxic effects on brain ATPase and enhances lipid peroxidation. ALA and vitamin E in combination may provide protection against lindane-induced acute toxicity.     

Phosphorylation of Enoyl-Acyl Carrier Protein Reductase InhA Impacts Mycobacterial Growth and Survival

InhA, the primary target for the first line anti-tuberculosis drug isoniazid, is a key enzyme of the fatty-acid synthase II system involved in mycolic acid biosynthesis in Mycobacterium tuberculosis. In this study, we show that InhA is a substrate for mycobacterial serine/threonine protein kinases. Using a novel approach to validate phosphorylation of a substrate by multiple kinases in a surrogate host (Escherichia coli), we have demonstrated efficient phosphorylation of InhA by PknA, PknB, and PknH, and to a lower extent by PknF. Additionally, the sites targeted by PknA/PknB have been identified and shown to be predominantly located at the C terminus of InhA. Results demonstrate in vivo phosphorylation of InhA in mycobacteria and validate Thr-266 as one of the key sites of phosphorylation. Significantly, our studies reveal that the phosphorylation of InhA by kinases modulates its biochemical activity, with phosphorylation resulting in decreased enzymatic activity. Co-expression of kinase and InhA alters the growth dynamics of Mycobacterium smegmatis, suggesting that InhA phosphorylation in vivo is an important event in regulating its activity. An InhA-T266E mutant, which mimics constitutive phosphorylation, is unable to rescue an M. smegmatis conditional inhA gene replacement mutant, emphasizing the critical role of Thr-266 in mediating post-translational regulation of InhA activity. The involvement of various serine/threonine kinases in modulating the activity of a number of enzymes of the mycolic acid synthesis pathway, including InhA, accentuates the intricacies of mycobacterial signaling networks in parallel with the changing environment.     

The C-terminal domain of dimeric serine hydroxymethyltransferase plays a key role in stabilization of the quaternary structure and cooperative unfolding of protein: domain swapping studies with enzymes having high sequence identity

The serine hydroxymethyltransferase from Bacillus subtilis (bsSHMT) and B. stearothermophilus (bstSHMT) are both homodimers and share approximately 77% sequence identity; however, they show very different thermal stabilities and unfolding pathways. For investigating the role of N- and C-terminal domains in stability and unfolding of dimeric SHMTs, we have swapped the structural domains between bs- and bstSHMT and generated the two novel chimeric proteins bsbstc and bstbsc, respectively. The chimeras had secondary structure, tyrosine, and pyridoxal-5'-phosphate microenvironment similar to that of the wild-type proteins. The chimeras showed enzymatic activity slightly higher than that of the wild-type proteins. Interestingly, the guanidium chloride (GdmCl)-induced unfolding showed that unlike the wild-type bsSHMT, which undergoes dissociation of native dimer into monomers at low guanidium chloride (GdmCl) concentration, resulting in a non-cooperative unfolding of enzyme, its chimera bsbstc, having the C-terminal domain of bstSHMT was resistant to low GdmCl concentration and showed a GdmCl-induced cooperative unfolding from native dimer to unfolded monomer. In contrast, the wild-type dimeric bstSHMT was resistant to low GdmCl concentration and showed a GdmCl-induced cooperative unfolding, whereas its chimera bstbsc, having the C- terminal domain of bsSHMT, showed dissociation of native dimer into monomer at low GdmCl concentration and a GdmCl-induced non-cooperative unfolding. These results clearly demonstrate that the C-terminal domain of dimeric SHMT plays a vital role in stabilization of the oligomeric structure of the native enzyme hence modulating its unfolding pathway.     

In vitro regeneration of roots of Phyla nodiflora and Leptadenia reticulata,and comparison of roots from cultured and natural plants for secondary metabolites

Adventitious roots, generated using leaf explants of P. nodiflora, and meristem explants of L. reticulata, were cultured on Murashige and Skoog (MS) medium supplemented with napthylacetic acid (2 microM) and indole butyric acid (3 microM) respectively. After 30 days, subculturing of roots in liquid MS medium with napthylacetic acid (1.5 microM) for P. nodiflora and indole butyric acid (3 microM) for L. reticulata afforded considerable increase in root mass. HPTLC profiles and microscopic examination of transverse sections of in vitro and naturally grown roots provided information on secondary metabolite accumulation vis-à-vis developmental stages of the root.