Catalysis by dihydrofolate reductase and other enzymes arises from electrostatic preorganization, not conformational motions

The proposal that enzymatic catalysis is due to conformational fluctuations has been previously promoted by means of indirect considerations. However, recent works have focused on cases where the relevant motions have components toward distinct conformational regions, whose population could be manipulated by mutations. In particular, a recent work has claimed to provide direct experimental evidence for a dynamical contribution to catalysis in dihydrofolate reductase, where blocking a relevant conformational coordinate was related to the suppression of the motion toward the occluded conformation. The present work utilizes computer simulations to elucidate the true molecular basis for the experimentally observed effect. We start by reproducing the trend in the measured change in catalysis upon mutations (which was assumed to arise as a result of a "dynamical knockout" caused by the mutations). This analysis is performed by calculating the change in the corresponding activation barriers without the need to invoke dynamical effects. We then generate the catalytic landscape of the enzyme and demonstrate that motions in the conformational space do not help drive catalysis. We also discuss the role of flexibility and conformational dynamics in catalysis, once again demonstrating that their role is negligible and that the largest contribution to catalysis arises from electrostatic preorganization. Finally, we point out that the changes in the reaction potential surface modify the reorganization free energy (which includes entropic effects), and such changes in the surface also alter the corresponding motion. However, this motion is never the reason for catalysis, but rather simply a reflection of the shape of the reaction potential surface.     

Converting structural information into an allosteric-energy-based picture for elongation factor Tu activation by the ribosome

The crucial process of aminoacyl-tRNA delivery to the ribosome is energized by the GTPase reaction of the elongation factor Tu (EF-Tu). Advances in the elucidation of the structure of the EF-Tu/ribosome complex provide the rare opportunity of gaining a detailed understanding of the activation process of this system. Here, we use quantitative simulation approaches and reproduce the energetics of the GTPase reaction of EF-Tu with and without the ribosome and with several key mutants. Our study provides a novel insight into the activation process. It is found that the critical H84 residue is not likely to behave as a general base but rather contributes to an allosteric effect, which includes a major transition state stabilization by the electrostatic effect of the P loop and other regions of the protein. Our findings have general relevance to GTPase activation, including the processes that control signal transduction.     

Laparoscopic Pyelolithotomy in a Pelvic Kidney: A Case Report and Review of the Literature

Background and Objectives:

Laparoscopic pyelolithotomy was performed in a pelvic kidney with a large renal pelvis calculus.

Methods and Results:

Laparoscopic pyelolithotomy was successfully performed in a pelvic kidney with an operative time of 310 minutes. The use of intraoperative fluoroscopy and a semi-automatic suturing device greatly facilitated the procedure. The patient''s operative pain was managed with 3 doses of ketorolac; she resumed a regular diet the day after surgery, and was discharged on the first postoperative day.

Conclusions:

For patients with a large stone in the renal pelvis of an ectopic kidney, laparoscopic pyelolithotomy provides an effective approach.     

Insulin resistance in patients with polycystic ovary syndrome is associated with elevated plasma homocysteine

BACKGROUND: Elevated levels of plasma homocysteine have recently been implicated as a significant risk factor for cardiovascular disease, pre-eclampsia, and recurrent pregnancy loss, and have been found to be associated with insulin resistance in a number of clinical situations. We examined the relationship between plasma homocysteine and insulin resistance in patients with polycystic ovary syndrome (PCOS). METHODS: A total of 155 infertile patients with PCOS as defined by clinical, biochemical and ultrasound criteria were screened for insulin resistance utilizing single-sample fasting insulin and glucose measurement, calculated by glucose:insulin ratio or homeostasis model assessment (HOMA) index. Total plasma homocysteine was measured by fluorescence polarization immunoassay. One hundred normo-ovulatory women with normal ovaries being treated for other infertility diagnoses served as a control group. RESULTS: Insulin resistance was found in the majority of PCOS patients: -53.5% (83/155), 60.6% (94/155) and 65.8% (102/155), when defined by fasting insulin, glucose:insulin ratio, or logHOMA respectively. Mean plasma homocysteine in the PCOS group was significantly higher than in the normal ovary group (11.5 +/- 7.4 versus 7.4 +/- 2.1 micromol/l, P < 0.001). Insulin-resistant PCOS patients had significantly higher plasma homocysteine (12.4 +/- 8.4 micromol/l) than non-insulin-resistant PCOS patients (9.6 +/- 4.4 micromol/l) regardless of body mass index (P = 0.003 by groups, P = 0.005 by correlation of single samples). Thirty-four per cent (53/155) of the PCO patients had homocysteine values >95th percentile of the controls (11.0 micromol/l, P < 0.0001). Statistically significant correlations were found between all insulin resistance indices and homocysteine levels. Multiple logistic regression defined insulin resistance as the major factor examined that influenced homocysteine levels. CONCLUSIONS: Insulin resistance and hyperinsulinaemia in patients with PCOS is associated with elevated plasma homocysteine, regardless of body weight. This finding may have important implications in the short term regarding reproductive performance, and in the long term regarding cardiovascular complications associated with insulin-resistant PCOS.     

Prevention of Pregnancy-Induced Hypertension in Twins by Early Administration of Low-Dose Aspirin: A Preliminary Report

PROBLEM: Effectiveness of early administered low-dose aspirin in prevention of pregnancy-induced hypertension (PIH) and fetal growth retardation in twin pregnancies was investigated in a randomized placebo controlled, double-blind trial in 47 twin pregnancies. METHOD: Twenty-four women received 100 mg of aspirin daily from mean gestational age of 17.7 wk, and 23 women ingested placebo from a mean gestational age of 18 wk until delivery. The placebo and aspirin group were similar in age, weight gain, zygosity, gravidity, parity, and obstetrical antecedents. Treatment lasted for a mean period of 16.8 wk and 18.3 wk in the placebo and aspirin groups, respectively. The mean gestational age at birth was 35.0 wk and 36.4 wk in the placebo and aspirin groups, respectively. RESULTS: PIH was noted in six women (26%) in the placebo group, but in only one woman (4%) in the aspirin treated patients (P<.05). The mean combined fetal weights of both twins, and the mean weight of the second twin at delivery were significantly higher in the aspirin treated mothers than in the placebo treated gravidas (mean difference of 781 g, P<.02 and mean difference of 488 g, P < .005, respectively). Intrauterine growth retardation (< 10th percentile) concerned 11 (24%) and six (13%) fetuses in the placebo and aspirin groups, respectively. No adverse effects of treatment to either the mothers or the infants were noted. CONCLUSION: Low-dose aspirin reduces the incidence of PIH and has a beneficial effect on fetal growth in twin pregnancies. Additional clinical trials are needed in order to define and select subgroups of twins where aspirin treatment is recommended.     

Exogenous Vimentin Supplementation Transiently Affects Early Steps during HPV16 Pseudovirus Infection

Understanding and modulating the early steps in oncogenic Human Papillomavirus (HPV) infection has great cancer-preventative potential, as this virus is the etiological agent of virtually all cervical cancer cases and is associated with many other anogenital and oropharyngeal cancers. Previous work from our laboratory has identified cell-surface-expressed vimentin as a novel HPV16 pseudovirus (HPV16-PsVs)-binding molecule modulating its infectious potential. To further explore its mode of inhibiting HPV16-PsVs internalisation, we supplemented it with exogenous recombinant human vimentin and show that only the globular form of the molecule (as opposed to the filamentous form) inhibited HPV16-PsVs internalisation in vitro. Further, this inhibitory effect was only transient and not sustained over prolonged incubation times, as demonstrated in vitro and in vivo, possibly due to full-entry molecule engagement by the virions once saturation levels have been reached. The vimentin-mediated delay of HPV16-PsVs internalisation could be narrowed down to affecting multiple steps during the virus’ interaction with the host cell and was found to affect both heparan sulphate proteoglycan (HSPG) binding as well as the subsequent entry receptor complex engagement. Interestingly, decreased pseudovirus internalisation (but not infection) in the presence of vimentin was also demonstrated for oncogenic HPV types 18, 31 and 45. Together, these data demonstrate the potential of vimentin as a modulator of HPV infection which can be used as a tool to study early mechanisms in infectious internalisation. However, further refinement is needed with regard to vimentin’s stabilisation and formulation before its development as an alternative prophylactic means.