Discordant Effects of Putative Lysine Acetyltransferase Inhibitors in Biochemical and Living Systems.

Lysine acetyltransferases (KATs) are exquisitely fine-tuned to target specific lysine residues on many proteins, including histones, with aberrant acetylation at distinct lysines implicated in different pathologies. However, researchers face a lack of molecular tools to probe the importance of site-specific acetylation events in vivo. Because of this, there can be a disconnect between the predicted in silico or in vitro effects of a drug and the actual observable in vivo response. We have previously reported on how an in vitro biochemical analysis of the site-specific effects of the compound C646 in combination with the KAT p300 can accurately predict changes in histone acetylation induced by the same compound in cells. Here, we build on this effort by further analyzing a number of reported p300 modulators, while also extending the analysis to correlate the effects of these drugs to developmental and phenotypical changes, utilizing cellular and zebrafish model systems. While this study demonstrates the utility of biochemical models as a starting point for predicting in vivo activity of multi-site targeting KATs, it also highlights the need for the development of new enzyme inhibitors that are more specific to the regulation of KAT activity in vivo.

The allosteric mechanism of substrate-specific transport in SLC6 is mediated by a volumetric sensor.

Neurotransmitter:sodium symporters (NSSs) in the SLC6 family terminate neurotransmission by coupling the thermodynamically favorable transport of ions to the thermodynamically unfavorable transport of neurotransmitter back into presynaptic neurons. Results from many structural, functional, and computational studies on LeuT, a bacterial NSS homolog, have provided critical insight into the mechanism of sodium-coupled transport, but the mechanism underlying substrate-specific transport rates is still not understood. We present a combination of molecular dynamics simulations, single-molecule fluorescence resonance energy transfer (smFRET) imaging, and measurements of Na+ binding and substrate transport that reveals an allosteric substrate specificity mechanism. In this mechanism, residues F259 and I359 in the substrate binding pocket couple the binding of substrate to Na+ release from the Na2 site by allosterically modulating the stability of a partially open, inward-facing state. We propose a model for transport selectivity in which residues F259 and I359 act as a volumetric sensor that inhibits the transport of bulky amino acids.

Synthesis, kinetics and inhibition of Escherichia coli Heptosyltransferase I by monosaccharide analogues of Lipid A.

Gram-negative bacteria comprise the majority of microbes that cause infections that are resistant to pre-existing antibiotics. The complex cell wall architecture contributes to their ability to form biofilms, which are often implicated in hospital-acquired infections. Biofilms promote antibiotic resistance by enabling the bacteria to survive hostile environments such as UV radiation, pH shifts, and antibiotics. The outer membrane of Gram-negative bacteria contains lipopolysaccharide (LPS), which plays a role in adhesion to surfaces and formation of biofilms. The main focus of this work was the synthesis of a library of glycolipids designed to be simplified analogues of the Lipid A, the membrane embedded portion component of LPS, to be tested as substrates or inhibitors of Heptosyltransferase I (HepI or WaaC, a glycosyltransferase enzyme involved in the biosynthesis of LPS). Fourteen analogues were synthesized successfully and characterized. While these compounds were designed to function as nucleophilic substrates of HepI, they all demonstrated mild inhibition of HepI. Kinetic characterization of inhibition mechanism identified that the compounds exhibited uncompetitive and mixed inhibition of HepI. Since both uncompetitive and mixed inhibition result in the formation of an Enzyme-Substrate-inhibitor complex, molecular docking studies (using AutoDock Vina) were performed, to identify potential allosteric binding site for these compounds. The inhibitors were shown to bind to a pocket formed after undergoing a conformational change from an open to a closed active site state. Inhibition of HepI via an allosteric site suggest that disruption of protein dynamics might be a viable mechanism for the inhibition of HepI and potentially other enzymes of the GT-B structural class.

The Stories Tryptophans Tell: Exploring Protein Dynamics of Heptosyltransferase I from Escherichia coli.

Heptosyltransferase I (HepI) catalyzes the addition of l-glycero-ß-d-manno-heptose to Kdo2-Lipid A, as part of the biosynthesis of the core region of lipopolysaccharide (LPS). Gram-negative bacteria with gene knockouts of HepI have reduced virulence and enhanced susceptibility to hydrophobic antibiotics, making the design of inhibitors of HepI of interest. Because HepI protein dynamics are partially rate-limiting, disruption of protein dynamics might provide a new strategy for inhibiting HepI. Discerning the global mechanism of HepI is anticipated to aid development of inhibitors of LPS biosynthesis. Herein, dynamic protein rearrangements involved in the HepI catalytic cycle were probed by combining mutagenesis with intrinsic tryptophan fluorescence and circular dichroism analyses. Using wild-type and mutant forms of HepI, multiple dynamic regions were identified via changes in Trp fluorescence. Interestingly, Trp residues (Trp199 and Trp217) in the C-terminal domain (which binds ADP-heptose) are in a more hydrophobic environment upon binding of ODLA to the N-terminal domain. These residues are adjacent to the ADP-heptose binding site (with Trp217 in van der Waals contact with the adenine ring of ADP-heptose), suggesting that the two binding sites interact to report on the occupancy state of the enzyme. ODLA binding was also accompanied by a significant stabilization of HepI (heating to 95 °C fails to denature the protein when it is in the presence of ODLA). These results suggest that conformational rearrangements, from an induced fit model of substrate binding to HepI, are important for catalysis, and the disruption of these conformational dynamics may serve as a novel mechanism for inhibiting this and other glycosyltransferase enzymes.

Effects of over-the-counter analgesic use on reproductive hormones and ovulation in healthy, premenopausal women.

STUDY QUESTION: Does use of commonly used over-the-counter (OTC) pain medication affect reproductive hormones and ovulatory function in premenopausal women? SUMMARY ANSWER: Few associations were found between analgesic medication use and reproductive hormones, but use during the follicular phase was associated with decreased odds of sporadic anovulation after adjusting for potential confounders. WHAT IS KNOWN ALREADY: Analgesic medications are the most commonly used OTC drugs among women, but their potential effects on reproductive function are unclear. STUDY DESIGN, SIZE, DURATION: The BioCycle Study was a prospective, observational cohort study (2005-2007) which followed 259 women for one (n = 9) or two (n = 250) menstrual cycles. PARTICIPANTS, SETTING, METHODS: Two hundred and fifty-nine healthy, premenopausal women not using hormonal contraception and living in western New York state. Study visits took place at the University at Buffalo. MAIN RESULTS AND THE ROLE OF CHANCE: During study participation, 68% (n = 175) of women indicated OTC analgesic use. Among users, 45% used ibuprofen, 33% acetaminophen, 10% aspirin and 10% naproxen. Analgesic use during the follicular phase was associated with decreased odds of sporadic anovulation after adjusting for age, race, body mass index, perceived stress level and alcohol consumption (OR 0.36 [0.17, 0.75]). Results remained unchanged after controlling for potential confounding by indication by adjusting for 'healthy' cycle indicators such as amount of blood loss and menstrual pain during the preceding menstruation. Moreover, luteal progesterone was higher (% difference = 14.0, -1.6-32.1, P = 0.08 adjusted) in cycles with follicular phase analgesic use, but no associations were observed with estradiol, LH or FSH. LIMITATIONS, REASONS FOR CAUTION: Self-report daily diaries are not validated measures of medication usage, which could lead to some classification error of medication use. We were also limited in our evaluation of aspirin and naproxen which were used by few women. WIDER IMPLICATIONS OF THE FINDINGS: The observed associations between follicular phase analgesic use and higher progesterone and a lower probability of sporadic anovulation indicate that OTC pain medication use is likely not harmful to reproduction function, and certain medications possibly improve ovulatory function. STUDY FUNDING/COMPETING INTERESTS: This work was supported by the Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health (contract # HHSN275200403394C). The authors have no conflicts of interest to disclose.

[Short stature treatment by lower limb lengthening--multicenter study from five centers]. / Leczenie niskiego wzrostu metoda Ilizarowa--zbiorcza analiza wyników z pieciu osrodków w Polsce.

MATERIAL: 26 patients (17 female, 9 male) from 5 centers were evaluated. The age at the beginning of treatment ranged from 6 to 29 years (mean 13.8). The cause of short stature in 19 patients was achondroplasia or pseudoachondroplasia, in next 2--other bone dysplasias. The other 5 patients had not bone pathology and were treated because of cosmetic indications. Preoperative body height ranged from 90 to 149 cm (mean 120). Axial deviations of the lower extremities were noted in 11 patients. Mean follow-up was 3.7 years. METHOD OF TREATMENT: Most of patients were treated with Ilizarov device using cross lengthening strategy (2 stages--opposite femur and tibia lengthening). Mean duration of treatment including interval between two stages (mean 12 months) was 29 months. Planned increase of body height ranged from 10 to 26 cm (mean 16.4). RESULTS: Planned or greater lengthening (mean 14.8 cm) was achieved in 14 patients. Partial planned lengthening (mean 65% of planned lengthening) was achieved in 8 patients (mean 11.8 cm) including two patients who resigned the second stage of treatment. In two patients lengthening was stopped during first month of treatment because of great complications. In 2 patients treatment was not completed (interval between first and second stage). Mean increase of body height of patients with complete treatment was 13.1 cm (from 2 to 28). Problems, obstacles and complications were analyzed according to Paley classification. PROBLEMS: There were 24 problems in 15 patient (inflammation process around K wires--15 patients, bone healing disturbances--3, regenerate fracture--2, transient foot equinus--2 and axial deviation of the lower extremity--1). OBSTACLES: There were 31 obstacles in 19 patients (regenerate's defect--7 patients, premature bone consolidation--6, foot equinus--4 and other--14). COMPLICATIONS: There were 26 complications in 18 patients (axial deviation of the lengthened segment--8, foot equinus--6, paresis of the peroneal nerve--3, fractures--2 and other--5). The most serious complication was hemiparesis after cerebral embolism (1 patient) and damaging of the femoral artery (1 patient) both disrupting bone lengthening. CONCLUSION: The risk of complication in surgical treatment of short stature patients is high. Qualification for short stature treatment because of cosmetic indication should be made very careful and after precise psychological and/or psychiatric investigation.

Growth factors and gene therapy: the prospects for treating limited cartilage defects.

The limited regenerative capacities of articular are well known. Among the many methods used to repair the joint surface, among the most innovative involve stimulating chondrocytes to grow and differentiate by local application of exogenous protein growth factors, and attempting to transfer the genes responsible for cell division and maturation. This article presents the current state of knowledge on the possibilities for the treatment of limited cartilage defects used these methods.

Ilizarow's external fixator in clinical use. Ten years of using the method at the Department of Orthopedics of Warsaw Medical University.

The Ilizarow's stabilizer is characterized by large functionality which enables performance of lengthening, compression, rotational and angular correction and also stabilization of joints. Between the years 1989 and 1998 at Orthopedic Department of Warsaw Medical University Ilizarow's device was used in the treatment of 410 patients during which 484 surgical procedures were performed. The most common among operations were elongations of limbs and treatment of pseudoarthroses. Authors present their own remarks and experience from over ten years using Ilizarow's method.

["Taurolin-Gel" and Taurolin-Trockengel" in treatment of osseous tissue inflammation]. / "Taurolin--Gel" i "Taurolin--Trockengel" w leczeniu zapalenia tkanki kostnej. Doniesienie wstepne.

The activity and principles of administration of Taurolin have been presented. That drug was used in 15 patients with chronic osteitis. Healing by first intention was obtained in 10 patients, necrosis of the skin occurred in 3 patients, recurrence of inflammation was found in one patient in whom accessory sequestroctomy was performed in the last case of osteitis and arteriosclerosis healing was obtained by second intention. The clinical observation of 15 patients has revealed: a) irritating influence of Taurolin on the soft tissue, b) increased phase of secretion of exudate since the 5 day after surgery, c) bactericidal influence of the drug. Sterilization of the focus occurred at 2 to 3 weeks after surgery. The use of Taurolin requires experience, complete abiding by the principles of use of this drug and equipment with a set of Charrier's drains. The authors are carrying on further observation of the use of Taurolin in the treatment of osteitis.