Refinement by linkage analysis in two large families of the candidate region of the third locus (SCA3) for autosomal dominant cerebellar ataxia type I

The autosomal dominant cerebellar ataxias (ADCA) are clinically and genetically heterogeneous. To date, several loci (SCAI-V) have been identified for ADCA type I. We have studied two large families from the northern part of The Netherlands with ADCA type I with a broad intra-familial variation of symptoms. In both families significant linkage is shown of the disease to the markers of the SCA3 locus on chromosome 14. Through recombinations, the candidate region for SCA3 could be refined to a 13-cM range between D14S256 and D14S81. No recombinations were detected with the markers D14S291 and D14S280, which suggests that the SCA3 gene lies close to these loci. This finding will benefit the individuals at risk in these two families who are seeking predictive testing or prenatal diagnosis.     

Cytotoxicity mechanisms of pyrazino[1,2-b]isoquinoline-4-ones and SAR studies

The cytotoxicity showed by 1b, an interesting representant of the title compounds, for HT-29 human colon cancer cells (CI₅₀ value of 1.95 × 10^?7 M) has been related to the induced cell death at the G2 phase and not to DNA damage. This compound promotes the degradation of components of the G2/M checkpoint machinery, in particular cdc2, Cyclin B1 and Wee1, which represents a novel mechanism of cytotoxicity. Degradation of Wee1 seems to be mediated by proteasome activity but degradation of cdc2 has to occur through a different mechanism. The activity of 1b on G2 cell cycle components suggests that tumor cells that are arrested in G2/M by anticancer drugs like cisplatin could be targeted by compound 1b, increasing the apoptosis induction, and that their optimized analogs might be useful in the treatment of colon cancer through combination therapies with cisplatin or other anticancer drugs that affect the cytoskeleton integrity such as taxol and taxotere.SAR studies with compounds obtained by manipulation of the N(2) and C(4)-functional groups and the C(6)-chain of compound 1b have confirmed the importance of these structural features in the in vitro antitumor activity. Fused oxazolidine derivatives as compound 5 were inactive, and the lack of activity found in the replacement of the C(4)-lactam by a cyanoamine function, as in compounds 8–10, could be explained considering that their all-syn relative configuration makes them too stable to generate alkylating iminium species.     

Characterization of a Novel Thermostable Carboxylesterase from Geobacillus kaustophilus HTA426 Shows the Existence of a New Carboxylesterase Family

The gene GK3045 (741 bp) from Geobacillus kaustophilus HTA426 was cloned, sequenced, and overexpressed into Escherichia coli Rosetta (DE3). The deduced protein was a 30-kDa monomeric esterase with high homology to carboxylesterases from Geobacillus thermoleovorans NY (99% identity) and Geobacillus stearothermophilus (97% identity). This protein suffered a proteolytic cut in E. coli, and the problem was overcome by introducing a mutation in the gene (K212R) without affecting the activity. The resulting Est30 showed remarkable thermostability at 65°C, above the optimum growth temperature of G. kaustophilus HTA426. The optimum pH of the enzyme was 8.0. In addition, the purified enzyme exhibited stability against denaturing agents, like organic solvents, detergents, and urea. The protein catalyzed the hydrolysis of p-nitrophenyl esters of different acyl chain lengths, confirming the esterase activity. The sequence analysis showed that the protein contains a catalytic triad formed by Ser93, Asp192, and His222, and the Ser of the active site is located in the conserved motif Gly91-X-Ser93-X-Gly95 included in most esterases and lipases. However, this carboxylesterase showed no more than 17% sequence identity with the closest members in the eight families of microbial carboxylesterases. The three-dimensional structure was modeled by sequence alignment and compared with others carboxylesterases. The topological differences suggested the classification of this enzyme and other Geobacillus-related carboxylesterases in a new α/β hydrolase family different from IV and VI.Esterases catalyze hydrolysis and synthesis of ester bonds. Even if the biological functions have not been fully described, they have been involved in catabolic pathways (3, 5). Essentially, carboxylesterases (CEs; EC 3.1.1.1) exhibit high regio- and stereospecificity, require no cofactor, and are active in organic solvents, which make them attractive in important industrial and medical roles in the synthesis and hydrolysis of stereospecific compounds, including the metabolic processing of drugs and antimicrobial agents (4, 24, 29).Due to their importance, CEs have been identified in a wide range of organisms, and several of these have been cloned, including those from several Bacillus stearothermophilus strains (20) and from Pseudomonas sp. strain S34 (19). The elucidation of many gene sequences and the resolution of some crystal structures have permitted a structural classification of these enzymes in several families within the α/β-hydrolase fold family (2, 8).Esterases from thermophiles have become objects of special interest for structural investigation and for a broad range of biotechnological applications. CE and lipase properties and applications have been reviewed recently by Bornscheuer (5) and Jaeger et al. (14-16).In the search for new CEs, the gene GK3045 (741 bp) of Geobacillus kaustophilus HTA426 is of particular interest since this microorganism can grow at up to 74°C (optimally at 60°C). It was isolated from the deep-sea sediment of the Mariana Trench (41, 42) at a depth of 10,897 m. The complete genome sequence of this strain, which is composed of a 3.54-Mb chromosome and a 47.9-kb plasmid, has been determined as the first thermophilic bacillus (43).In this paper, we describe for the first time the cloning and characterization of a thermostable CE from G. kaustophilus HTA426 (CE_Gk). In addition, a plausible three-dimensional structure is proposed and compared with known structures.     

β-thymosins and interstitial lung disease: study of a scleroderma cohort with a one-year follow-up

Background

β-thymosins play roles in cytoskeleton rearrangement, angiogenesis, fibrosis and reparative process, thus suggesting a possible involvement in the pathogenesis of systemic sclerosis. The aim of the study was to investigate the presence of thymosins β₄, β₄ sulfoxide, and β₁₀ in bronchoalveolar lavage fluid of scleroderma patients with interstitial lung disease and the relation of these factors with pulmonary functional and radiological parameters.

Methods

β-thymosins concentrations were determined by Reverse Phase-High Performance Liquid Chromatography-Electrospray-Mass Spectrometry in the bronchoalveolar lavage fluid of 46 scleroderma patients with lung involvement and of 15 controls.

Results

Thymosin β_4, β₄ sulfoxide, and β₁₀ were detectable in bronchoalveolar lavage fluid of patients and controls. Thymosin β₄ levels were significantly higher in scleroderma patients than in controls. In addition, analyzing the progression of scleroderma lung disease at one-year follow-up, we have found that higher thymosin β₄ levels seem to have a protective role against lung tissue damage. Thymosin β₄ sulfoxide levels were higher in the smokers and in the scleroderma patients with alveolitis.

Conclusions

We describe for the first time β-thymosins in bronchoalveolar lavage fluid and their possible involvement in the pathogenesis of scleroderma lung disease. Thymosin β₄ seems to have a protective role against lung tissue damage, while its oxidation product mirrors an alveolar inflammatory status.     

Lipophilic Prodrugs and Formulations of Conventional (Deoxy)Nucleoside and Fluoropyrimidine Analogs in Cancer

Many drugs that are currently used for the treatment of cancer have limitations, such as induction of resistance and/or poor biological half-life, which reduce their clinical efficacy. To overcome these limitations, several strategies have been explored. Chemical modification by the attachment of lipophilic moieties to (deoxy)nucleoside analogs should enhance the plasma half-life, change the biodistribution, and improve cellular uptake of the drug. Attachment of a lipophilic moiety to a phosphorylated (deoxy)nucleoside analog will improve the activity of the drugs by circumventing the rate-limiting activation step of (deoxy)nucleoside analogs. Encapsulating drugs in nanoparticles or liposomes protects the drug against enzymatic breakdown in the plasma and makes it possible to get lipophilic compounds to the tumor site. In this review, we discuss the considerable progress that has been made in increasing the efficacy of classic (deoxy)nucleoside and fluoropyrimidine compounds by chemical modifications and alternative delivery systems.     

Gait retraining to reduce the knee adduction moment through real-time visual feedback of dynamic knee alignment

Varus knee alignment is a risk factor for medial knee osteoarthritis and is associated with high knee adduction moments. Therefore, reducing the knee adduction moment in varus-aligned individuals with otherwise healthy knees may reduce their risk for developing osteoarthritis. A gait modification that improves dynamic knee alignment may reduce the adduction moment, and systematic training may lead to more natural-feeling and less effortful execution of this pattern. To test these hypotheses, eight healthy, varus-aligned individuals underwent a gait modification protocol. Real-time feedback of dynamic knee alignment was provided over eight training sessions, using a fading paradigm. Natural and modified gait were assessed post-training and after 1 month, and compared to pre-training natural gait. The knee adduction moment, as well as hip adduction, hip internal rotation and knee adduction angles were evaluated. At each training session, subjects rated how effortful and natural-feeling the modified pattern was to execute. Post-training, the modified pattern demonstrated an 8° increase in hip internal rotation and 3° increase in hip adduction. Knee adduction decreased 2°, and the knee adduction moment decreased 19%. Natural gait did not differ between the three visits, nor did the modified gait pattern between the post-training and 1 month visits. The modified pattern felt more natural and required less effort after training. Based on these results, gait retraining to improve dynamic knee alignment resulted in significant reductions in the knee adduction moment, primarily through hip internal rotation. Further, systematic training led to more natural-feeling and less effortful execution of the gait pattern.