Structure-based design of inhibitors specific for bacterial thymidylate synthase

Thymidylate synthase is an attractive target for antiproliferative drug design because of its key role in the synthesis of DNA. As such, the enzyme has been widely targeted for anticancer applications. In principle, TS should also be a good target for drugs used to fight infectious disease. In practice, TS is highly conserved across species, and it has proven to be difficult to develop inhibitors that are selective for microbial TS enzymes over the human enzyme. Using the structure of TS from Lactobacillus casei in complex with the nonsubstrate analogue phenolphthalein, inhibitors were designed to take advantage of features of the bacterial enzyme that differ from those of the human enzyme. Upon synthesis and testing, these inhibitors were found to be up to 40-fold selective for the bacterial enzyme over the human enzyme. The crystal structures of two of these inhibitors in complex with TS suggested the design of further compounds. Subsequent synthesis and testing showed that these second-round compounds inhibit the bacterial enzyme at sub-micromolar concentrations, while the human enzyme was not inhibited at detectable levels (selectivities of 100-1000-fold or greater). Although these inhibitors share chemical similarities, X-ray crystal structures reveal that the analogues bind to the enzyme in substantially different orientations. Site-directed mutagenesis experiments suggest that the individual inhibitors may adopt multiple configurations in their complexes with TS.     

Pharmacological preconditioning of primary rat cardiac myocytes by FK506

Pre-treatment with the immunosuppressant FK506 is shown to protect primary cardiocytes against a subsequent severe thermal or ischaemic stress. This effect is not observed with the related compounds cyclosporin A or rapamycin. It does not involve induction of the FK506 binding, heat inducible protein hsp56 or of the other heat shock proteins. In addition over-expression of hsp56 does not protect cardiac cells from severe stress in contrast to our previous results with hsp70 and hsp90. These results suggest the FK506 is acting via a novel mechanism to protect cardiac cells against cellular ischaemia which may not be related to its immunosuppressant action.     

Cytotoxic activity of platelets and peripheral blood mononuclear cells from oncology patients and healthy donors

Testicular germ cell tumour is said to be a model for curable neoplasm. However, the prognosis of primary extragonadal germ cell tumour does not appear to be as promising. Though similar in histology, the biology of primary extragonadal germ cell tumour is different as exemplified by the patients in this review. Eight patients with primary mediastinal germ cell tumours were treated with intensive cisplatin-based chemotherapy. All, except one, had non-seminomatous components. The poor prognosis of mediastinal germ cell tumour is due to a combination of poor treatment results with the cisplatin-based regimen and the development of non-germ cell and haematological malignancies.     

Chain length of the polylysine in receptor-targeted gene transfer complexes affects duration of reporter gene expression both in vitro and in vivo

Complexes composed of peptide ligand for the serpin enzyme complex receptor covalently coupled to poly-L-lysine condensed by charge interaction with plasmid DNA direct gene transfer into receptor bearing cells. We compared intensity and duration of reporter gene expression in vitro and in vivo from serpin-enzyme receptor-directed gene transfer complexes prepared with poly-L-lysine of different chain lengths. When substituted with linker and ligand to comparable extents, DNA complexes containing short chain poly-L-lysine were larger and gave higher peak expression but significantly shorter duration of expression than those containing long chain poly-L-lysine. Both peak expression and duration of expression exceeded that observed with Lipofectin. Neither naked DNA nor DNA complexed with unsubstituted polylysine was effective in gene transfer. For in vivo experiments, complexes containing optimal ligand and degree of substitution (based on in vitro data, peptide C105Y, 11 ligands/plasmid DNA molecule) were prepared with either short chain or long chain polylysine and a beta-galactosidase expression plasmid. Following injection into the tail veins of mice, longer chain complexes gave significantly higher expression of reporter gene in lung and spleen that lasted for a significantly longer period of time than the shorter chain complexes. The short chain poly-L-lysine-DNA complexes were larger in diameter, as assessed by electron microscopy or atomic force microscopy, and gave less protection against DNase digestion in vitro than longer chain complexes. Thus, for gene transfer complexes directed at the serpin enzyme complex receptor, longer chain poly-L-lysine gave a much longer duration of expression both in vitro and in vivo. We speculate that this may be due to protection against degradation afforded the plasmid DNA by the tighter compaction produced by long chain poly-L-lysine.     

The asymmetric synthesis and in vitro characterization of succinyl mercaptoalcohol and mercaptoketone inhibitors of matrix metalloproteinases

A series of succinyl based mercaptoketones and diastereomeric mercaptoalcohols were prepared and evaluated in vitro as inhibitors of the matrix metalloproteinases collagenase-1 (MMP-1), stromelysin (MMP-3), and gelatinase-B (MMP-9).     

Differential regulation of discrete apoptotic pathways by Ras

The products of the ras genes are known to regulate cell proliferation and differentiation; recently, they have been found to play a role in apoptosis. The expression of oncogenic p21(ras) in a number of cell types, including Jurkat (a human T lymphoblastoid cell line) and murine fibroblasts, makes the cells susceptible to apoptosis following suppression of protein kinase C (PKC) activity (PKC/Ras-mediated apoptosis). Engagement of Fas antigen, a potent effector of apoptosis, activates cellular p21(ras), which may be required for completion of the cell death program. To further investigate the role of p21(ras) in the regulation of apoptosis, the cellular mechanisms employed in these two apoptotic processes in which Ras activity is involved (PKC/Ras-related and Fas-triggered apoptosis), was explored. Increasing p21(ras) activity by expressing v-ras or by treatment with an antisense oligonucleotide to the GTPase-activating protein was found to accelerate the Fas-mediated apoptotic process in Jurkat and mouse LF cells. PKC/Ras-related apoptosis was associated with, and required, cell cycle progression, accompanied by the expression of the G1/S cyclins. In contrast, Fas engagement, although inducing a vigorous and PKC-independent activation of endogenous p21(ras), did not alter cell cycle progression, nor did it require such progression for apoptosis. Both the protein synthesis inhibitor cycloheximide and cyclin E antisense oligonucleotides partially abolished PKC/Ras-mediated apoptosis but had only a moderate effect on Fas-induced apoptosis. In contrast, the CED-3/interleukin-1beta-converting enzyme (ICE) protease inhibitor Z-VADfmk efficiently suppressed Fas-induced apoptosis and only marginally inhibited PKC/Ras-mediated apoptosis. Induction of both pathways resulted in activation of the Jun NH2-terminal kinase/JUN signaling system. These results suggest that different cell death programs, such as PKC/Ras-mediated and Fas-mediated apoptosis, may be interconnected via p21(ras) and perhaps Jun NH2-terminal kinase/JUN. In response to various death stimuli, p21(ras) may act as a common intermediate regulator in the transduction of apoptotic signals.     

Asymptomatic myocardial ischemia before and after surgical revascularization in patients with multivessel occlusive coronary disease

INTRODUCTION: The objective of this study was to examine the occurrence of asymptomatic myocardial ischemia prior to and after myocardial revascularization in patients with multivessel occlusive coronary disease. Asymptomatic ischemia can be described as real ischemia without anginal pain or other ischemic symptoms in patients with coronary disease or coronary artery spasm. Our study examined silent ischemia after myocardial revascularization. Early detection of silent ischemia is important for prevention of cardiac incidents. MATERIAL AND METHODS: We have examined patients with multivessel coronary disease with occurrence of continued preoperative silent ischemia. All patients have undergone ECG examination, exercise stress test and Holter-monitoring prior to and after myocardial revascularization. RESULTS: The investigation comprised 27 patients and their average age was 54.5 years. All patients with silent ischemia had a multivessel occlusive coronary disease and have undergone myocardial revascularization managed with triple or quadruple aortocoronary bypass surgery. Exercise stress test was performed postoperatively in elder patients, as well as ECG and Holter-monitoring. Silent ischemia was established in 21.6% of patients, while in 87.5% untreated diabetes mellitus was diagnosed. Silent ischemia most often occurred in the early morning hours and it was frequently associated with heart rhythm disturbances (VES) whereas these rhythm disturbances depended on the length of the ischemic episode. Intermittent 2nd degree atrioventricular block was found in one patient. CONCLUSION: Silent myocardial ischemia occurred in 21% of patients after myocardial revascularization. It is most often detected in the early morning hours and is associated with ventricular rhythm disorders. Silent ischemia is easily detected by simple examination procedures providing adequate therapy and prevention of cardiac incidents.     

The clinical complexity of splenic vein thrombosis

Upper gastrointestinal hemorrhage secondary to splenic vein thrombosis is a curable form of localized portal hypertension when treated with splenectomy. A high index of suspicion is necessary in order to promptly diagnose and treat this underrecognized condition that is most commonly caused by inflammation or neoplasm of the pancreas. The triad of isolated gastric varices, splenomegaly, and normal hepatic function is classic; it is not uncommon, however, for patients to have only some or even none of these conditions. Mesenteric angiography with venous phase imaging is the gold standard of diagnosis. Ultrasound and CT may identify splenic vein thrombosis, but are most helpful in delineating concomitant upper abdominal pathology. Early recognition and intervention allow associated underlying conditions to be treated under the same anesthetic with minimal morbidity and mortality.     

The development and morphofunctional characteristics of the human lateral pterygoid muscle

Peculiarities of the development of superior and inferior heads of lateral pterygoid muscle (LPM) as well as their micro- and ultramicroscopic structure were studied as related to the temporomandibular joint elements in human fetuses and newborns. As a result, heterogeneity and asynchronism of the development of myogenic elements of certain LPM heads were demonstrated. The process of muscle fibres differentiation in LPM superior head leads to more mature structural organization and runs faster than in the inferior head. By the moment of birth average section area of superior head muscle fibres is significantly higher than the inferior head similar parameter.