The additivity of substrate fragments in enzyme-ligand binding

BACKGROUND: Enzymes have evolved to recognise their target substrates with exquisite selectivity and specificity. Whether fragments of the substrate--perhaps never available to the evolving enzyme--are bound in the same manner as the parent substrate addresses the fundamental basis of specificity. An understanding of the relative contributions of individual portions of ligand molecules to the enzyme-binding interaction may offer considerable insight into the principles of substrate recognition. RESULTS: We report 12 crystal structures of Escherichia coli thymidylate synthase in complexes with available fragments of the substrate (dUMP), both with and without the presence of a cofactor analogue. The structures display considerable fidelity of binding mode and interactions. These complexes reveal several interesting features: the cofactor analogue enhances the localisation of substrate and substrate fragments near the reactive thiol; the ribose moiety reduces local disorder through additional specific enzyme-ligand interactions; the pyrimidine has multiple roles, ranging from stereospecificity to mechanistic competence; and the glycosidic linkage has an important role in the formation of a covalent attachment between substrate and enzyme. CONCLUSIONS: The requirements of ligand-protein binding can be understood in terms of the binding of separate fragments of the ligand. Fragments which are subsystems of the natural substrate for the enzyme confer specific contributions to the binding affinity, orientation or electrostatics of the enzymatic mechanism. This ligand-binding analysis provides a complementary method to the more prevalent approaches utilising site-directed mutagenesis. In addition, these observations suggest a modular approach for rational drug design utilising chemical fragments.     

Peritraumatic dissociation and posttraumatic stress in male Vietnam theater veterans

OBJECTIVE: The aim of this study was to determine the reliability and validity of a proposed measure of peritraumatic dissociation and, as part of that effort, to determine the relationship between dissociative experiences during disturbing combat trauma and the subsequent development of posttraumatic stress disorder (PTSD). METHOD: A total of 251 male Vietnam theater veterans from the Clinical Examination Component of the National Vietnam Veterans Readjustment Study were examined to determine the relationship of war zone stress exposure, retrospective reports of dissociation during the most disturbing combat trauma events, and general dissociative tendencies with PTSD case determination. RESULTS: The total score on the Peritraumatic Dissociation Experiences Questionnaire--Rater Version was strongly associated with level of posttraumatic stress symptoms, level of stress exposure, and general dissociative tendencies and weakly associated with general psychopathology scales from the MMPI-2. Logistic regression analyses supported the incremental value of dissociation during trauma, over and above the contributions of level of war zone stress exposure and general dissociative tendencies, in accounting for PTSD case determination. CONCLUSIONS: These results provide support for the reliability and validity of the Peritraumatic Dissociation Experiences Questionnaire--Rater Version and for a trauma-dissociation linkage hypothesis: the greater the dissociation during traumatic stress exposure, the greater the likelihood of meeting criteria for current PTSD.     

Involvement of regional lymph nodes after penetration of Schistosoma mansoni cercariae in naive and infected mice

The very low density lipoprotein receptor (VLDL-r) is a cell-surface molecule specialized for the internalization of multiple diverse ligands, including apolipoprotein E (apoE)-containing lipoprotein particles, via clathrin-coated pits. Its structure is similar to the low-density lipoprotein receptor (LDL-r), although the two have substantially different systemic distributions and regulatory pathways. The present work examines the distribution of VLDL-r in the central nervous system (CNS) and in relation to senile plaques in Alzheimer disease (AD). VLDL-r is present on resting and activated microglia, particularly those associated with senile plaques (SPs). VLDL-r immunoreactivity is also found in cortical neurons. Two exons of VLDL-r mRNA are differentially spliced in the mature receptor mRNA. One set of splice forms gives rise to receptors containing (or lacking) an extracellular O-linked glycosylation domain near the transmembrane portion of the molecule. The other set of splice forms appears to be brain-specific, and is responsible for the presence or absence of one of the cysteine-rich repeat regions in the binding region of the molecule. Ratios of the receptor variants generated from these splice forms do not differ substantially across different cortical areas or in AD. We hypothesize that VLDL-r might contribute to metabolism of apoE and apoE/A beta complexes in the brain. Further characterizations of apoE receptors in Alzheimer brain may help lay the groundwork for understanding the role of apoE in the CNS and in the pathophysiology of AD.     

Non-hierarchical logistic models and case-only designs for assessing susceptibility in population-based case-control studies

Mechanisms for the abnormal copper (Cu) accumulation in the liver of LEC rats were examined using primary cultured liver parenchymal cells prepared from mutant LEC rats and those from control LEA rats (original strain). The Cu and metallothionein (MT) mRNA levels in the liver of LEC rats were caused to decrease to the same levels as those of LEA rats by removing Cu in vivo selectively with tetrathiomolybdate. Cu was taken up by LEC rat cells to the same extent as LEA rat cells by exposure to low medium Cu and to a higher extent by exposure to high medium Cu, while the MT mRNA level in LEC rat cells increased dose-dependently at a much higher rate than that in LEA rats. MT mRNA levels in both cells were comparable by exposure to cadmium, zinc and dexamethasone. The results indicate that expression of MT mRNA is selectively enhanced by Cu in LEC cells despite the fact that uptake of Cu is comparable with normal cells.     

New energy transfer dyes for DNA sequencing

We have synthesized a set of four energy transfer dyes and demonstrated their use in automated DNA sequencing. The donor dyes are the 5- or 6-carboxy isomers of 4'-aminomethylfluorescein and the acceptor dyes are a novel set of four 4,7-dichloro-substituted rhodamine dyes which have narrower emission spectra than the standard, unsubstituted rhodamines. A rigid amino acid linker, 4-aminomethylbenzoic acid, was used to separate the dyes. The brightness of each dye in an automated sequencing instrument equipped with a dual line argon ion laser (488 and 514 nm excitation) was 2-2.5 times greater than the standard dye-primers with a 2 times reduction in multicomponent noise. The overall improvement in signal-to-noise was 4- to 5-fold. The utility of the new dye set was demonstrated by sequencing of a BAC DNA with an 80 kb insert. Measurement of the extinction coefficients and the relative quantum yields of the dichlororhodamine components of the energy transfer dyes showed their values were reduced by 20-25% compared with the dichlororhodamine dyes alone.     

Post-traumatic stress disorder and functioning and quality of life outcomes in female Vietnam veterans

OBJECTIVE: This investigation assessed whether current post-traumatic stress disorder (PTSD) was associated with impaired functioning in a nationally representative sample of female Vietnam veterans. METHODS: Logistic models were used to determine the association between PTSD and outcome while adjusting for demographic characteristics and medical and psychiatric co-morbidities. RESULTS: PTSD was associated with significantly elevated odds of poorer functioning in five of the six outcome domains; only the association between perpetration of violence in the past year and PTSD did not achieve statistical significance. After adjusting for demographics and medical and psychiatric co-morbidities, PTSD remained associated with significantly elevated odds of bed days, poorer physical health, and currently not working. CONCLUSIONS: Among female Vietnam veterans PTSD is associated with a broad profile of functional impairment. The significantly increased odds of impaired functioning and diminished quality of life suggest that PTSD may be the core problem of the set of problems afflicting female Vietnam veterans.     

Subanesthetic concentrations of isoflurane suppress learning as defined by the category-example task

It has been shown that chronic oral steroid therapy (ST) does not induce respiratory muscle dysfunction in normal and asthmatic subjects. As corticosteroids are sometimes chronically used in the treatment of the patients with chronic obstructive pulmonary disease (COPD), the aim of our study was to verify whether ST could cause respiratory muscle impairment and, since ST also affects the central nervous system, whether ST could influence the ventilatory pattern. We retrospectively studied 12 COPD patients (group A), on long-term therapy (for at least 4 consecutive months, range 4-18 months) with an oral steroid, deflazacort, 15 mg.d-1. The subjects were strictly matched, with regard to age, sex, height, weight, forced expiratory volume in one second (FEV1), residual volume (RV), arterial oxygen tension (PaCO2), arterial carbon dioxide tension (PaCO2) and pH, with 12 COPD patients (Group B) who had never taken oral steroids. To assess respiratory muscle strength, we measured maximal inspiratory (MIP) and expiratory (MEP) pressures, while mouth occlusion pressure (P0.1) was employed to assess neuromuscular drive; ventilatory pattern and airway impedence were also evaluated. Effectiveness of ST was confirmed by the plasmatic levels of endogenous cortisol. No significant differences were observed between the two groups with regard to MIP (A 72.2 +/- 9.7 vs B: 70 +/- 7.2 cmH2O) and MEP (A 91.6 +/- 10.5 vs B 94.4 +/- 7.6 cmH2O) whilst P0.1 was significantly higher in group A (2.6 +/- 0.3 cmH2O) than in group B (1.8 +/- 0.1 cmH2O). No significant differences were found among all the ventilatory parameters, but the impedence was significantly higher in group A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Localization by immunoelectron microscopy of Schistosoma mansoni antigens in the glomerulus of the hamster (Mesocricetus auratus) kidney

Gene therapy has the potential to provide cancer treatments based on novel mechanisms of action with potentially low toxicities. This therapy may provide more effective control of loco-regional recurrence in diseases such as non-small cell lung cancer (NSCLC), as well as systemic control of micrometastases. Despite current limitations, retroviral and adenoviral vectors can in certain circumstances provide an effective means of delivering therapeutic genes to tumour cells. Although multiple genes are involved in the process of carcinogenesis, mutations of the p53 gene are the most frequent abnormality identified in human tumours. Pre-clinical studies both in vitro and in vivo have shown that restoration of p53 function can induce apoptosis in cancer cells. Phase I clinical trials now show that p53 gene replacement therapy is feasible and safe using both retroviral and adenoviral vectors, and that it induces tumour regression in patients with advanced NSCLC and recurrent head and neck cancer. Other pre-clinical studies indicate that gene therapy may have useful synergy with cytotoxic and radiation therapy. This paper describes the different gene therapy strategies under investigation and the pre-clinical data that provides a rationale for the gene replacement approach, reviews clinical trial data and presents novel ideas for improving current vectors and gene delivery to tumours.     

Interdisciplinary collaboration and discharge planning communication for elders

The effects of personal characteristics and perceptions of interdisciplinary collaboration on discharge planning communication were examined for nurses, physicians, and social workers in two hospitals. The model for the study explained 61.7% of the variance in discharge planning communication for nurses. For all 142 health professionals, communication openness with social workers, problem solving between nurses and physicians, and collaboration with social workers were important to discharge planning communication. For nurses, communication satisfaction with patients and families also was important.