Organ perfusion and mass spectrometry: a timely merger for drug development

Organ perfusion techniques bridge the methodological gap between in vivo studies on the one hand and in vitro studies on the other. In drug candidate selection and subsequent development the differences between these systems should be considered carefully in study design, as one approach may be more suitable than the other depending on the question(s) being asked and, in particular, how the data will be used. This article is not concerned with the mechanics, the surgery or composition of perfusates as there are numerous reviews/books covering these aspects. Instead, using perfused gut, liver, lung, kidney and brain as examples, the emphasis is on the usefulness (or otherwise) of the data generated with respect to drug absorption, metabolism, pharmacokinetics (PK) and the factors which affect these parameters. Perfusion systems are not difficult to set up but do require 'high maintenance' for routine use. For this reason they have been used sparingly by the pharmaceutical industry mainly for problem solving or mechanistic studies. The latter part of this article shows how simultaneous dosing of numerous compounds followed by multiple--component analysis using LC/MS/MS has proved to be an effective way to improve the throughput of absorption, pharmacokinetics and metabolism screening ex vivo.     

Cloning and characterization of the bleomycin biosynthetic gene cluster from Streptomyces verticillus ATCC15003

Bleomycin (BLM) biosynthesis has been studied as a model for hybrid peptide-polyketide natural product biosynthesis. Cloning, sequencing, and biochemical characterization of the blm biosynthetic gene cluster from Streptomyces verticillus ATCC15003 revealed that (1) the BLM hybrid peptide-polyketide aglycon is assembled by the BLM megasynthetase that consists of both nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) modules; (2) BlmIX/BlmVIII/BlmVII constitute a natural hybrid NRPS/PKS/NRPS system, serving as a model for both hybrid NRPS/PKS and PKS/NRPS systems; (3) the catalytic sites appear to be conserved in both hybrid NRPS/PKS and nonhybrid NRPS or PKS systems, with the exception of the KS domains in the hybrid NRPS/PKS systems that are unique; (4) specific interpolypeptide linkers may play a critical role in intermodular communication to facilitate the transfer of the growing intermediates between the interacting NRPS and/or PKS modules; (5) post-translational modification of the BLM megasynthetase has been accomplished by a single PPTase with broad carrier protein specificity; and (6) BlmIV/BlmIII-templated assembly of the BLM bithiazole moiety requires intriguing protein juxtaposition and modular recognition. These results lay the foundation to investigate the molecular basis for intermodular communication between NRPS and PKS in hybrid peptide-polyketide natural product biosynthesis and set the stage for engineering novel BLM analogues by genetic manipulation of genes governing BLM biosynthesis.     

Tec regulates platelet activation by GPVI in the absence of Btk

The Tec family kinase Btk plays an important role in the regulation of phospholipase C gamma 2 (PLC gamma 2) downstream of the collagen receptor glycoprotein VI (GPVI) in human platelets. Platelets also express a second member of this family, Tec; however, its function has not been analyzed. To address the role of Tec, we analyzed Btk-/-, Tec-/-, and Btk/Tec double-deficient (Btk-/-/Tec-/-) platelets. Tec-/- platelets exhibit a minor reduction in aggregation to threshold concentrations of collagen or the GPVI-specific agonist collagen-related peptide (CRP), whereas responses to higher concentrations are normal. Tyrosine phosphorylation of PLC gamma 2 by collagen and CRP is not altered in Tec-/- platelets. However, Btk-/-/Tec-/- platelets exhibit a greater reduction in PLC gamma 2 phosphorylation than is seen in the absence of Btk, thus revealing an important role for Tec in this situation. Furthermore, Btk-/-/Tec-/- platelets fail to undergo an increase in Ca2+, aggregation, secretion, and spreading in response to collagen or CRP, whereas they aggregate normally to adenosine diphosphate (ADP) and spread on fibrinogen. A residual GPVI signal exists in the Btk-/-/Tec-/- platelets as CRP synergizes with ADP to mediate aggregation. These results demonstrate an essential requirement for Tec and Btk in platelet activation by GPVI and reveal a functional role for Tec in the regulation of PLC gamma 2 in the absence of Btk.     

Investigation of white matter structure in velocardiofacial syndrome: a diffusion tensor imaging study

OBJECTIVE: Velocardiofacial syndrome, caused by a deletion on chromosome 22q11.2, is often accompanied by cognitive, behavioral, and psychiatric impairments. Specifically, velocardiofacial syndrome has been proposed as a disease model for a genetically mediated subtype of schizophrenia. Velocardiofacial syndrome is also known to affect brain structure. The most prominent structural findings in velocardiofacial syndrome are reduced white matter volumes. However, the structure of white matter and extent of specific regional involvement in this syndrome have never been investigated. The current study used diffusion tensor imaging to investigate white matter structure in children and young adults with velocardiofacial syndrome. METHOD: Nineteen participants with velocardiofacial syndrome and 19 age- and gender-matched comparison subjects underwent diffusion-weighted magnetic resonance imaging scans. Whole brain voxel-by-voxel analyses were conducted to investigate white matter fractional anisotropy differences between the groups. RESULTS: Relative to the comparison group, the velocardiofacial syndrome group had reduced white matter anisotropy in the frontal, parietal, and temporal regions as well as in tracts connecting the frontal and temporal lobes. CONCLUSIONS: This study demonstrates that alterations of white matter tract structure occur in velocardiofacial syndrome. Reduced white matter anisotropy was observed in individuals with velocardiofacial syndrome in areas previously implicated in the neurocognitive phenotype of velocardiofacial syndrome. The finding of aberrant parietal white matter tracts as well as aberrant frontotemporal connectivity in velocardiofacial syndrome and in previous schizophrenia studies may be associated with increased vulnerability for development of psychotic symptoms.     

Architectonics and cortical connections of the upper bank of the superior temporal sulcus in the rhesus monkey: an analysis in the tangential plane

Area TPO in the upper bank of the superior temporal sulcus (STS) of macaque monkeys is thought to correspond to the superior temporal polysensory (STP) cortex, but has been shown to have neurochemical/connectional subdivisions. To examine directly the relationship between chemoarchitecture and cortical connections of area TPO, the upper bank of the STS was sectioned tangential to the cortical surface. Three subdivisions of area TPO (TPOr, TPOi, and TPOc) were examined with cytochrome oxidase (CO) histochemistry and neurofilament protein (NF) immunoreactivity and architectonic patterns were compared with connections on the same or adjacent sections. Area TPOc, which may partly overlap with the location of the medial superior temporal area MST, exhibited regular patchy staining for CO in layers III/IV and a complementary pattern in the NF stain. Area TPOr, but not TPOi, also had a patchy pattern of complementary staining in CO and neurofilament similar to TPOc, although not as distinct. Tracer injections within cortex including the frontal eye fields (areas 46 and 8) labeled areas TPOc, TPOi, and TPOr. The caudal inferior parietal lobule (IPL) projected to all three areas. The projections from prearcuate and posterior parietal cortices showed both overlap and nonoverlap with each other within TPOc, TPOi, and TPOr. Projections were to all neurochemical components within the subdivisions of TPO. The findings support the parcellation of area TPO into three subdivisions and extend findings of chemoarchitectonic modules within high-order association cortices.     

Integrin beta cytoplasmic domain interactions with phosphotyrosine-binding domains: a structural prototype for diversity in integrin signaling

The cytoplasmic domains (tails) of heterodimeric integrin adhesion receptors mediate integrins' biological functions by binding to cytoplasmic proteins. Most integrin beta tails contain one or two NPXYF motifs that can form beta turns. These motifs are part of a canonical recognition sequence for phosphotyrosine-binding (PTB) domains, protein modules that are present in a wide variety of signaling and cytoskeletal proteins. Indeed, talin and ICAP1-alpha bind to integrin beta tails by means of a PTB domain-NPXY ligand interaction. To assess the generality of this interaction we examined the binding of a series of recombinant PTB domains to a panel of short integrin beta tails. In addition to the known integrin-binding proteins, we found that Numb (a negative regulator of Notch signaling) and Dok-1 (a signaling adaptor involved in cell migration) and their isolated PTB domain bound to integrin tails. Furthermore, Dok-1 physically associated with integrin alpha IIb beta 3. Mutations of the integrin beta tails confirmed that these interactions are canonical PTB domain-ligand interactions. First, the interactions were blocked by mutation of an NPXY motif in the integrin tail. Second, integrin class-specific interactions were observed with the PTB domains of Dab, EPS8, and tensin. We used this specificity, and a molecular model of an integrin beta tail-PTB domain interaction to predict critical interacting residues. The importance of these residues was confirmed by generation of gain- and loss-of-function mutations in beta 7 and beta 3 tails. These data establish that short integrin beta tails interact with a large number of PTB domain-containing proteins through a structurally conserved mechanism.     

Crystal structure of the MurG:UDP-GlcNAc complex reveals common structural principles of a superfamily of glycosyltransferases

MurG is an essential glycosyltransferase that forms the glycosidic linkage between N-acetyl muramyl pentapeptide and N-acetyl glucosamine in the biosynthesis of the bacterial cell wall. This enzyme is a member of a major superfamily of NDP-glycosyltransferases for which no x-ray structures containing intact substrates have been reported. Here we present the 2.5-A crystal structure of Escherichia coli MurG in complex with its donor substrate, UDP-GlcNAc. Combined with genomic analysis of other superfamily members and site-specific mutagenesis of E. coli MurG, this structure sheds light on the molecular basis for both donor and acceptor selectivity for the superfamily. This structural analysis suggests that it will be possible to evolve new glycosyltransferases from prototypical superfamily members by varying two key loops while maintaining the overall architecture of the family and preserving key residues.     

The Mid America Heart Institute: part II

The Mid America Heart Institute (MAHI) is one of the first and largest hospitals developed and designed specifically for cardiovascular care. The MAHI hybrid model, which is a partnership between the not-for-profit Saint Luke's Health System, an independent academic medical center, and a private practice physician group, has been extremely successful in providing high-quality patient care as well as developing strong educational and research programs. The Heart Institute has been the leader in providing cardiovascular care in the Kansas City region since its inception in 1975. Although challenges in the future are substantial, it is felt that the MAHI is in an excellent position to deal with the serious issues in health care because of the Heart Institute, its facility, organization, administration, dedicated medical and support staff, and its unique business model of physician management. In part I, the authors described the background and infrastructure of the Heart Institute. In part II, cardiovascular research and benefits of physician management are addressed.     

Dermatomyositis and malignancy in Tunisia: a multicenter national retrospective study of 20 cases

OBJECTIVE AND METHODS: The aim of our study was to report the epidemiologic, clinical, and biologic profiles of dermatomyositis (DM) associated with malignancy in patients from Tunisia. From January 1982 to January 2000, we collected retrospectively 20 case reports of DM associated with cancer from the different university hospital centers of Tunisia. Initial workup included anamnesis, clinical examination, cancer staging and classification, serum muscle enzymes (creatine phosphokinase, lactate dehydrogenase, aldolase, and transaminases), electromyography, and muscular biopsy. We calculated the median survival and mean value of all the variables. Comparisons of statistical tests were done with the Kruskal-Wallis test. RESULTS: Among the 130 DM cases of our study, 20 were associated with cancer (15.38%). The mean age of our patients was 49.6 years and the sex ratio (female/male) was equal to 3. Cancers were mainly those of the breast (35%) and nasopharynx (25%). DM followed a paraneoplastic course in 90% of the cases. The profile of seric muscular enzymes showed a significant statistical difference (P =.05) between a group of patients with severe muscular weakness and a group with moderate muscle weakness only for creatine kinase. The median survival was 36.5 months after diagnosis of DM and 48.6 months after that of cancer. The 5-year actuarial survival was 38% as related to cancer and 16% as related to DM. Mortality was 45%, in 90% as a result of cancer. CONCLUSIONS: In our study, nasopharyngeal carcinoma represents the second cancer associated with DM, after breast neoplasm, demonstrating the frequency of these 2 cancers in our country. Despite our reduced number of study samples, our study also suggests a relationship between severe muscle weakness and high seric muscle enzymes.