Chimeric analysis of fibroblast growth factor receptor-1 (Fgfr1) function: a role for FGFR1 in morphogenetic movement through the primitive streak

Fibroblast growth factor (FGF) signaling has been implicated in the patterning of mesoderm and neural lineages during early vertebrate development. In the mouse, FGF receptor-1 (FGFR1) is expressed in an appropriate spatial and temporal manner to be orchestrating these functions. Mouse embryos homozygous for a mutated Fgfr1 allele (fgfr1(delta tmk)) die early in development, show abnormal growth and aberrant mesodermal patterning. We have performed a chimeric analysis to further study FGFR1 function in the morphogenesis and patterning of the mesodermal germ layer at gastrulation. At E9.5, fgfr1(delta tmk)/fgfr1(delta tmk) cells showed a marked deficiency in their ability to contribute to the extra-embryonic, cephalic, heart, axial and paraxial mesoderm, and to the endoderm of chimeric embryos. Analysis at earlier stages of development revealed that fgfr1(delta tmk)/fgfr1(delta tmk) cells accumulated within the primitive streak of chimeric embryos, and consequently failed to populate the anterior mesoderm and endodermal lineages at their inception. We suggest that the primary defect associated with the fgfr1(delta tmk) mutation is a deficiency in the ability of epiblast cells to traverse the primitive streak. fgfr1(delta tmk)/fgfr1(delta tmk) cells that accumulated within the primitive streak of chimeric embryos tended to form secondary neural tubes. These secondary neural tubes were entirely fgfr1(delta tmk)/fgfr1(delta tmk) cell derived. The adoption of ectopic neural fate suggests that normal morphogenetic movement through the streak is essential not only for proper mesodermal patterning but also for correct determination of mesodermal/neurectodermal cell fates.     

A dose-dependent increase in mortality with vesnarinone among patients with severe heart failure. Vesnarinone Trial Investigators

BACKGROUND: Vesnarinone, an inotropic drug, was shown in a short-term placebo-controlled trial to improve survival markedly in patients with severe heart failure when given at a dose of 60 mg per day, but there was a trend toward an adverse effect on survival when the dose was 120 mg per day. In a longer-term study, we evaluated the effects of daily doses of 60 mg or 30 mg of vesnarinone, as compared with placebo, on mortality and morbidity. METHODS: We enrolled 3833 patients who had symptoms of New York Heart Association class III or IV heart failure and a left ventricular ejection fraction of 30 percent or less despite optimal treatment. The mean follow-up was 286 days. RESULTS: There were significantly fewer deaths in the placebo group (242 deaths, or 18.9 percent) than in the 60-mg vesnarinone group (292 deaths, or 22.9 percent) and longer survival (P=0.02). The increase in mortality with vesnarinone was attributed to an increase in sudden death, presumed to be due to arrhythmia. The quality of life had improved significantly more in the 60-mg vesnarinone group than in the placebo group at 8 weeks (P<0.001) and 16 weeks (P=0.003) after randomization. Trends in mortality and in measures of the quality of life in the 30-mg vesnarinone group were similar to those in the 60-mg group but not significantly different from those in the placebo group. Agranulocytosis occurred in 1.2 percent of the patients given 60 mg of vesnarinone per day and 0.2 percent of those given 30 mg of vesnarinone. CONCLUSIONS: Vesnarinone is associated with a dose-dependent increase in mortality among patients with severe heart failure, an increase that is probably related to an increase in deaths due to arrhythmia. A short-term benefit in terms of the quality of life raises issues about the appropriate therapeutic goal in treating heart failure.     

A nonlethal conditioning approach to achieve engraftment of xenogeneic rat bone marrow in mice and to induce donor-specific tolerance

BACKGROUND: The supply of solid organs for transplantation will never meet the growing demand. Xenotransplantation is considered to be a potential solution for the critical shortage of allografts. However, xenograft rejection is currently not controlled by conventional immunosuppressive agents. Bone marrow chimerism induces donor-specific tolerance without the requirement for chronic immunosuppressive therapy. The aim of this study was to develop a nonlethal recipient-conditioning approach to achieve mixed bone marrow chimerism and donor-specific tolerance. METHODS: C57BL/10SnJ mice were conditioned with total body irradiation followed by a single injection of cyclophosphamide on day +2. On day 0, mice were reconstituted with untreated bone marrow cells from Fischer 344 rats. Recipients were analyzed by flow cytometry for donor bone marrow engraftment and multilineage chimerism. Donor-specific tolerance was tested by skin grafting. RESULTS: One hundred percent of recipients engrafted after irradiation with 600 cGy total body irradiation, transplantation with 80 x 10(6) Fischer 344 bone marrow cells, and injection with 50 mg/kg cyclophosphamide intraperitoneally. Donor chimerism was detectable in all engrafted animals for up to 11 months. This conditioning was nonlethal, because conditioned untransplanted animals survived indefinitely. Mixed xenogeneic chimeras were tolerant to donor-specific skin grafts but rejected third-party (Wistar Furth) grafts as rapidly as naive C57BL/10SnJ mice. In contrast, animals that received less efficacious conditioning regimens and did not exhibit detectable chimerism showed prolonged graft survival, but delayed graft rejection occurred in all animals within 10 weeks. CONCLUSION: The induction of bone marrow chimerism and donor-specific tolerance after nonlethal conditioning might be useful to prevent the vigorous cellular and humoral rejection response to xenografts.     

HIV-1 seroconversion in a prospective study of female sex workers in northern Thailand: continued high incidence among brothel-based women

OBJECTIVES: To determine the incidence of HIV-1 infection, temporal trends in incidence, and risk factors for seroconversion in a cohort of female commercial sex workers (CSW) in upper northern Thailand, the region of Thailand with the highest rates of HIV-1 infection. METHODS: CSW were enrolled from 1991 through 1994 and evaluated prospectively with interviews, physical examination, testing for sexually transmitted diseases (STD), and serologic testing for HIV-1 infection. RESULTS: The incidence of HIV-1 seroconversion in the first year of follow-up was 20.3 per 100 person-years among 126 brothel-based CSW and 0.7 per 100 person-years among 159 other CSW who worked in other venues such as bars or massage parlors. Incidence remained elevated among brothel-based CSW who were enrolled later in the study compared with those who enrolled earlier. Through 1996, 30 women seroconverted. In a multivariable proportional hazards model, seroconversion was significantly associated (P < 0.05) with brothel-based sex work (adjusted risk ratio, 7.3) and Chlamydia trachomatis cervical infection (adjusted risk ratio, 3.3). CONCLUSION: Despite national HIV control efforts and declining rates of infection among young men in Thailand, brothel-based CSW may continue to be at high risk for HIV-1 infection. Additional efforts are needed to provide alternative economic choices for young women, to ensure universal condom use during commercial sex, and to develop new prevention technologies.     

Evolution of the cytochrome P450 superfamily: sequence alignments and pharmacogenetics

The evolution of the cytochrome P450 (CYP) superfamily is described, with particular reference to major events in the development of biological forms during geological time. It is noted that the currently accepted timescale for the elaboration of the P450 phylogenetic tree exhibits close parallels with the evolution of terrestrial biota. Indeed, the present human P450 complement of xenobiotic-metabolizing enzymes may have originated from coevolutionary 'warfare' between plants and animals during the Devonian period about 400 million years ago. A number of key correspondences between the evolution of P450 system and the course of biological development over time, point to a mechanistic molecular biology of evolution which is consistent with a steady increase in atmospheric oxygenation beginning over 2000 million years ago, whereas dietary changes during more recent geological time may provide one possible explanation for certain species differences in metabolism. Alignment between P450 protein sequences within the same family or subfamily, together with across-family comparisons, aid the rationalization of drug metabolism specificities for different P450 isoforms, and can assist in an understanding of genetic polymorphisms in P450-mediated oxidations at the molecular level. Moreover, the variation in P450 regulatory mechanisms and inducibilities between different mammalian species are likely to have important implications for current procedures of chemical safety evaluation, which rely on pure genetic strains of laboratory bred rodents for the testing of compounds destined for human exposure.     

Use of a Mycobacterium tuberculosis H37Rv bacterial artificial chromosome library for genome mapping, sequencing, and comparative genomics

The bacterial artificial chromosome (BAC) cloning system is capable of stably propagating large, complex DNA inserts in Escherichia coli. As part of the Mycobacterium tuberculosis H37Rv genome sequencing project, a BAC library was constructed in the pBeloBAC11 vector and used for genome mapping, confirmation of sequence assembly, and sequencing. The library contains about 5,000 BAC clones, with inserts ranging in size from 25 to 104 kb, representing theoretically a 70-fold coverage of the M. tuberculosis genome (4.4 Mb). A total of 840 sequences from the T7 and SP6 termini of 420 BACs were determined and compared to those of a partial genomic database. These sequences showed excellent correlation between the estimated sizes and positions of the BAC clones and the sizes and positions of previously sequenced cosmids and the resulting contigs. Many BAC clones represent linking clones between sequenced cosmids, allowing full coverage of the H37Rv chromosome, and they are now being shotgun sequenced in the framework of the H37Rv sequencing project. Also, no chimeric, deleted, or rearranged BAC clones were detected, which was of major importance for the correct mapping and assembly of the H37Rv sequence. The minimal overlapping set contains 68 unique BAC clones and spans the whole H37Rv chromosome with the exception of a single gap of approximately 150 kb. As a postgenomic application, the canonical BAC set was used in a comparative study to reveal chromosomal polymorphisms between M. tuberculosis, M. bovis, and M. bovis BCG Pasteur, and a novel 12.7-kb segment present in M. tuberculosis but absent from M. bovis and M. bovis BCG was characterized. This region contains a set of genes whose products show low similarity to proteins involved in polysaccharide biosynthesis. The H37Rv BAC library therefore provides us with a powerful tool both for the generation and confirmation of sequence data as well as for comparative genomics and other postgenomic applications. It represents a major resource for present and future M. tuberculosis research projects.     

Identification of race and sex from palate dimensions

Measurements of the width and depth of the palate were used to predict the race (American black or white) or sex or both of an individual. The sample consisted of 332 living subjects with permanent dentitions, and measurements were made between cusp tips, so palate size includes bony and dental components. Blacks, with a more square palate, were distinguished from whites primarily by greater interpremolar widths and P1-to-M2 depths. Simultaneous prediction of race and sex had a correct classification of 48%, which is about twice that expected from chance. Pooling the two sex increases correct classification of race to 83%. Formulas also are provided for each variable separately to accommodate fragmentary remains. Resilience of palatal structures to traumatic and natural forces makes this method practical in several forensic situations.     

Synthesis of the four isomers of 4-aminopyrrolidine-2,4-dicarboxylate: identification of a potent, highly selective, and systemically-active agonist for metabotropic glutamate receptors negatively coupled to adenylate cyclase

The four isomers of 4-aminopyrrolidine-2,4-dicarboxylate (APDC) were prepared and evaluated for their effects at glutamate receptors in vitro. (2R,4R)-APDC (2a), an aza analog of the nonselective mGluR agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate (1S,3R)-ACPD, 1), was found to possess relatively high affinity for metabotropic glutamate receptors (mGluRs) (ACPD-sensitive [3H]glutamate binding IC50 = 6.49 +/- 1.21 microM) with no effects on radioligand binding to NMDA, AMPA, or kainate receptors up to 100 microM. None of the other APDC isomers showed significant mGluR binding affinity, indicating that this interaction is highly stereospecific. Both 1 and 2a were effective in decreasing forskolin-stimulated cAMP formation in the adult rat cerebral cortex (EC50 = 8.17 +/- 2.21 microM for 1; EC50 = 14.51 +/- 5.54 microM for 2a); however, while 1 was also effective in stimulating basal tritiated inositol monophosphate production in the neonatal rat cerebral cortex (EC50 = 27.7 +/- 5.2 microM), 2a (up to 100 microM) was ineffective in stimulating phosphoinositide hydrolysis in this tissue preparation, further supporting our previous observations that 2a is a highly selective agonist for mGluRs negatively coupled to adenylate cyclase. Microelectrophoretic application of either 1 or 2a to intact rat spinal neurons produced an augmentation of AMPA-induced excitation (95 +/- 10% increase for 1, 52 +/- 6% increase for 2a). Intracerebral injection of 1 (400 nmol) produced characteristic limbic seizures in mice which are not mimicked by 2a (200-1600 nmol, ic). However, the limbic seizures induced by 1 were blocked by systemically administered 2a in a dose-dependent manner (EC50 = 271 mg/kg, ip). It is concluded that (2R,4R)-APDC (2a) is a highly selective, systemically-active agonist of mGluRs negatively coupled to adenylate cyclase and that selective activation of these receptors in vivo can result in anticonvulsant effects.