Crystals of an ATPase fragment of bovine clathrin uncoating ATPase

A 44,000 Mr amino-terminal, clathrin-independent ATPase fragment of the bovine clathrin uncoating ATPase has been crystallized in a form suitable for X-ray diffraction studies. The crystals are orthorhombic, space group P2(1)2(1)2(1), a = 145.3 A, b = 65.0 A, c = 46.9 A, with one protein molecule per asymmetric unit (1 A = 0.1 nm).     

Relative stability of human respiration during progressive hypoxia

We have systematically studied the relationship between the relative stability (R) of respiration and the loop gain (LG) of the CO2 control system in 15 healthy awake adult males during progressive hypoxia. R was measured by the ventilatory oscillations after brief (less than 10 s) CO2 challenges. Control theory suggests that such oscillations are completely governed by LG. A significant positive correlation was found between R and LG (r = 0.74, P less than 0.01, n = 85). A minimal mathematical model of respiratory control was used to predict R as a function of LG. Serial correlation analysis (r = 0.09, P greater than 0.1) of the residuals indicated statistical agreement between predictions and observations. The mean residual (0.011) was not significantly different from zero (P greater than 0.1). Also, as the model predicted, sustained periodic breathing (PB) occurred whenever the estimated LG was greater than unity. The mean LG breathing room air was 0.51 and for the 13 epochs of PB was 1.17 (range 0.71-1.65). It is concluded that PB is a quantitative extension of the relative stability continuum and corresponds to unstable operation of the CO2 control system. Furthermore, relative stability can be quantitatively predicted for each subject by a minimal mathematical model.     

Seroreactivity to HPV-16 proteins in women with early cervical neoplasia

Summary Although serological reactivity to human papillomavirus type 16 (HPV-16) proteins has been demonstrated in patients with invasive cervical carcinoma, the degree of seroreactivity to these proteins in women with preinvasive disease and its relationship to the HPV type associated with the disease are unclear. We obtained sera from 27 women undergoing cone biopsy for cervical precursor lesions and 22 controls and analyzed seroreactivity by Western blot to fusion proteins containing portions of the HPV-16 E4, L1 and L2 open-reading frames (ORFs). Positives were analyzed by scanning densitometry and intensity values for each case plotted relative to controls. Cervical biopsy specimens from patients were analyzed for HPV-16 nucleic acids by DNA · DNA in situ hybridization. Mean intensity values for seroreactivity to the pATH-E4 protein approached significance (P = 0.058) and a significantly higher proportion of cases vs controls registered values over 4.0 for pATH-E4 (26% vs 4.5%;P = 0.04) and pATH-L2 (48% vs 18%;P = 0.03) proteins. A significantly higher mean intensity value for E4 was observed for cases containing HPV-16 DNA vs HPV-16 negative cases or controls. Thus, seroreactivity to HPV-16-derived proteins may be more common in women with preinvasive cervical disease, and for some protein targets (E4) may indicate a relatively type-specific response.Supported in part by grants from the National Cancer Institute [CA 47676 (C.P.C.)], American Cancer Society [MV-395 (C.P.C.)] and an institutional support grant (J.K.R.). Dr. Crum is a recipient of a Physician Scientist Award from the National Institute of Allergy and Infectious Disease (AI00628)     

Evidence implicating heterozygous deletion of chromosome 7 in the pathogenesis of familial leukemia associated with monosomy 7.

Complete or partial monosomy 7 is a recurring cytogenetic abnormality in acute myelogenous leukemia (AML) and myeloproliferative syndromes (MPS) and is particularly common in patients with Fanconi's anemia and in secondary AML. A familial form of monosomy 7 has been recognized in which two or more siblings develop MPS or AML before age 20. We tested the hypothesis that a recessive cancer susceptibility locus on chromosome 7 was important in the pathogenesis of leukemia in familial monosomy 7 by determining the parental origins of the chromosome 7 retained in the bone marrows of three pairs of affected siblings. We found no overlapping region where all three pairs retained DNA derived from the same paternal or maternal chromosome. These data suggest that inactivation of a single allele of a putative tumor-suppressor gene may be sufficient to contribute to leukemic transformation in familial monosomy 7.     

Cellular targets and trophic functions of neurotrophin-3 in the developing rat hippocampus.

The expression of the neurotrophins and trk receptors in the hippocampus has directed attention toward their roles in the development and maintenance of this region. We have examined the effects of the neurotrophins NT-3, BDNF, and NGF in cultures of developing rat hippocampal cells by two criteria: rapid induction of c-fos and neurotrophic responses. The selective induction of c-fos mRNA suggests the presence of functional receptors for NT-3 and BDNF, but not NGF, in embryonic hippocampal cultures. The NT-3-responsive cells were localized in pyramidal neurons of areas CA1 through CA3 and dentate granular and hilar cells of postnatal organotypic slices, as detected by c-Fos immunocytochemistry. In addition to immediate early responses, NT-3 caused a 10-fold increase in the number of cells expressing the neuronal antigen calbindin-D28k. This increase was dose dependent, with maximal stimulation at 10 ng/ml. In contrast, BDNF elicited small but significant calbindin responses. These results indicate biological responses to NT-3 in the CNS and suggest roles for for this neurotrophin during hippocampal neurogenesis.     

Region-specific differentiation of the hippocampal stem cell line HiB5 upon implantation into the developing mammalian brain

Proliferating precursors to the distinct cell types constituting the mammalian brain can be identified by the presence of the nestin intermediate filament. We report the establishment of a nestin-positive cell line, HiB5, from embryonic precursor cells to the rat hippocampus. Since it was immortalized using the temperature-sensitive allele tsA58 of SV40 large T antigen, these cells grow continuously at 33 degrees C, but not at 39 degrees C, the body temperature of rodents. To test their developmental capacity, HiB5 cells were implanted into both the neonatal hippocampus and cerebellum. The cells integrated into the host tissue and acquired morphologies characteristic of the neurons and glial cells found at the implant site. HiB5 cells might thus be useful in characterizing the signals regulating cell type determination in the mammalian brain.     

Identification, DNA sequence, and distribution of IS981, a new, high-copy-number insertion sequence in lactococci.

An insertion in the lactococcal plasmid pGBK17, which inactivated the gene(s) encoding resistance to the prolate-headed phage c2, was cloned, sequenced, and identified as a new lactococcal insertion sequence (IS). IS981 was 1,222 bp in size and contained two open reading frames, one large enough to encode a transposase. IS981 ended in imperfect inverted repeats of 26 of 40 bp and generated a 5-bp direct repeat of target DNA at the site of insertion. IS981 was present on the chromosome of Lactococcus lactis subsp. lactis LM0230 from where it transposed to pGBK17 during transformation. Twenty-three strains of lactococci examined for the presence of IS981 by Southern hybridization showed 4 to 26 copies per genome, with L. lactis subsp. cremoris strains containing the highest number of copies. Comparison of the DNA sequence and the amino acid sequence of the long open reading frame to other known sequences showed that IS981 is related to a family of IS elements that includes IS2, IS3, IS51, IS150, IS600, IS629, IS861, IS904, and ISL1.     

Isolation of spontaneous mutant strains of Flavobacterium spp.

Flavobacterium sp. ATCC 27551 is prototrophic, streptomycin-resistant and contains plasmid DNA. Two strains of this Flavobacterium sp. with altered growth requirements and antibiotic sensitivity have been isolated. Unlike the parental strain, both isolates are sensitive to the antibiotic streptomycin. Isolate YE requires yeast extract supplementation of the medium and contains no plasmid DNA. Isolate AHC contains plasmid DNA and requires aspartic acid, histidine and cysteine for growth.