Vam7p, a SNAP-25-like molecule, and Vam3p, a syntaxin homolog, function together in yeast vacuolar protein trafficking

A genetic screen to isolate gene products required for vacuolar morphogenesis in the yeast Saccharomyces cerevisiae identified VAM7, a gene which encodes a protein containing a predicted coiled-coil domain homologous to the coiled-coil domain of the neuronal t-SNARE, SNAP-25 (Y. Wada and Y. Anraku, J. Biol. Chem. 267:18671-18675, 1992; T. Weimbs, S. H. Low, S. J. Chapin, K. E. Mostov, P. Bucher, and K. Hofmann, Proc. Natl. Acad. Sci. USA 94:3046-3051, 1997). Analysis of a temperature-sensitive-for-function (tsf) allele of VAM7 (vam7(tsf)) demonstrated that the VAM7 gene product directly functions in vacuolar protein transport. vam7(tsf) mutant cells incubated at the nonpermissive temperature displayed rapid defects in the delivery of multiple proteins that traffic to the vacuole via distinct biosynthetic pathways. Examination of vam7(tsf) cells at the nonpermissive temperature by electron microscopy revealed the accumulation of aberrant membranous compartments that may represent unfused transport intermediates. A fraction of Vam7p was localized to vacuolar membranes. Furthermore, VAM7 displayed genetic interactions with the vacuolar syntaxin homolog, VAM3. Consistent with the genetic results, Vam7p physically associated in a complex containing Vam3p, and this interaction was enhanced by inactivation of the yeast NSF (N-ethyl maleimide-sensitive factor) homolog, Sec18p. In addition to the coiled-coil domain, Vam7p also contains a putative NADPH oxidase p40(phox) (PX) domain. Changes in two conserved amino acids within this domain resulted in synthetic phenotypes when combined with the vam3(tsf) mutation, suggesting that the PX domain is required for Vam7p function. This study provides evidence for the functional and physical interaction between Vam7p and Vam3p at the vacuolar membrane, where they function as part of a t-SNARE complex required for the docking and/or fusion of multiple transport intermediates destined for the vacuole.     

Measurement of intravariceal pressure by fine needle direct puncture in hepatitis B surface antigen-positive cirrhotic patients: the effect of vasopressin

A trp E fusion protein containing a C-terminal portion of the rat substance P receptor (SPR) was expressed in bacteria and used to produce an antibody. The antibody specifically reacted with SPR expressed in a mammalian cell line and rat striatum. Light and electron microscope analyses of the rat striatum revealed intense SPR-like immunoreactivity in neuronal somata and dendrites. These immunoreactive neurons constituted approximately 3% of the total population of striatal neurons; they were putative interneurons of large and medium-sized aspiny type.     

Sulphation requirement for GlyCAM-1, an endothelial ligand for L-selectin

L-selectin participates in the initial attachment of leukocytes to the vascular endothelium. On lymphocytes, it mediates binding to high endothelial venules of lymph nodes. As a selectin it functions as a calcium-dependent lectin recognizing carbohydrate-bearing ligands on endothelial cells. Two lymph node ligands for L-selectin have been identified as sulphated glycoproteins of M(r) approximately 50K and approximately 90K, called Sgp50 and Sgp90 (ref. 10). The recently cloned Sgp50 (ref. 12), now designated GlyCAM-1, is a high endothelial venule-associated, mucin-like glycoprotein containing predominantly O-linked carbohydrate chains. Sialylation of GlyCAM-1 is necessary for its ligand activity and a role for fucosylation is suspected. We have used chlorate as a metabolic inhibitor of sulphation, and report here that GlyCAM-1 has an additional requirement for sulphate.     

Pertussis in Missouri: evaluation of nasopharyngeal culture, direct fluorescent antibody testing, and clinical case definitions in the diagnosis of pertussis

No diagnostic test for pertussis in routine use in the United States has both high sensitivity and high specificity. During a statewide increase in the incidence of pertussis in Missouri, we studied the clinical features of 153 patients with suspected pertussis in the Greater St. Louis area from whom a specimen for pertussis culture had been taken between 15 May and 19 September 1989. In this cross-sectional study, nasopharyngeal cultures were more likely to be positive for persons whose specimens were collected < 21 days after cough onset (adjusted rate ratio [RRa] and 95% confidence interval = 3.4; 1.5-8.0) and who were not receiving erythromycin/sulfamethoxazole prior to the culture [RRa = 5.8; 0.8-40.6], who had received fewer than three prior doses of pertussis vaccine [RRa = 1.8; 0.8-4.2], and whose specimen was in transit to the laboratory for < 4 days [RRa = 2.0; 0.8-5.5]. Among children < 5 years of age, spasmodic cough plus a lymphocytosis of > 10,000/mm3 was the acute symptom complex associated with the highest predictive value for a positive culture result (67%). Cough for > or = 14 days plus whoop was sensitive (81%) and specific (58%) for identifying children with culture-confirmed pertussis. Direct fluorescent antibody staining performed well as a screening test for pertussis but requires substantial commitment of personnel and resources. In the absence of a positive culture result, clinical case definitions should be used for decision making (e.g., initiation of antimicrobial therapy and routine case reporting).     

Establishment and characterization of a megakaryoblast cell line with amplification of MLL

A new cell line with megakaryoblastic features, designated UoC-M1, was established from the malignant cells of a 68-year-old patient with acute myeloid leukemia. The patient's leukemic cells reacted with alpha-naphthyl acetate esterase and acid phosphatase and expressed CD7, CD24, CD34, CD38, CD45, HLA-DR and CD61. Cytogenetic analysis of the patient's malignant cells (and of the UoC-M1 cells) showed a human, male hypodiploid karyotype with many chromosome rearrangements and marker chromosomes. Spectral karyotyping (SKY) analysis complemented the G-banded karyotyping and clarified several chromosomal translocations and identified the marker chromosomes. Fluorescence in situ hybridization (FISH) and SKY analysis demonstrated that one marker chromosome contained three segments of chromosome 9 interspersed with three segments of chromosome 11, as well as a portion of chromosome 19. FISH analysis with a probe for MLL revealed that the UoC-M1 cells contained four copies of the MLL gene. Southern blot analysis determined that the MLL gene had a germline profile while Northern and Western analyses showed that the MLL mRNAs and protein were of the appropriate sizes. This is the first report of amplification of the MLL gene which may be an additional mechanism of leukemogenesis or disease progression.