A new genetic isolate of gray platelet syndrome (GPS): clinical, cellular, and hematologic characteristics.

Gray platelet syndrome (GPS) is a disorder characterized by thrombocytopenia and large platelets that lack alpha granules and their contents. We describe two siblings with GPS who are members of a Moslem Bedouin genetic isolate. The children, an 8-year-old girl and a 5-year-old boy, had characteristic pale blue platelets lacking alpha granules on electron microscopy. Platelet aggregation studies were normal. The girl underwent a bone marrow aspiration and biopsy which showed mild myelofibrosis and extensive emperipolesis, i.e., the passage of other hematopoietic cells through megakaryocytes. Both children lacked high-molecular-weight multimers of von Willebrand factor (vWF) and had reduced activity and antigens of vWf. Platelet activation was approximately normal when ADP was employed as agonist, but significantly impaired using the thrombin receptor-activating peptide (TRAP). These findings are explained in light of the mechanism of action of each agonist. In addition, we propose that the emperipolesis was caused by increased P-selectin in megakaryocytes, and resulted in release of fibroblastic growth factors, explaining the myelofibrosis. The detailed description of these cases provides a basis for future differentiation of the various types of GPS, and for our current attempts to isolate the gene causing GPS in this genetic isolate.

Structural characterization of erythroid and megakaryocytic differentiation in Friend erythroleukemia cells.

OBJECTIVE: The aim of this study was to examine the structural characterization of erythroid and megakaryocytic cell differentiation in Friend erythroleukemic cells using spectral imaging and electron microscopy. MATERIALS AND METHODS: Two variants of Friend erythroleukemia cells were treated with hexamethylene bisacetamide (HMBA) to induce differentiation: 1) MEL, which exhibit the normal phenotype and are susceptible to differentiation; and 2) the resistant R1 cells. The cells were analyzed by spectral imaging along with transmission and scanning electron microscopy. The expression of cell cycle regulatory proteins was analyzed by Western blotting. RESULTS: Spectral imaging of HMBA-treated MEL and R1 cells stained by May-Grünwald-Giemsa and subjected to spectral similarity mapping revealed five morphologic cell types: proerythroblast-like cells, normoblast-like cells, reticulocyte-like cells, megakaryocytes, and apoptotic cells. In MEL cells, both megakaryocytic differentiation characterized by nuclear lobes and erythroid differentiation characterized by accumulation of hemoglobin were detected; R1 cells were not committed to terminal differentiation. HMBA-induced cell cycle arrest at G(1) affected the expression of regulatory proteins in a similar manner in both types of cells. Expression of cyclin-dependent kinase 4 decreased and expression of p21(WAF1) increased. The level of the underphosphorylated form of phosphorylated retinoblastoma protein increased, inducing a decrease in the level of c-myc. In addition, we detected a decrease in the expression of the anti-apoptotic regulator, Bcl-2, and an increased expression of the pro-apoptotic regulator, Bax. CONCLUSIONS: Spectral imaging provides new insight for the morphologic characterization of erythroid and megakaryocytic cell differentiation as well as apoptosis. Image analysis was well correlated to cell cycle arrest and the expression of regulatory proteins.

Papillomavirus type 16 oncogenes downregulate expression of interferon-responsive genes and upregulate proliferation-associated and NF-kappaB-responsive genes in cervical keratinocytes.

Infection with high-risk human papillomaviruses (HPV) is a major risk factor for development of cervical cancer. Expression of the HPV E6 and E7 oncoproteins increases in differentiating keratinocytes, resulting in inactivation of the p53 and retinoblastoma proteins, two important transcriptional regulators. We used cDNA microarrays to examine global alterations in gene expression in differentiating cervical keratinocytes after infection with retroviruses encoding HPV type 16 (HPV-16) E6 and E7. Expression of 80 cellular genes (approximately 4% of the genes on the array) was altered reproducibly by E6 and/or E7. Cluster analysis classified these genes into three functional groups: (i) interferon (IFN)-responsive genes, (ii) genes stimulated by NF-kappaB, and (iii) genes regulated in cell cycle progression and DNA synthesis. HPV-16 E6 or a dominant negative p53 protein downregulated multiple IFN-responsive genes. E6 decreased expression of IFN-alpha and -beta, downregulated nuclear STAT-1 protein, and decreased binding of STAT-1 to the IFN-stimulated response element. E7 alone was less effective; however, coexpression of E6 and E7 downregulated IFN-responsive genes more efficiently than E6. The HPV-16 E6 protein also stimulated expression of multiple genes known to be inducible by NF-kappaB and AP-1. E6 enhanced expression of functional components of the NF-kappaB signal pathway, including p50, NIK, and TRAF-interacting protein, and increased binding to NF-kappaB and AP-1 DNA consensus binding sites. Secretion of interleukin-8, RANTES, macrophage inflammatory protein 1alpha, and 10-kappaDa IFN-gamma-inducible protein were increased in differentiating keratinocytes by E6. Thus, high-level expression of the HPV-16 E6 protein in differentiating keratinocytes directly alters expression of genes that influence host resistance to infection and immune function.

Stu2 promotes mitotic spindle elongation in anaphase.

During anaphase, mitotic spindles elongate up to five times their metaphase length. This process, known as anaphase B, is essential for correct segregation of chromosomes. Here, we examine the control of spindle length during anaphase in the budding yeast Saccharomyces cerevisiae. We show that microtubule stabilization during anaphase requires the microtubule-associated protein Stu2. We further show that the activity of Stu2 is opposed by the activity of the kinesin-related protein Kip3. Reexamination of the kinesin homology tree suggests that KIP3 is the S. cerevisiae orthologue of the microtubule-destabilizing subfamily of kinesins (Kin I). We conclude that a balance of activity between evolutionally conserved microtubule-stabilizing and microtubule-destabilizing factors is essential for correct spindle elongation during anaphase B.

Involvement of PKR in the regulation of myogenesis.

The involvement of the double-stranded RNA-activated protein kinase PKR in the regulation of the myogenic process was investigated. For this purpose, the murine myogenic cell line C2C12 was used. The cells were first cultivated in either growth medium or differentiation medium (DM), and the activation of PKR during differentiation was determined by monitoring its enzymatic activity and by immunoblot analysis. A significant increase in both parameters was detected already at 24 h in DM, whereas in cells grown in growth medium, the increase was evident only after 96 h, when spontaneous differentiation was observed in highly crowded cultures. Consequently, we established the direct effect of PKR activation on the myogenic process. C2C12 cells were transfected with an expression vector harboring a cDNA molecule encoding human PKR fused to the inducible metallothionein promoter. One of the clones (clone 8) expressing high levels of PKR was selected and further analyzed. In the presence of ZnCl2, which activates the promoter, the rate of cell growth of the transfected cells was clearly reduced compared to that of wild-type C2C12 cells transfected with only the neomycin-resistant gene (C2-NEO). In addition, altered morphology with partial fusion was observed. Biochemically, an increase in creatine kinase activity accompanied by an increased rate of expression of the myogenic protein troponin T and the myogenic transcription factors myoD and myogenin was detected in clone 8 cells exposed to ZnCl2. Most importantly, an induction in the level of cyclin-dependent kinase inhibitor p21WAF1 and an increase in the level of the underphosphorylated active form of the tumor suppressor protein pRb concomitant with the down-regulation of cyclin D1 and c-myc were also evident in the transfected clones. These changes were similar to those observed in normal C2C12 cells cultivated in DM. We conclude that PKR is an important regulatory protein participating in the myogenic process.

Dissection of cell division processes in the one cell stage Caenorhabditis elegans embryo by mutational analysis.

To identify novel components required for cell division processes in complex eukaryotes, we have undertaken an extensive mutational analysis in the one cell stage Caenorhabditis elegans embryo. The large size and optical properties of this cell permit observation of cell division processes with great detail in live specimens by simple differential interference contrast (DIC) microscopy. We have screened an extensive collection of maternal-effect embryonic lethal mutations on chromosome III with time-lapse DIC video microscopy. Using this assay, we have identified 48 mutations in 34 loci which are required for specific cell division processes in the one cell stage embryo. We show that mutations fall into distinct phenotypic classes which correspond, among others, to the processes of pronuclear migration, rotation of centrosomes and associated pronuclei, spindle assembly, chromosome segregation, anaphase spindle positioning, and cytokinesis. We have further analyzed pronuclear migration mutants by indirect immunofluorescence microscopy using antibodies against tubulin and ZYG-9, a centrosomal marker. This analysis revealed that two pronuclear migration loci are required for generating normal microtubule arrays and four for centrosome separation. All 34 loci have been mapped by deficiencies to distinct regions of chromosome III, thus paving the way for their rapid molecular characterization. Our work contributes to establishing the one cell stage C. elegans embryo as a powerful metazoan model system for dissecting cell division processes.

Ectopic expression of 2-5A synthetase in myeloid cells induces growth arrest and facilitates the appearance of a myeloid differentiation marker.

Two variants of the human myeloid leukemia cell line HL-60 were used to study the possible involvement of the IFN-induced protein 2-5A synthetase in cell growth arrest and differentiation. The two variants, HL-205 and HL-525, are equally susceptible to differentiation to the granulocyte lineage by exposure to DMSO, but only HL-205 cells acquire the macrophage phenotype following exposure to phorbol esters. The kinetics of 2-5A synthetase activity was established in both variants exposed to either DMSO or phorbol 12-myristate 13-acetate. With DMSO treatment, 2-5A synthetase activity was markedly induced in both variants, although with slightly different kinetics. With phorbol 12-myristate 13-acetate treatment, 2-5A enzymatic activity increased only in HL-205; no activity was detected up to 96 h after treatment in HL-525. The induction of 2-5A synthetase activity is apparently alpha/beta-IFN dependent, because only antibodies directed against a mixture of alpha- and beta-IFN completely abolished the increase in activity detected during differentiation of HL-205 cells. To directly establish the role of 2-5A synthetase in differentiation, HL-205 cells were transfected with an expression vector harboring the cDNA for the 43-kDa isoform of murine 2-5A synthetase fused to the inducible metallothionein promoter. Two clones, clone 6, which yielded a low level of 2-5A synthetase activity in response to ZnCl2 (which activates the promoter), and clone 7, which was a high responder, were further analyzed and compared with the control clone, neo. Reductions in the rates of cell growth and thymidine incorporation were observed with both clone 6 and clone 7 cells exposed to ZnCl2; clone 7 was more responsive. In addition, the level of c-myc-specific RNA transcript was greatly reduced in ZnCl2 or beta-IFN-treated clone 7 cells, whereas the neo cells responded similarly only after beta-IFN treatment. Treatment of clone-neo cells with beta-IFN resulted in conversion of pRb protein from the phosphorylated to the underphosphorylated form within 24 h; ZnCl2 had no effect, even after 72 h. In contrast, the accumulation of the underphosphorylated form of pRb was observed in clone 7 cells treated either with beta-IFN or ZnCl2. Finally, a significant increase in nitro blue tetrazolium-positive cells, an indication of differentiation, was evident with ZnCl2-treated clone 6 and clone 7 cells; no such increase was observed with clone-neo cells under similar conditions. We conclude that ectopic expression of 2-5A synthetase in HL-205 cells results in cell growth arrest and facilitates the appearance of a myeloid differentiation marker.

Peru-15, an improved live attenuated oral vaccine candidate for Vibrio cholerae O1.

Cholera vaccine candidate Peru-15 was derived from a Vibrio cholerae O1 El Tor Inaba strain by deleting the cholera toxin genetic element, introducing the gene encoding cholera toxin B subunit into recA, and screening for nonmotility. In a controlled study, Peru-15 (2 x 10(8) cfu) was administered to 11 volunteers. No vaccinee developed diarrhea, and 10 of 11 had > 4-fold rises in vibriocidal antibody titers. One month later, 5 vaccinees and 5 control volunteers were challenged with wild type V. cholerae O1. Four of 5 controls developed diarrhea (mean, 1.9 L). Two Peru-15 vaccinees developed diarrhea, 1 with < 0.3 L and 1 with approximately 1.0 L; this latter volunteer had not developed a significant vibriocidal immune response to vaccination. Peru-15 shows promise as a single-dose, oral cholera vaccine that is safe, immunogenic, and protective.

Heterologous antigen expression in Vibrio cholerae vector strains.

Live attenuated vector strains of Vibrio cholerae were derived from Peru-2, a Peruvian El Tor Inaba strain deleted for the cholera toxin genetic element and attRS1 sequences, which was developed as a live, oral vaccine strain. A promoterless gene encoding the Shiga-like toxin I B subunit (slt-IB) was inserted in the V. cholerae virulence gene irgA by in vivo marker exchange, such that slt-IB was under transcriptional control of the iron-regulated irgA promoter. slt-IB was also placed under transcriptional control of the V. cholerae heat shock promoter, htpGp, and introduced into either the irgA or lacZ locus, or both loci, on the chromosome of Peru-2, generating JRB10, JRB11, or JRB12, respectively. A new technique was used to perform allelic exchange with lacZ. This method uses plasmid p6891MCS, a pBR327 derivative containing cloned V. cholerae lacZ, to insert markers of interest into the V. cholerae chromosome. Recombinants can be detected by simple color screening and antibiotic selection. In vitro measurements of Slt-IB produced by the vector strains suggested that expression of Slt-IB from the irgA and htpG promoters was synergistic and that two copies of the gene for Slt-IB increased expression over a single copy. The V. cholerae vectors colonized the gastrointestinal mucosa of rabbits after oral immunization, as demonstrated by very high serum antibody responses to V. cholerae antigens. Comparison of the serologic responses to the B subunit of cholera toxin (CtxB) following orogastric inoculation either with the wild-type C6709 or with Peru-10, a strain containing ctxB regulated by htpGp, suggested that both the cholera toxin and heat shock promoters were active in vivo, provoking comparable immunologic responses. Orogastric inoculation of rabbits with vector strains evoked serum immunoglobulin G (IgG) responses to Slt-IB in two of the four strains tested; all four strains produced biliary IgA responses. No correlation was observed between the type of promoter expressing slt-IB and the level of serum IgG or biliary IgA response, but the vector strain containing two copies of the gene for slt-IB evoked greater serum IgG responses than strains containing a single copy, consistent with the increased expression of Slt-IB from this strain observed in vitro. A comparison of the serum and biliary antibody responses to Slt-IB expressed from htpGp versus CtxB expressed from the same promoter suggested that CtxB is a more effective orally delivered immunogen.

Parameter uncertainty and sensitivity in a liquid-effluent dose model.

Radioactive materials which are released into streams on the Savannah River Site (SRS) eventually flow into the Savannah River. Tritium, (90)Sr, (137)Cs, and (239)Pu account for the majority of the radiation dose received by users of the Savannah River. This paper focuses on the dose uncertainties originating from variability in parameters describing the transport and uptake of these nuclides. Parameter sensitivity has also been determined for each liquid pathway exposure model. The models used here to estimate radiation dose to an exposed individual provide a range of possible dose estimates that span approximately one order of magnitude. A pathway analysis reveals that aquatic food and water consumption account for more than 95% of the total dose to an individual.

Development of a live, oral, attenuated vaccine against El Tor cholera.

Vibrio cholerae El Tor strains from Peru, Bangladesh, and Bahrain were attenuated by deletion of a genetic element that encodes virulence factors and RS1. The B subunit of ctx (ctxB) was reintroduced into the recA gene of the deletion mutants, rendering them unable to recombine with exogenous genetic elements and generating Peru-3, Bang-3, and Bah-3. Fifteen volunteers received one dose of various vaccine strains at 4 x 10(6) to 1 x 10(8) cfu. All strains colonized the gut. A > or = 4-fold rise in vibriocidal titer was observed in 14 volunteers, with titers of > or = 1600 in 13. Peru-3 was the least reactogenic, but 2 of 6 volunteers had loose stools. Peru-14, a filamentous motility-deficient mutant of Peru-3, was well tolerated and colonized 18 of 21 volunteers at doses of 2 x 10(6) to 1 x 10(9) cfu. Also, when 8 Peru-3 or Peru-5 vaccinees, 5 Peru-14 vaccinees, and 8 controls were challenged with 2 x 10(6) cfu V. cholerae El Tor Inaba (N16961), 11 vaccinees were protected compared with no controls. Peru-14 shows promise as a safe, effective, single-dose oral vaccine against El Tor cholera.

Characterization of normal brain tissue using seven calculated MRI parameters and a statistical analysis system.

A statistical analysis system for classifying normal brain tissue has been applied to the analysis of MRI scans on 45 volunteers. The Bayes Maximum Likelihood method was used to achieve a discrimination accuracy of 84% for 13 tissue types among three age group sets, with classification accuracies for individual regions ranging from 50 to 100%. In order to attain this level of discrimination a set of seven derived relaxation-type parameters was used to categorize the tissue types. Values for these experimentally estimated parameters were derived from the MRI intensities of eight images in the following pulse sequences: (1) a Carr-Purcell-Meiboom-Gill (CPMG) four-echo train, (2) a single-echo inversion recovery, and (3) three single-echo sequences with varying repetition times, TR, and echo delays, TE. The T2 values derived from ratios of single-echo intensities showed better discrimination power than those from the four-echo CPMG train. The general precision of the seven estimated parameters was excellent, with percentage standard deviations ranging from 4 to 18% for the various regions studied. The tissue discrimination achieved by use of just three relaxation parameters, T1, T2, and proton density, calculated from intensities of images from a four-echo sequence, an inversion recovery sequence, and a short TR single-echo sequence, was not as good, being only 55%.

13C NMR assignments of the bases in oligodeoxynucleotides: an automated procedure using Bayesian statistics.

A statistical method, Bayes Maximum Likelihood, has been applied to the classification of base 13C NMR resonances in DNA oligomers. An accuracy of 100% for carbon class discrimination was achieved for a preliminary training set of four oligomers using the following four parameters: (1) the chemical shift; (2) the temperature at which the spectrum was obtained; (3) the difference in chemical shift from the C5 resonances; and (4) a sequence factor representing the neighboring nucleotides. Classification of a fifth oligomer, previously assigned and not contained in the original training set, gave reasonable carbon class assignments.