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Biomedical subjects

J R Wells

Publications and source records attributed to J R Wells.

At least 91 records · Page 5Linked to original sources
PubMed1979

Isolation, cryopreservation, and autotransplantation of human stem cells.

We have used cryopreserved autologous hematopoietic cells to repopulate bone marrow of patients receiving intensive chemotherapy and radiation. We employed the technique of density step centrifugation to concentrate stem cells from whole bone marrow. The concentrate of cells was frozen in vials using dimethyl sulphoxide (DMSO) as a cryo-protective agent and was stored in the vapor phase of liquid nitrogen. Test vials were thawed and assayed for myeloid stem cells (CFU-C) to determine the potential of the larger marrow aliquot. These test values were compared to the recovery of cells and CFU-C when the marrow was used for autotransplantation. The return of hematopoietic function after autotransplantation was evaluated and correlated with the dosage of cells and CFU-C infused.

Bone Marrow Cells↗
PubMed1977

Characterization of functionally distinct lymphoid and myeloid cells from human blood and bone marrow. I. Separation by a buoyant density gradient technique.

A buoyant density gradient procedure for mammalian cell separation is described. Gradients of Ficoll and Hypaque in balanced salt solution were used in a zonal rotor to separate distinct leukocyte populations from human peripheral blood and bone marrow. This method possesses the advantages of uniform osmolarity, density determination by refractometry, ease of preparation of gradient solutions, large sample capacity, and low viscosity with consequent ease of sterilization. Granulocyte-monocyte colony-forming units (CFU-C) from blood and bone marrow banded as a homogeneous peak with a density of 1.056 g cm-3 on steep gradients. Recovery of CFU-C was 36% with a 7- to 9-fold enrichment on shallow gradients. Both fractionated and unfractionated peripheral blood cells were stimulated by phytohemagglutinin (PHA), Concanavalin A (Con A), and allogeneic lymphocytes. In contrast, cells from unfractionated bone marrow and cells from the light density fraction of marrow were inhibited by Con A and responded variably to PHA.

Blood Cells↗
PubMed1977

Characterization of functionally distinct lymphoid and myeloid cells from human blood and bone marrow. II. Separation by velocity sedimentation.

Velocity sedimentation in a zonal rotor using gradients of uniform osmolarity was used to separate leukocyte subpopulations from human blood and bone marrow. The separations were performed at high sedimentation rates having the advantage of rapidity over conventional unit gravity separations. Myeloid stem cells (CFU-C) and cells reactive with phytohemagglutinin (PHA), Concanavalin A (Con A), and in mixed leukocyte culture (MLC) were separated and their sedimentation profiles obtained. CFU-C sedimented ahead of lymphoid cells and behind mature myeloid elements. Two distinct marrow subpopulations separated by velocity sedimentation were consistently stimulated by Con A, and variably stimulated by PHA and MLC reactions. Both large cells (predominantly myeloid) and small cells (predominantly lymphoid) from bone marrow were stimulated by Con A in [3H]thymidine incorporation assays. When separated subpopulations showing stimulation by Con A were mixed, inhibition of [3H]thymidine incorporation resulted.

Blood Cells↗
PubMed1977

Organization of sequences of avian globin mRNA.

Formamide polyacrylamide gel electrophoresis shows that chicken globin mRNA contains about 6.50 nucleotides, and since only 435 of these code for globin, a further 215 are not translated, and their function and position are not known. This work has produced the following conclusions. 1. 45-50 of these untranslated nucleotides are present as poly (A) at the 3' terminus. 2. The 3' untranslated region of chicken globin mRNA is at least 90 nucleotides in length. This minimal estimate is based on data derived from hybridization of defined lenghts of chicken globin cDNA to rabbit globin mRNA. The percentage of avian globin cDNA sequences which hybridize to rabbit globin mRNA is directly proportional to the length of the cDNA in each case. This relationship holds for lengths of cDNA from 115 up to 620 nucleotides. The low percentage homology for short cDNA molecules is not due to their being short per se. In homologous mRNA excess hybridizations (chicken cDNA/chicken mRNA), all cDNA preparations were completely protected from S1 nuclease digestion. 3. It is probable that there is greater evolutionary divergence in the 3' untranslated region of chicken and rabbit globin mRNA when compared with the coding regions of these molecules; The combined data is sued to formulate a regional map of chicken globin mRNA,

Animals↗
PubMed1976

The distirubtion of active genes (globin) and inactive genes (keratin) in fractionated chicken erythroid chromatin.

Repeatable fractionation of sheared chromatin from purified populations of chicken erythroid cells has been achieved, based on the Bio-Gel procedure of Janowski et al. ((1972) Karolinska Symp. 5, 112). For reticulocytes, 3-5% of chromatin DNA is excluded from Bio-Gel A-50 m (peak I) and over 90% elutes in the included volume of the column(peak II). Peak I material has a higher protein/DNA ratio than peak II chromatin and the two fractions have melting profiles characteristic of "active" and "inactive" chromatin, respectively. In cells prelabeled with [3H]uridine or [3H]leucine there was very pronounced preferential association of radioactivity with the "active" peak I chromatin. The distribution of "active" (globin) and "inactive" (keratin) gene sequences in the DNA of fractions from peak I and peak II chromatin was determined with complimentary DNA (cDNA) probes to chicken globin mRNA and chicken feather keratin mRNA. While slight enrichment for globin gene sequences was found in peak I (relative to DNA in these fractions), some 80% of the total globin hybrid formed was found in peak II fractions. Experiments with the keratin cDNA probe showed that these genes were equally distributed in both chromatin fractions rather than being confined to the "inactive" peak II material. The hybridization data in particular question the validity of claims for fractionation of chromatin into "active" and "inactive" material.

Anemia↗
PubMed1976

Reiteration frequency of the gene for tissue-specific histone H5 in the chicken genome.

Chicken erythroid cells contain a tissue specific histone known as H5 in addition to the five major histone species found in other organisms. The mRNA coding for this histone has been isolated by indirect immunoprecipitation from immature, non-dividing reticulocytes in which this is the only histone synthesised. The mRNA has been modified by the enzymatic addition of a 3' polyadenylic acid tract, and transcribed into complementary DNA (cDNA) using the RNA-dependent DNA-polymerase from avian myeloblastosis virus. Studies on the hybridisation of this cDNA indicate that the gene coding for the H5 histone is reiterated 10 times in the chicken genome.

Alleles↗
PubMed1976

Distribution of globin genes in chicken reticulocyte chromatin fractionated on urografin gradients.

Buoyant density centrifugation in Urografin solutions resolved French Pressure Cell-sheared, and micrococcal nuclease-digested avian reticulocyte chromatin into a broad profile of two peaks. Hybridization experiments using a globin cDNA probe suggested minimal fractionation of transcriptionally active and inactive components with chromatin sheared at 6000 psi, while no evidence was obtained for any fractionation with chromatin sheared at lower or higher pressures, or with chromatin digested to various extents with micrococcal nuclease, despite a considerable spread of chromatin material across gradients.

Animals↗
PubMed1976

Gene specific priming of complementary DNA synthesis.

DNA, complementary to chicken globin mRNA was synthesized using either Avian Myeloblastosis virus reverse transcriptase, or E. coli DNA polymerase I. Transcriptase cDNA sediments at 9 S on sucrose gradients, and is 620 nucleotides in length, representing a complete copy of globin mRNA template. In contrast, Polymerase I cDNA sediments at 4 S, is 100 to 200 nucleotides in length, and is a copy of a small region at the 3'(poly A) end of globin mRNA. Similarly, Transcriptase cDNA and Polymerase I cDNA hybridize to globin mRNA template with characteristic, individual Crot1/2 values. The Crot1/2 value for Transcriptase cDNA hybridization is 7 X 10(-4) mol s 1(-1), and that for Polymerase I cDNA is 5 X 10(-3). This work shows that Avian Myeloblastosis virus reverse transcriptase can use Polymerase I cDNA to prime further cDNA synthesis along the mRNA template. The product of extended cDNA synthesis is identical in length and hybridization properties to oligo (dT) primed transcriptase cDNA.

Avian Myeloblastosis Virus↗
PubMed1976

Preservation of granulopoietic precursors in nonfrozen, stored human bone marrow.

We have examined procedures for maintaining human bone marrow in a viable state for a period of 1 to 3 days. Various fractionation methods and environmental conditions were compared, and these results indicate that unfractionated, washed bone marrow cells stored at 25 C in a CO2-charged incubator gave the highest total recovery of granulopoietic stem cells capable of colony formation in vitro. After 24- and 48-hr incubation, a mean of 93 and 89% were preserved. Room temperature storage of bone marrow may have clinical applicability in autologous marrow infusion after high-dose chemoradiotherapy.

Bone Marrow↗
PubMed1975

Preparation of membrane-free chromatin bodies from avian erythroid cells and analysis of chromatin acidic proteins.

A primary objective, realized in this study, was the preparation from avian erythroid cells of chromatin free of contaminating membrane, as a prerequisiste to the study of chromatin acidic proteins from cells throughout the maturation pathway. Cells are lysed in saponin (S), washed in Nonidet-P40 (N), and plasma membrane removed by high-speed rotating knives (K). Purified SNK nuclear bodies are recovered free of membrane after centrifugation through 2.35 M sucrose. The chromatin acidic proteins from such preparations of the three major avian erythroid cell types were studied. Reticulocyte SNK chromatin was compared with reticulocyte chromatin derived from saponin lysis of cells and subsequent dispersion in EDTA solutions (Harlow et al. (1972), Cell Differ. 2, 341). The dispersed preparation has a lower acidic protein/DNA ratio, but the pattern of these proteins is more complex, presumably due to the contaminating membrane. In examining SNK acidic proteins throughout the maturation pathway it is clear that there are quantitative and qualitative differences. In the dividing erythroblast, the pattern of proteins is complex and the amount relative to DNA is 1.25:1.0. For nondividing, but transcriptionally active reticulocytes and also for transcriptionally inactive erythrocytes, the pattern is very much simpler, being dominated by three bands visible on sodium dodecyl sulfate polyacrylamide gels. The ratios of chromatin acidic proteins in these preparations relative to DNA are 0.69:1.0 and 0.36:1.0, respectively. These results, obtained with purified populations of cells from a single cell line, indicate a strong positive correlation between the transcriptional activity of the cell and both the amount and complexity of non-histone proteins associated with chromatin. The correlation does not indicate whether the proteins are the cause or result of increased transcription.

Amino Acids↗
PubMed1975

Histone proteases of avian erythroid cells.

Protease activity associated with avian erythroid chromatin has been studied by gel electrophoresis of histones. Histone degradation is minimal at neutral pH, but is readily detected when chromatin is incubated at pH 3, and is evident to a lesser extent at pH 9. As a result of the pH 3 activity, the f1 and f2 chistones are preferentially degraded when the histone complement is DNA-bound, but these histones are relatively resistant to attack when present as free histone. The pH 3 activity reported here has properties similar to those of neutral histone proteases from other tissues, except that it is not inhibited by bisulphite. Added exogenous proteins are not degraded. The activity of avian erythroid histone protease decreases as maturation of the cells proceeds. Since we have previously shown that turnover of DNA-bound f2c histone occurs in reticulocytes and histone synthesis is absent in erythrocytes, it is possible that the histone protease described here may be involved in f2c histone turnover.

Animals↗
PubMed1974

The amino acid sequence of plastocyanin from French bean (Phaseolus vulgaris).

The amino acid sequence of the plastocyanin from French bean (Phaseolus vulgaris) was determined. The protein consists of a single polypeptide chain of 99 residues, and the sequence was determined by characterization of CNBr, tryptic, chymotryptic and thermolysin peptides. When the sequence is compared with that from the plastocyanin of the unicellular green alga Chlorella fusca, the French-bean protein shows the deletion of the N-terminal residue, a two residue insertion and 53 identical residues. Detailed evidence for the sequence of the protein has been deposited as Supplementary Publication SUP 50037 (16pp., 1 microfiche) at the British Library (Lending Division) (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1973) 131, 5.

Amino Acid Sequence↗