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S D Patterson

Publications and source records attributed to S D Patterson.

At least 73 records · Page 4Linked to original sources
PubMed1997

The C-terminal domain of RNA polymerase II couples mRNA processing to transcription.

Messenger RNA is produced by RNA polymerase II (pol II) transcription, followed by processing of the primary transcript. Transcription, splicing and cleavage-polyadenylation can occur independently in vitro, but we demonstrate here that these processes are intimately linked in vivo. We show that the carboxy-terminal domain (CTD) of the pol II large subunit is required for efficient RNA processing. Splicing, processing of the 3' end and termination of transcription downstream of the poly(A) site, are all inhibited by truncation of the CTD. We found that the cleavage-polyadenylation factors CPSF and CstF specifically bound to CTD affinity columns and copurified with pol II in a high-molecular-mass complex. Our demonstration of an association between the CTD and 3'-processing factors, considered together with reports of a similar interaction with splicing factors, suggests that an mRNA 'factory' exists which carries out coupled transcription, splicing and cleavage-polyadenylation of mRNA precursors.

Animals↗
PubMed1997

Cytosolic proteins from neutrophilic granulocytes: a comparison between patients with severe chronic neutropenia and healthy donors.

A modified technique of two-dimensional gel electrophoresis (2-DE) was used to investigate differences in the pattern of cytosolic proteins of neutrophilic granulocytes from patients with severe congenital neutropenia, cyclic neutropenia, and idiopathic neutropenia in comparison with healthy donors. At the time of study, all patients tested received treatment with a recombinant human granulocyte colony-stimulating factor (r-metHuG-CSF; Filgrastim, Amgen). Using the Investigator 2-D Electrophoresis System (Millipore) we were able to detect more than 1000 protein spots in the cytosol of neutrophilic granulocytes from both patients and healthy controls. We investigated six patients with severe congenital neutropenia, five patients with cyclic neutropenia, four patients with idiopathic neutropenia, and 13 healthy donors. In the cytosol of neutrophilic granulocytes from patients we found an additional protein spot. This protein spot (molecular mass approximately 32.4 kDa, pI about 5.5) could be identified by internal sequencing after in-gel digestion with endoproteinase Lys-C as tropomyosin. The importance of the overexpression of tropomyosin in neutrophilic granulocytes from patients with severe chronic neutropenia is not yet understood.

Amino Acid Sequence↗
PubMed1996

Application of combined mass spectrometry and partial amino acid sequence to the identification of gel-separated proteins.

The combined use of peptide mass information with amino acid sequence information derived by chemical sequencing or mass spectrometry (MS)-based approaches provides a powerful means of protein identification. We have used a two-part strategy to identify proteins from nerve growth factor (NGF)-stimulated rat adrenal pheochromocytoma cell line PC-12 cell lysates that associate with the adaptor protein Shc (Shc homologous and collagen protein). Initial experiments with metabolically radiolabeled cell extracts separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a number of proteins that coimmunoprecipitated with anti-Shc antibody compared with control (unstimulated) cell extracts. The experiment was scaled up and cell lysate from NGF-stimulated PC-12 cells was applied to a glutathione-S-transferase (GST)-Shc affinity column, eluted, separated by SDS-PAGE and blotted to Immobilon-CD. The blotted proteins were proteolytically digested in situ, and the masses obtained from the extracted peptides were used in a peptide-mass search program in an attempt to identify the protein. Even if a strong candidate was found using this search, an additional step was performed to confirm the identification. The mixtures were fractionated by reversed-phase high-performance liquid chromatography (RP-HPLC) and subjected to chemical sequencing to obtain (partial) sequence information, or post-source decay (PSD-) matrix-assisted laser-desorption ionization (MALDI)-MS to obtain sequence-specific fragment ions. This data was used in a peptide-sequence tag search to confirm the identity of the proteins. This combined approach allowed identification of four proteins of M(r) 43,000 to 200,000. In one case the identified protein clearly did not correspond to the radiolabeled band, but to a protein contaminant from the column. The advantages and pitfalls of the approach are discussed.

Actinin↗
PubMed1996

Intrafocal (Kapandji) pinning of unstable distal radius fractures: a preliminary report.

Seventeen consecutive patients (17 wrists) who underwent intrafocal pinning of unstable distal radius fractures, as described by Kapandji, were retrospectively reviewed at a mean of 42 weeks after surgery (range, 13-88 weeks). The patients were immobilized for 6 weeks postoperatively. All patients were assessed by a single physician and were asked a set of questions regarding subjective and functional status. All patients underwent physical examination of their upper extremities, and bilateral wrist PA and lateral x-rays were taken. There were 13 females and four males; the average age was 49 years. Ten dominant and seven nondominant extremities were involved. Average volar tilt on follow-up lateral wrist radiography was 7 degrees. This compares with -20 degrees at initial presentation, -12 degrees preoperatively, 6 degrees immediately postoperatively, and 10 degrees in the normal wrists. Radial shortening (average) was -2 mm at initial presentation, -1mm preoperatively, 1 mm postoperatively, 0 mm at follow-up, and 1 mm in the normal wrists. Radial inclination was 17 degrees initially, 20 degrees preoperatively, 23 degrees immediately postoperatively, 23 degrees on follow-up, and 24 degrees in the normal wrists. There was a trend for patients with osteopenic bone to lose their postoperative reduction. However, this was not statistically significant. Patients older than 65 years of age had significantly inferior radiologic results. Loss of pronation and supination averaged 2 degrees (range 0-10 degrees) compared with the uninjured wrist. Loss of dorsiflexion averaged 6.5 degrees, and palmar flexion averaged 7.6 degrees. The patients' subjective complaints were minimal. Average pain on visual analog scale (VAS) was 0.44/10. Function measured 8.64/10 (VAS). Sixteen of the patients were happy with the surgery and the outcome of their wrists. Complications included extensor tendon rupture (one patient), pin migration requiring premature removal (one patient), and initial loss of reduction requiring reoperation (one patient). Intrafocal pinning of unstable distal radius fractures provides an effective means to stabilize these complex injuries. Early follow-up suggests that the patients have a satisfactory functional outcome. The complications in this series were preventable. Intrafocal pinning should be added to the surgical armamentarium in treating distal radius fractures.

Adolescent↗
PubMed1995

Src homologous and collagen (Shc) protein binds to F-actin and translocates to the cytoskeleton upon nerve growth factor stimulation in PC12 cells.

Immunoprecipitates of metabolically labeled PC12 cells consistently contained a 43-kDa protein that was associated with Shc, a signal-transducing protein with a single SH2 domain. Following affinity chromatography with immobilized recombinant glutathione S-transferase (GST)-Shc fusion protein, the 43-kDa protein was identified as actin by mass spectrometry and immunoblotting. Cosedimentation experiments using purified actin and GST-Shc showed that Shc binds directly to F-actin, confirming Shc-actin interaction in vivo. Various GST-truncated Shc fusion proteins were prepared and used in actin cosedimentation assays. Constructs containing the SH2 and collagen homology domains were not precipitated, and those containing the amino-terminal domain were. Thus, Shc-actin interactions do not occur in the region of tyrosine phosphorylation and leave the SH2 domain free to bind to other tyrosine-phosphorylated molecules. Although the major pool of Shc in unstimulated PC12 cells is soluble, two other pools are associated with the cytoskeleton and the submembranous cytoskeleton. Upon nerve growth factor stimulation, approximately 50% of the soluble Shc translocates to both cytoskeleton environments within 2 min, decreasing thereafter. When cells were pretreated with cytochalasin D, a drug that disrupts actin filaments, Shc translocation to the cytoskeleton was abolished. However, in the submembranous fraction, the Shc level was elevated in resting cells following cytochalasin D treatment. The kinetics of translocation, compared to mitogen-activated protein kinase activation, and the nature of the Shc-actin interaction suggest that the cytoskeletal association of Shc, induced by growth factors, may be related to membrane ruffling and actin fiber reorganization.

Actins↗
PubMed1995

Reduced numatrin/B23/nucleophosmin labeling in apoptotic Jurkat T-lymphoblasts.

Jurkat T-lymphoblasts were induced to undergo apoptosis by treatment with either EGTA (5 mM/24 h) or a high concentration of lovastatin (100 microM/48 h) to identify proteins that exhibited coordinate regulation between the two treatments and thus provide candidate proteins in the common apoptotic induction pathway. A pure population of apoptotic cells, as determined by morphology, "DNA laddering," and flow cytometry, was obtained by Percoll density gradient centrifugation. Cells of increased buoyant density were clearly apoptotic by all criteria. Following this gradient centrifugation, the cells were labeled with [35S]methionine/cysteine, and lysates were separated by two-dimensional polyacrylamide gel electrophoresis. Surprisingly, the two-dimensional polyacrylamide gel electrophoresis patterns generated from the apoptotic cells did not differ dramatically from that of control cells. Thus, apoptotic Jurkat cells are able to synthesize new proteins and do not exhibit extensive proteolysis. Subsequent quantitative analysis revealed that only five proteins exhibited decreases in turnover that were common to the two treatments. No increases in protein turnover were able to be confirmed across the replicate experiments. One of the proteins that showed decreased labeling by both apoptotic inductions was an abundant nuclear protein with a pI of 5.1 and M(r) 40,000. This protein was identified as numatrin/B23/nucleophosmin (NPM) based on internal amino acid sequence, and this identity was confirmed by immunoblotting and mass spectrometry. NPM is implicated in a range of diverse cellular functions, but its role in apoptosis is unclear.

Apoptosis↗
PubMed1995

Matrix-assisted laser-desorption/ionization mass spectrometric approaches for the identification of gel-separated proteins in the 5-50 pmol range.

The ability to identify and characterize low picomole quantities of gel-separated proteins has greatly benefited from recent advancements in mass spectrometric analysis methods, particularly peptide-mass search routines. We are investigating the use of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS)) to gain as much mass information as possible from a single gel-separated protein species. This report details results obtained from one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separations, under nonreducing conditions, of known quantities of three proteins, followed by blotting to Immobilon-CD. Three methods were used to obtain MALDI-MS data from a single blotted protein band: (i) direct MALDI-MS of approximately 10% of the band, (ii) cyanogen bromide (CNBr) cleavage of another approximately 10% of the band, and (iii) enzymatic (endoproteinase Lys-C) digestion of the remaining approximately 70-80% of the band followed by MALDI-MS. At the level of 50 pmol of protein loaded onto the gel, data was obtained from all three approaches. At levels down to 5 pmol of protein loaded onto the gel, MALDI-MS data was obtained from the latter two methods, CNBr and Lys-C digestions, but not direct MALDI-MS. Sufficient peptide masses were obtained from the 5 pmol loads to identify two of the three test proteins using four mass search programs. Only limited peptide mass data was obtained from fetuin, a sialylated glycoprotein with six disulfides and no methionines, but it was identified.

Amino Acid Sequence↗
PubMed1995

The midline posterior elbow incision. An anatomical appraisal.

The formation of a painful neuroma after operations on the medial or lateral sides of the elbow is a common problem. Our aim was to determine the relationship of the cutaneous nerves to the three usual skin incisions around the elbow. In 18 freshly frozen cadaver arms we made three standard 16 cm incisions in the skin medially, laterally, and posteriorly and explored them using loupe magnification. The number of nerves crossing each incision was determined by gross observation and their diameter measured by electronic microcallipers. In ten arms, biopsies of the nerves in each incision were sent for histological examination. We found significantly more cutaneous nerves crossing the medial and lateral incisions than the posterior. The diameter of the nerves crossing the posterior incision was significantly smaller than those crossing the lateral incision. Cutaneous nerves are at considerable risk of injury when medial or lateral incisions are used to approach the elbow, but the posterior approach carries less hazard. The routine use of the posterior incision may reduce the incidence of symptomatic paraesthesia and the formation of a painful neuroma after operation.

Analysis of Variance↗
PubMed1994

Prompt fragmentation of disulfide-linked peptides during matrix-assisted laser desorption ionization mass spectrometry.

During the analysis of an Asp-N digest of a recombinant hematopoietic growth factor by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), we observed pseudomolecular ions corresponding to reduced forms of peptides known to be present only in single disulfide linkages. Chromatographic fractionation of the peptide digest, followed by MALDI-MS and electrospray ionization (ESI) MS, confirmed that the reduced peptides were not present in the map. Fragmentation of the disulfide-linked peptides into their reduced forms occurred upon ionization from different matrices (alpha-cyano-4-hydroxycinnamic acid,2,5-dihydroxybenzoic acid, and in some instances sinapinic acid) but only after increasing the laser fluence to above threshold. Analysis of the disulfide-linked peptide fractions by ESI-MS, before and after mixing and drying with matrix, indicated that the matrix did not cause reduction. In a low-energy tandem mass spectrometric experiment with one of the cystinyl peptides, fragmentation did not occur preferentially at the disulfide bond. The pseudomolecular ions exhibited the same m/z values by MALDI-MS as their chemically reduced counterparts, indicating that they arose due to prompt fragmentation or "in-source decay" rather than "post-source decay". This finding is important for MALDI-MS analysis of peptide maps of proteins and peptide fractions with intact disulfides.

Amino Acid Sequence↗
PubMed1994

Early changes in protein synthesis induced by basic fibroblast growth factor, nerve growth factor, and epidermal growth factor in PC12 pheochromocytoma cells.

Nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) stimulate neuronal differentiation, whereas epidermal growth factor (EGF) promotes only mitogenic responses in PC12 pheochromocytoma cells. The early changes in protein synthesis induced by bFGF, NGF, and EGF in these cells have been determined by two-dimensional PAGE of [35S]methionine-labeled proteins and computerized image analysis. The rate of synthesis of only 29 proteins (out of approximately 1500 identified) was found to be modulated during the first several hours of growth factor stimulation. Individually, 12 were affected by EGF, 23 were affected by bFGF, and 20 were affected by NGF. Eight of these were regulated by all three growth factors, while 10 proteins were commonly induced by bFGF and NGF, in accordance with the essentially identical morphological responses induced by these two factors. In addition, the effects of bFGF and NGF were about equally divided between increases and decreases in the rate of synthesis of individual proteins, whereas EGF caused significantly more positive (increased) responses. All proteins modulated by NGF or FGF alone were negative in their response and those induced by only EGF were positive. Of particular interest, the rate of synthesis of two proteins of 55 kDa and pI 5.45 and 5.50 was dramatically and transiently induced during the first 2 hr of bFGF and NGF treatment and was not affected by EGF. This study indicates that all three factors elicit early increases and decreases in the synthesis of a quite limited number of proteins and provides molecular evidence for the specificity of a differentiative vs. a proliferative growth factor-induced signaling pathway in these cells.

Adrenal Gland Neoplasms↗
PubMed1994

Expression, purification, crystallization, and preliminary x-ray analysis of casein kinase-1 from Schizosaccharomyces pombe.

The catalytic domain of Schizosaccharomyces pombe casein kinase-1 (the product of the cki1 gene) has been overexpressed in Escherichia coli, purified by chromatographic methods, characterized in vitro, and crystallized in the presence and absence of nucleotide substrate. The best crystals belong to the trigonal space group P3(1)21 or its enantiomorph, have unit cell parameters a = b = 79 A, c = 121 A, and diffract x-rays to 2.0-A resolution. Kinetic characterization of the purified catalytic domain and other C-terminal deletion mutants of Cki1 suggests that it is subject to two forms of regulation. One mechanism involves autophosphorylation, and results in a 4-fold decrease in the affinity for protein substrate. In contrast, truncation of intact Cki1 results in a 3-fold activation in its catalytic rate. This activation may arise from the removal of an inhibitory domain present in the intact enzyme.

Amino Acid Sequence↗
PubMed1993

Isolation and properties of YCK2, a Saccharomyces cerevisiae homolog of casein kinase-1.

A soluble fragment of YCK2, a casein kinase-1 isoform from Saccharomyces cerevisiae, has been purified and characterized in vitro. The procedure enriches enzyme activity to a final specific activity of 4.7 mumol min-1 mg-1 (when assayed with casein as substrate). Structural analysis reveals that the preparation arises from N-terminal modification and C-terminal proteolysis of the initially synthesized 546-residue protein, consisting of residues 2-495 +/- 1. Kinetic analysis demonstrates that YCK2 is similar to casein kinase-1 isolated from other organisms in its inability to use GTP as nucleotide substrate, in its sensitivity to heparin and ribofuranosyl-benzimidazole inhibitors, and in its peptide substrate selectivity. The enzyme is unusual, however, in that it is insensitive to the potent mammalian casein kinase-1 inhibitor N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide.

Amino Acid Sequence↗
PubMed1993

A special plate for arthrodesis of the wrist: design considerations and biomechanical testing.

The purpose of this investigation was to determine the strength and mode of failure of a plate specifically designed for wrist arthrodesis. An eight-hole plate was constructed with a solid central portion and a distal taper. Four 2.7 mm screws were used in the distal (metacarpal) portion of the plate. Four 3.5 mm screws were used in the proximal (radius) portion. The strength of the wrist arthrodesis plate was compared with that of a conventional 10-hole 3.5 dynamic compression plate by gap testing and a paired cadaver wrist arthrodesis construct. The strength of the wrist arthrodesis plate was similar to the strength of the conventional dynamic compression plate when applied to cadaver wrist arthrodesis constructs. Failure of the wrist arthrodesis constructs occurred through bone without deformation of either plate.

Arthrodesis↗