scholarly journals Differential localization of tropomyosin isoforms in cultured nonmuscle cells.

1988 ◽  
Vol 107 (2) ◽  
pp. 563-572 ◽  
Author(s):  
J J Lin ◽  
T E Hegmann ◽  
J L Lin
Keyword(s):  
Molecular Mass ◽  
Chicken Embryo Fibroblast ◽  
Polyclonal Antiserum ◽  
Western Blots ◽  
Tropomyosin Isoforms ◽  
Lower Molecular Mass ◽  
Nonmuscle Cells ◽  
Double Label ◽  
Sequential Absorption ◽  
The Stability

We have previously shown that chicken embryo fibroblast (CEF) cells and human bladder carcinoma (EJ) cells contain multiple isoforms of tropomyosin, identified as a, b, 1, 2, and 3 in CEF cells and 1, 2, 3, 4, and 5 in human EJ cells by one-dimensional SDS-PAGE (Lin, J. J.-C., D. M. Helfman, S. H. Hughes, and C.-S. Chou. 1985. J. Cell Biol. 100: 692-703; and Lin, J. J.-C., S. Yamashiro-Matsumura, and F. Matsumura. 1984. Cancer Cells 1:57-65). Both isoform 3 (TM-3) of CEF and isoforms 4,5 (TM-4,-5) of human EJ cells are the minor isoforms found respectively in normal chicken and human cells. They have a lower apparent molecular mass and show a weaker affinity to actin filaments when compared to the higher molecular mass isoforms. Using individual tropomyosin isoforms immobilized on nitrocellulose papers and sequential absorption of polyclonal antiserum on these papers, we have prepared antibodies specific to CEF TM-3 and to CEF TM-1,-2. In addition, two of our antitropomyosin mAbs, CG beta 6 and CG3, have now been demonstrated by Western blots, immunoprecipitation, and two-dimensional gel analysis to have specificities to human EJ TM-3 and TM-5, respectively. By using these isoform-specific reagents, we are able to compare the intracellular localizations of the lower and higher molecular mass isoforms in both CEF and human EJ cells. We have found that both lower and higher molecular mass isoforms of tropomyosin are localized along stress fibers of cells, as one would expect. However, the lower molecular mass isoforms are also distributed in regions near ruffling membranes. Further evidence for this different localization of different tropomyosin isoforms comes from double-label immunofluorescence microscopy on the same CEF cells with affinity-purified antibody against TM-3, and monoclonal CG beta 6 antibody against TM-a, -b, -1, and -2 of CEF tropomyosin. The presence of the lower molecular mass isoform of tropomyosin in ruffling membranes may indicate a novel way for the nonmuscle cell to control the stability and organization of microfilaments, and to regulate the cell motility.

scholarly journals The purification of tissue inhibitor of metalloproteinases-2 from its 72 kDa progelatinase complex. Demonstration of the biochemical similarities of tissue inhibitor of metalloproteinases-2 and tissue inhibitor of metalloproteinases-1

1991 ◽  
Vol 278 (1) ◽  
pp. 179-187 ◽  
Author(s):  
R V Ward ◽  
R M Hembry ◽  
J J Reynolds ◽  
G Murphy
Keyword(s):  
Molecular Mass ◽  
Polyclonal Antiserum ◽  
High Molecular Mass ◽  
U937 Cells ◽  
Tissue Inhibitor Of Metalloproteinases ◽  
Gingival Fibroblasts ◽  
Tissue Inhibitor ◽  
Lower Molecular Mass ◽  
Inhibitory Activities

Human gingival fibroblasts in culture were shown to secrete a 72 kDa progelatinase, of which a proportion in the medium was found to be complexed with tissue inhibitor of metalloproteinases-2 (TIMP-2). A purification procedure was devised to purify free enzyme and inhibitor. We also describe the purification of both 95 kDa progelatinase bound to TIMP-1 and free 95 kDa progelatinase from the medium of U937 cells. A polyclonal antiserum to TIMP-2 was prepared and it was shown that TIMP-1 and TIMP-2 are antigenically distinct. The ability to form stable complexes and the relative inhibitory activities of TIMP-1 and TIMP-2 towards 95 kDa and 72 kDa gelatinases, collagenase, stromelysins 1 and 2 and punctuated metalloproteinase were determined; only minor differences were found. Complex-formation between TIMP-2 and 72 kDa progelatinase was demonstrated not to reduce the metalloproteinase-inhibitory activity of TIMP-2, a finding that led to the characterization of high-molecular-mass TIMP activity. Competition experiments between progelatinases and active gelatinases for TIMPs indicated that the affinity of TIMPs for progelatinases is weaker than that for active gelatinases. In a study of the effects of TIMP-1 and TIMP-2 on progelatinase self-cleavage we found that both TIMP-1 and TIMP-2 inhibit the conversion of 95 kDa and 72 kDa progelatinases and prostromelysin into lower-molecular-mass forms. TIMP capable of complexing with progelatinase was shown to be no more efficient an inhibitor of gelatinase self-cleavage than TIMP not able to complex with progelatinase.

scholarly journals Alternative RNA splicing of the human endothelin-A receptor generates multiple transcripts

1996 ◽  
Vol 313 (3) ◽  
pp. 795-801 ◽  
Author(s):  
Yoshihiro MIYAMOTO ◽  
Takaaki YOSHIMASA ◽  
Hiroshi ARAI ◽  
Kazuhiko TAKAYA ◽  
Yoshihiro OGAWA ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Rna Splicing ◽  
Cdna Clones ◽  
Human Tissues ◽  
Rt Pcr ◽  
Endothelin A Receptor ◽  
Lower Molecular Mass ◽  
Novel Transcripts ◽  
Exon 4 ◽  
Endothelin A

In order to elucidate the regulatory mechanisms of expression of the human endothelin-A receptor (hET-AR) gene, we characterized hET-AR transcripts using reverse transcriptase (RT)-PCR analysis in a variety of human tissues. RT-PCR of lung mRNA using a set of primers from exons 2 and 5 showed two lower-molecular-mass transcripts in addition to the expected fragment. When RT-PCR with primers from exons 4 and 8 was performed, no transcripts other than the expected one were detected. PCR cloning utilizing a set of primers from exons 2 and 8 which covered the entire coding sequence revealed that the cDNA clones corresponding to the two novel transcripts contained deletions of 199 bp and 327 bp respectively compared with the previously described hET-AR cDNA. Comparison of their sequences with that of the hET-AR gene showed that the deleted sequences correspond exactly to exon 4 and exons 3 and 4 respectively, indicating that these lower-molecular-mass ET-AR transcripts result from alternative RNA splicing (designated ET-AR∆4 and ET-AR∆3,4 respectively). Alternative splicing of exon 4 results in a transcript which would be translated into a C-terminal truncated protein containing the first, second and third transmembrane domains, while the splicing out of exons 3 and 4 would produce a protein with five membrane-spanning domains but lacking the third and fourth domains present in the ET-AR protein. An RNase protection assay revealed that ET-AR∆4 and ET-AR∆3,4, as well as ET-AR, transcripts were observed in various human tissues, including the lung, aorta, atrium, kidney and placenta, which are known to express ET-AR abundantly. Thus we have isolated the cDNAs of novel transcripts of hET-AR which are generated by alternative RNA splicing, and these results suggest that this alternative RNA splicing might contribute to the regulation of ET-AR gene expression.

scholarly journals Cloning and characterization of a Schistosoma mansoni 1H and 30S clones as two tegumental vaccine candidate antigens.

2003 ◽  
Vol 50 (1) ◽  
pp. 269-278
Author(s):  
Amr M Shabaan ◽  
Magdy M Mohamed ◽  
Mohga S Abdallah ◽  
Hayat M Ibrahim ◽  
Amr M Karim
Keyword(s):  
Amino Acid ◽  
Fusion Protein ◽  
Schistosoma Mansoni ◽  
Molecular Mass ◽  
Vaccine Development ◽  
Complete Sequence ◽  
Cdna Clones ◽  
Western Blots ◽  
Calculated Molecular Mass ◽  
Reading Frame

Two Schistosoma mansoni cDNA clones 30S and 1H were identified by immunoscreening of sporocyst lambdagt11 library and by random sequencing of clones from lambdaZap libraries, respectively. Clone 30S was one of 30 clones identified by an antibody raised against tegument of 3-h schistosomules. The clone was found to encode an 81 amino-acid protein fragment. It was expressed in Escherichia coli as a fusion protein of calculated molecular mass of about 35 kDa with C-terminus of Schistosoma japonicum glutathione-S-transferase (Sj26; about 26 kDa). The recombinant fusion protein was specifically recognized by serum of rabbits immunized with irradiated cercariae. Clone 1H is one of 76 expressed sequence tags derived from an adult worm library. It encodes the complete sequence of a tegumental membrane protein, Sm13. The 104 amino-acid open reading frame encodes a protein with a calculated molecular mass of about 11.9 kDa. Clone 1H was expressed in E. coli as an insoluble fusion protein with Sj26 of about 40 kDa. In Western blots, the fusion protein was recognized by serum from rabbits vaccinated with irradiated cercariae but not by preimmune rabbit sera. The cloning, characterization and expression of those proteins are therefore potentially usefull for vaccine development.

scholarly journals Temperature Regulation of the Hemin Storage (Hms+) Phenotype of Yersinia pestis Is Posttranscriptional

2004 ◽  
Vol 186 (6) ◽  
pp. 1638-1647 ◽  
Author(s):  
Robert D. Perry ◽  
Alexander G. Bobrov ◽  
Olga Kirillina ◽  
Heather A. Jones ◽  
Lisa Pedersen ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Yersinia Pestis ◽  
Growth Temperature ◽  
Temperature Regulation ◽  
Iron Status ◽  
Outer Membrane Proteins ◽  
Western Blots ◽  
Protein Levels ◽  
The Stability ◽  
In Cells

ABSTRACT In Yersinia pestis, the Congo red (and hemin) binding that is characteristic of the Hms+ phenotype occurs at temperatures up to 34°C but not at higher temperatures. Manifestation of the Hms+ phenotype requires at least five proteins (HmsH, -F, -R, -S, and -T) that are organized into two separate operons: hmsHFRS and hmsT. HmsH and HmsF are outer membrane proteins, while HmsR, HmsS, and HmsT are predicted to be inner membrane proteins. We have used transcriptional reporter constructs, RNA dot blots, and Western blots to examine the expression of hms operons and proteins. Our studies indicate that transcription from the hmsHFRS and hmsT promoters is not regulated by the iron status of the cells, growth temperature, or any of the Hms proteins. In addition, the level of mRNA for both operons is not significantly affected by growth temperature. However, protein levels of HmsH, HmsR, and HmsT in cells grown at 37°C are very low compared to those in cells grown at 26°C, while the amounts of HmsF and HmsS show only a moderate reduction at the higher growth temperature. Neither the Pla protease nor a putative endopeptidase (Y2360) encoded upstream of hmsH is essential for temperature regulation of the Hms+ phenotype. However, HmsT at 37°C is sensitive to degradation by Lon and/or ClpPX. Thus, the stability of HmsH, HmsR, and HmsT proteins likely plays a role in temperature regulation of the Hms+ phenotype of Y. pestis.

scholarly journals The neuropeptide proctolin induces phosphorylation of a 30 kDa protein associated with the thin filament in crustacean muscle

2001 ◽  
Vol 204 (15) ◽  
pp. 2627-2635 ◽  
Author(s):  
Berit Brüstle ◽  
Sabine Kreissl ◽  
Donald L. Mykles ◽  
Werner Rathmayer
Keyword(s):  
Molecular Mass ◽  
Thin Filament ◽  
Troponin I ◽  
Extensor Muscle ◽  
Apparent Molecular Mass ◽  
Muscle Fibres ◽  
Single Pair ◽  
Myofibrillar Proteins ◽  
Single Muscle ◽  
Western Blots

SUMMARYIn the isopod Idotea emarginata, the neuropeptide proctolin is contained in a single pair of motoneurones located in pereion ganglion 4. The two neurones supply dorsal extensor muscle fibres of all segments. Proctolin (1μmoll−1) potentiates the amplitude of contractures of single extensor muscle fibres elicited by 10mmoll−1 caffeine. In western blots of myofibrillar proteins isolated from single muscle fibres and treated with an anti-phosphoserine antibody, a protein with an apparent molecular mass of 30kDa was consistently found. The phosphorylation of this protein was significantly increased by treating the fibres with proctolin. After separation of myofibrillar filaments, a 30kDa protein was found only in the thin filament fraction. This protein is phosphorylated and detected by an antiserum against crustacean troponin I.

scholarly journals A soluble NADH dehydrogenase (NADH: ferricyanide oxidoreductase) from Thermus aquaticus strain T351

1983 ◽  
Vol 209 (2) ◽  
pp. 427-433 ◽  
Author(s):  
K A J Walsh ◽  
R M Daniel ◽  
H W Morgan
Keyword(s):  
Absorption Coefficient ◽  
Molecular Mass ◽  
Half Life ◽  
Nadh Dehydrogenase ◽  
Molar Absorption Coefficient ◽  
Effect Of Temperature ◽  
Thermus Aquaticus ◽  
Membrane Bound ◽  
The Stability

A soluble NADH dehydrogenase (NADH:ferricyanide oxidoreductase) has been obtained by simple disruption of cells of Thermus aquaticus strain T351, and purified. The enzyme is of low molecular mass, 50 000 Da, and displays many of the properties of the membrane-bound enzyme, including inhibition by both NADH and ferricyanide, and the same Km for ferricyanide. The enzyme contains 0.05 mol of FMN, 0.16 mol of labile sulphur and 2.2 mol of iron per mol of protein. The enzyme is inhibited by NAD and cupferron competitively with ferricyanide, and by ATP (but not ADP) competitively with NADH. The enzyme is particularly thermostable, having a half-life at 95 degrees C of 35 min. The effect of temperature on the molar absorption coefficient and the stability of NADH was determined.

Na(+)-Ca2+ exchanger-like immunoreactivity and regulation of intracellular Ca2+ levels in fish retinal ganglion cells

1994 ◽  
Vol 72 (1) ◽  
pp. 47-55 ◽  
Author(s):  
V. P. Bindokas ◽  
M. Yoshikawa ◽  
A. T. Ishida
Keyword(s):  
Intracellular Calcium ◽  
Retinal Ganglion Cells ◽  
Ganglion Cells ◽  
Apparent Molecular Weight ◽  
Polyclonal Antiserum ◽  
Retinal Ganglion ◽  
Western Blots ◽  
Outer Segments ◽  
Teleost Retina ◽  
Intracellular Calcium Ion

1. We have used two experimental approaches to examine regulation of intracellular calcium ion levels in fish retinal ganglion cells. In the first set of experiments, we ratio-imaged fura-2 emission intensity to estimate the concentration of free intracellular calcium ions ([Ca2+]i) in isolated goldfish retinal ganglion cells depolarized by increases in extracellular levels of potassium ions ([K+]o), in the presence and absence of extracellular sodium ions (Na+). Stepwise increases in [K+]o from 5 mM to as high as 60 mM produced stepwise increases in [Ca2+]i. These increases were sustained in the absence of external Na+, but transient and smaller in the presence of external Na+. The decline of [Ca2+]i in high-K, Na(+)-containing saline could be reversed by application of the ionophore monensin, or by replacement of external Na+ with either N-methyl-D-glucamine or lithium. In Na(+)-containing saline, [Ca2+]i fell to control levels after [K+]o was restored to control levels. 2. In the second set of experiments, we assessed Na(+)-Ca2+ exchanger-like immunoreactivity in goldfish retinal ganglion cells with the use of a polyclonal antiserum directed against Na(+)-Ca2+,K+ exchanger purified from bovine rod outer segments. This antiserum specifically stained the somata, neurites, and growth cones of isolated ganglion cells, the outer segments of rod photoreceptors, and (on Western blots prepared from mechanically isolated rods) protein displaying an apparent molecular mass of 210 kDa. 3. These results provide measurements of changes in [Ca2+]i of retinal ganglion cells depolarized in Na(+)-containing saline, and the distribution and apparent molecular weight of Na(+)-Ca2+ exchanger-like immunoreactivity in teleost retina.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Effect of diosgenin on biliary cholesterol transport in the rat

1993 ◽  
Vol 291 (3) ◽  
pp. 793-798 ◽  
Author(s):  
A Thewles ◽  
R A Parslow ◽  
R Coleman
Keyword(s):  
Bile Salt ◽  
Molecular Mass ◽  
Bile Salts ◽  
Mass Fraction ◽  
Gel Permeation Chromatography ◽  
High Molecular Mass ◽  
Cholesterol Transport ◽  
Biliary Cholesterol ◽  
Lower Molecular Mass ◽  
Salt Depletion

Biliary cholesterol output in rats was stimulated over 3-fold by feeding diosgenin for 5 days, whereas biliary outputs of phospholipid and bile salts were not changed by diosgenin feeding. Isolating and perfusing the liver without bile salts resulted in a rapid and substantial decrease in biliary bile salt output; bile salt depletion abolished the diosgenin-induced increment in biliary cholesterol output, showing that the diosgenin-elevated biliary cholesterol output was bile-salt-dependent. Diosgenin treatment also produced a significant decrease in biliary alkaline phosphodiesterase I. Fresh bile obtained from control and diosgenin-fed rats was subjected to gel-permeation chromatography in order to separate different-sized biliary cholesterol carriers. Two major peaks of cholesterol were eluted, with cholesterol also being eluted between the peaks. The cholesterol peak eluted at the lower molecular mass (20-30 kDa) was observed in all bile samples. The higher-molecular-mass peak, which was eluted at the void volume, was not observed in all biles; control biles contained very little high-molecular-mass form of cholesterol, whereas biles from the diosgenin group contained up to 47% of cholesterol in the high-molecular-mass fraction. Diosgenin treatment produced a range of elevated biliary cholesterol values which positively correlated with the proportion of cholesterol contained in the high-molecular-mass fraction (r = 0.98). The results show that diosgenin induced a marked bile-salt-dependent increase in biliary cholesterol output and a shift in biliary cholesterol transport to higher-molecular-mass structures.

scholarly journals Continuous s.c. infusion rather than twice-daily injections of IGF-I more effectively increases serum IGF binding protein-3 in female monkeys

1999 ◽  
pp. 303-312 ◽  
Author(s):  
ME Wilson ◽  
SL Lackey
Keyword(s):  
Molecular Mass ◽  
Ternary Complex ◽  
Binding Protein ◽  
Serum Insulin ◽  
Size Exclusion ◽  
Constant Infusion ◽  
Serum Concentrations ◽  
Lower Molecular Mass ◽  
Igf I ◽  
Igfbp 3

OBJECTIVE: In order to better understand how the IGF-I axis is affected by exogenous IGF-I, this study compared the effects of a constant s.c. infusion of IGF-I with that of twice-daily injections of IGF-I in young adult female rhesus monkeys. Clinical studies suggest that circulating concentrations of insulin-like growth factor binding protein-3 (IGFBP-3) are decreased or unaffected by IGF-I administration, whereas acute increases in IGF-I may increase serum IGFBP-1. However, studies in monkeys indicate that acute or continuous infusion of IGF-I effectively increases serum IGFBP-3. DESIGN AND METHODS: Female monkeys were studied for 5 days with no IGF-I supplementation (baseline) and for 5 days of IGF-I treatment by either constant infusion (120 microg/kg per day s.c., n = 5) or twice-daily injections of IGF-I (60 microg/kg per injection s.c., n = 5). Serum samples were collected daily at 0800 h and at 0800, 0900, 1100, 1500, and 2000 h on days 1 and 4 for each condition. Samples were assayed for IGF-I, IGFBPs-1 and -3, insulin, and glucose. RESULTS: Serum IGF-I was consistently increased above baseline within 24 h of the initiation of constant infusion, but was delayed until the second day of treatment in the injection group. Serum IGFBP-3 followed the pattern of IGF-I, with concentrations increased by day 1 during constant infusion and by day 2 during intermittent injections. Although both treatments effectively increased serum IGFBP-3, the increase was greater during constant infusion (31% above baseline) compared with injection (17%). Immunoblotting revealed that the constant infusion of IGF-I resulted in quantitatively more lower-molecular-mass fragments of IGFBP-3 than were observed during baseline or intermittent injections. Size-exclusion chromatography and ultrafiltration indicated that most IGFBP-3 was found in the ternary complex, with a greater percentage found in the ternary complex during baseline (90%) than during constant infusion (86%) or intermittent injections of IGF-I (87%). In contrast, serum concentrations of IGFBP-1 were increased on day 1 of both treatments, but declined towards baseline values as treatment progressed. Serum concentrations of insulin and glucose were unaffected by either mode of IGF-I treatment. Serum concentrations of IGF-I and IGFBP-3 were increased within 3h of the injection, before declining towards the pre-injection level. In contrast, the daily pattern of serum hormone concentrations was similar between the baseline condition and during constant infusion of IGF-I. Although higher during the treatment phase, serum IGF-I and IGFBP-3 concentrations decreased significantly from 0800 h until the afternoon meal, reaching a nadir in the evening before increasing again the next morning. Serum insulin decreased also after the morning meal and increased significantly immediately after the afternoon meal. Although serum IGFBP-1 also decreased initially after the morning meal, concentrations reached a peak before the afternoon meal as serum insulin reached its nadir. CONCLUSION: The results of the present analysis indicate that the constant infusion of IGF-I more effectively sustains serum concentrations of IGF-I and IGFBP-3 than do twice-daily injections. Although the percentage of IGF-I and IGFBP-3 in the ternary complex was similar during both treatments, the constant infusion regimen produced lower-molecular-mass fragments of IGFBP-3. In addition, serum IGF-I and IGFBP-3 appeared to be regulated diurnally, even during IGF-I infusion, whereas IGFBP-1 and insulin were affected by the timing of food intake. Taken together, these data suggest that, in the monkey, IGFBP-3 is regulated by factors in addition to GH, and that IGF-I can affect its own bioavailability by increasing circulating concentrations of IGFBP-3.

Sign in / Sign up

Export Citation Format

Share Document