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Biomedical subjects

R M Robson

Publications and source records attributed to R M Robson.

At least 37 records · Page 2Linked to original sources
PubMed1995

Effects of postmortem aging time, animal age, and sex on degradation of titin and nebulin in bovine longissimus muscle.

This study was conducted to determine degradation of the giant myofibrillar proteins titin and nebulin in postmortem aged beef, with known tenderness values, from animals differing in sex (steers vs bulls) and age (cows vs steers and bulls). Ten bulls and 10 steers (both groups were approximately 14 mo old) and 10 cows (44 to 108 mo old) were slaughtered. Longissimus muscle samples were removed for determination of Warner-Bratzler shear force, sensory panel tenderness evaluation, and SDS-PAGE analysis at 3, 7, 14, and 28 d postmortem. The SDS-PAGE analysis of titin and nebulin revealed that titin often migrated as three closely-spaced bands (T1, T1-2, T2, in increasing order of migration) in 3-d postmortem samples. With increasing time post-mortem, intact titin (T1) decreased and degraded titin (T2) increased in all samples. Within a class (i.e., steers, bulls, or cows) the rate of conversion of T1 to T2 was slower in the less-tender samples. The T1 to T2 conversion postmortem was slower in the intact males (bulls) than in the castrated males (steers). The T1 to T2 conversion postmortem also was slower in the older animals (cows) in comparison to the younger steers, but not in comparison to the younger bulls. Nebulin was degraded by 3 d postmortem in tender samples from steers, but some nebulin remained in the less-tender 3-d samples from steers and in all of the 3-d samples from bulls and older animals (cows). Intact nebulin was absent in all 7-d samples, regardless of the class of animal. Our results suggest that titin and nebulin are degraded at faster rates in more tender beef samples within each of the three classes of animals examined. The rate of degradation seems to differ when sex and age classifications are compared.

Aging↗
PubMed1994

The hypE gene completes the gene cluster for H2-oxidation in Azotobacter vinelandii.

The nucleotide sequence was obtained for the hypE gene in the cluster of structural and accessory genes required for the assembly and functioning of the membrane-bound, dimeric, (NiFe)hydrogenase in Azotobacter vinelandii. The hypE gene encodes a polypeptide of 341 amino acid residues which is rich in alanine, glycine, valine and proline and appears to be involved in maturation of the enzyme because chromosomal mutations in hypE block O2-dependent H2-oxidation and affect the amount, processing and localization of the (NiFe) hydrogenase alpha-subunit. The complete nucleotide sequence for the hydrogenase gene cluster in A. vinelandii has now been assembled into a contiguous sequence of 13,914 bp containing 16 potential genes which appear to be transcribed undirectionally. They are arranged in the order hoxK, hoxG, hoxZ, hoxM, hoxL, hoxO, hoxQ, hoxR, hoxT, hoxV, hypA, hypB, hypF, hypC, hypD and hypE. This cluster closely resembles those described for comparable (NiFe) hydrogenases in other bacteria.

Amino Acid Sequence↗
PubMed1994

Substructure of cytoplasmic dense bodies and changes in distribution of desmin and alpha-actinin in developing smooth muscle cells.

The substructure of assembling cytoplasmic dense bodies (CDBs) and changes in the distribution of desmin and alpha-actinin during development of smooth muscle were studied in gizzard samples from 10- and 16-day embryos and from 1- and 7-day post-hatch chickens. CDBs in these cells lack the density of CDBs in mature or adult smooth muscle cells and, thus, allow observations of the changes inside CDBs. The random filament orientation seen in younger embryonic cells is first modified to include relatively small patches of IFs that are somewhat straighter and are approaching a side-by-side arrangement. As development proceeds, the IFs in these arrays become straighter, are parallel over longer lengths of the IFs and later acquire the density characteristic of mature CDBs. Anti-desmin labeling in embryonic 10- and 16-day cells showed that desmin intermediate filaments (IFs) were located in the myofilament compartment but were concentrated in or near assembling CDBs. Anti-desmin labeling shifted to the perimeter of CDBs after hatching. Cross sections, longitudinal sections, and stereo pairs all show that IF profiles are present inside unlabeled assembling CDBs. Anti-alpha-actinin labeling was directly on CDBs and was often associated with the cross-connecting filaments (CCFs) (average diameter of 2-3nm) inside CDBs. We propose, based on these data, that desmin IFs, alpha-actinin-containing CCFs, and actin filaments are the principal components of the substructure of assembling CDBs. We also present a proposed model for CDB assembly.

Actinin↗
PubMed1994

Identification of the 30 kDa polypeptide in post mortem skeletal muscle as a degradation product of troponin-T.

Although a 30 kDa polypeptide frequently is seen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of post mortem (pm) skeletal muscle and in turn is used as an indicator of proteolysis, its origin has not been conclusively identified. We used antibodies to selected myofibrillar proteins, including some known to be degraded pm, to identify this polypeptide. The left side of eight beef carcasses was electrically stimulated (ES) within 1 h after slaughter, and the right side served as the non-stimulated (NS) control. The longissimus lumborum (LL) muscle was removed from the carcass at 24 h pm and was stored at 2 degrees C. Myofibrils were prepared from the LL muscle immediately after stimulation (0 day) and from the stored muscle sample at 1, 3, 7, 14 and 28 days pm for analysis of SDS-PAGE and Western blots. By SDS-PAGE, troponin-T (TN-T) decreased in amount more rapidly pm in ES samples than in NS samples. By SDS-PAGE, a 30 kDa band increased and became a prominent band by 7 days pm in both NS and ES samples. A monoclonal antibody (mAb) to TN-T labeled purified TN-T, as well as the TN-T in myofibrils, a prominent 30 kDa polypeptide and a family of lower molecular mass polypeptides in pm muscle. This mAb also labeled a 30 kDa band that had been electrophoretically purified from pm muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

ADP-ribosylation of the intermediate filament protein desmin and inhibition of desmin assembly in vitro by muscle ADP-ribosyltransferase.

Arginine-specific mono(ADP-ribosyl)transferase purified from rabbit skeletal muscle catalyzes stoichiometric ADP-ribosylation of the intermediate filament protein, desmin. In contrast, cholera toxin catalyzes a much lower level of ADP-ribosylation of desmin. Modification results in potent inhibition of desmin's ability to assemble into filaments. Phosphorylation of desmin by the catalytic subunit of cAMP dependent protein kinase is also inhibited by ADP-ribosylation. ADP-ribosylation site(s) are located within the N-terminal head domain of desmin.

ADP Ribose Transferases↗
PubMed1993

The marked pH dependence of the talin-actin interaction.

The effect of pH on the interaction between talin and actin was examined by using cosedimentation assays, low shear viscometry, and electron microscopy. When the pH at which the interaction was tested was lowered from 7.5 to 7.0 and below, the ability of talin to cosediment with F-actin was greatly enhanced, with maximal binding at pH 6.6. Talin's ability to increase the low shear viscosity of F-actin solutions was also significantly elevated as pH was decreased from 7.4 to 6.6. Electron microscope observations of negatively stained actin controls and talin-actin mixtures supported these results by demonstrating an increase in the degree of crosslinking of actin filaments in the presence of talin at pH values of 7 and lower.

Actins↗
PubMed1993

Molecular characterization of avian muscle titin.

Titin is an approximately 3000-kDa polypeptide that constitutes a set of elastic filaments that connect thick filaments to the Z-line in vertebrate striated muscle myofibrils. To characterize the primary structure of titin, three overlapping cDNA clones comprising 2.4 kilobases of avian muscle titin coding sequence were obtained from a cDNA library constructed from embryonic chick cardiac muscle RNA size-selected for large transcripts. Expression of one cDNA clone in Escherichia coli produced a fusion protein that reacted specifically with titin antibodies, and titin antiserum affinity-purified against this fusion protein reacted specifically with titin on immunoblots of chicken cardiac and skeletal muscle myofibrils. Indirect immunofluorescence localization with the fusion protein-specific antibodies demonstrated that the cDNA sequence was from the region of titin located in the myofibrillar A-band adjacent to the A/I junction. Derived amino acid sequences demonstrated a repeating pattern of fibronectin type III and immunoglobulin C2 motifs, as shown previously for a portion of rabbit skeletal muscle titin located in the central region of the A-band and for other myofibrillar proteins that bind to myosin. Differences between the rabbit and chicken titin sequences included a unique, serine-rich region in one motif, which represents a potential phosphorylation site. This is the first report of sequence information for avian titin from a previously uncharacterized portion of the titin molecule.

Amino Acid Sequence↗
PubMed1993

Organization of potential alternative nitrogenase genes from Clostridium pasteurianum.

A 3.3 kb HindIII genomic DNA fragment from Clostridium pasteurianum ATCC 6013 which hybridized to the anfDGK genes for the Fe-only 'alternative' nitrogenase from Azotobacter vinelandii was cloned. Open reading frames (ORFs D, G, and K) with high sequence identity to anfD, anfG, and part of anfK were located in the nucleotide sequence obtained for 2494 bp of this fragment. In C. pasteurianum, ORFD maps approximately 1.8 kb downstream of nifH3 and is transcribed in the same direction. There was no evidence for additional copies of ORFDGK-like sequences in the genome of C. pasteurianum, other than those encoding the Mo-nitrogenase. Physiological and biochemical studies suggest that a nitrogenase not requiring molybdenum may occur in C. pasteurianum. This enzyme is probably encoded by nifH3 and ORFs D, G, and K identified here.

Amino Acid Sequence↗
PubMed1992

Assembly of contractile and cytoskeletal elements in developing smooth muscle cells.

Specific developmental changes in smooth muscle were studied in gizzards obtained from 6-, 8-, 10-, 12-, 14-, 16-, 18-, and 20-day chick embryos and from 1- and 7-day posthatch chicks. Myoblasts were actively replicating in tissue from 6-day embryos. Cytoplasmic dense bodies (CDBs) first appeared at Embryonic Day 8 (E8) and were recognized as patches of increased electron density that consisted of actin filaments (AFs), intermediate filaments (IFs), and cross-connecting filaments (CCFs). Although the assembly of CDBs was not synchronized within a cell, the number, size, and electron density of CDBs increased as age increased. Membrane-associated dense bodies (MADBs) also could be recognized at E8. The number and size of MADBs increased as age increased, especially after E16. Filaments with the diameter of thick filaments first appeared at E12. Smooth muscle cells were able to divide as late as E20. The axial intermediate filament bundle (IFB) could first be identified in 1-day posthatch cells and became larger and more prominent in 7-day posthatch cells. Immunogold labeling of 1- and 7-day posthatch cells with anti-desmin showed that the IFB contained desmin IFs. The developmental events during this 23-day period were classified into seven stages, based primarily on the appearance and the growth of contractile and cytoskeletal elements. These stages are myoblast proliferation, dense body appearance, thick filament appearance, dense body growth, muscle cell replication, IFB appearance, and appearance of adult type cells. Smooth muscle cells in each stage express similar developmental characteristics. The mechanism of assembly of myofilaments and cytoskeletal elements in smooth muscle in vivo indicates that myofilaments (AFs and thick filaments) and filament attachment sites (CDBs and MADBs) are assembled before the axial IFB, a major cytoskeletal element.

Animals↗
PubMed1992

Effect of porcine stress syndrome on the solubility and degradation of myofibrillar/cytoskeletal proteins.

This study examined the effect of stress classification (stress-positive, stress-carrier, stress-negative) of pigs on selected properties of postmortem muscle, including protein solubility and degradation of proteins such as titin. Longissimus muscle samples were removed 45 min postslaughter, divided into samples, and stored at 0 to 2 degrees C for analysis at 0, 1, 3, 5, and 7 d postmortem. Whole-muscle samples (homogenates) and purified myofibrils were prepared from each sample for analysis by SDS-PAGE. A portion of each muscle sample also was extracted 1) with a low-ionic-strength solution to obtain a sarcoplasmic protein fraction and 2) with two different high-ionic-strength solutions to obtain a myofibrillar/cytoskeletal protein fraction for measurement of protein solubility and for analysis of extracts by SDS-PAGE. No significant differences were observed between muscle from stress-negative and stress-carrier animals in this study. Sarcoplasmic (P less than .05) and myofibrillar/cytoskeletal (P less than .01) protein solubility was lower in muscle samples from stress-positive animals than in muscle samples from stress-carrier and stress-negative animals at all postmortem times studied. The high molecular weight protein titin was degraded more slowly postmortem in muscle from stress-positive than in muscle from stress-negative animals, as observed by SDS-PAGE analysis of whole-muscle samples (homogenates) an myofibrils. The combination of lowered protein solubility and reduced rate of postmortem degradation of structural proteins such as titin may explain, at least in part, the reduced quality and protein functionality of muscle from stress-positive pigs.

Animals↗
PubMed1991

Studies of the alpha-actinin/actin interaction in the Z-disk by using calpain.

Both mu- and m-calpain (the micro- and millimolar Ca(2+)-requiring Ca(2+)-dependent proteinases) can completely remove Z-disks from skeletal muscle myofibrils and leave a space devoid of filaments in the Z-disk area. alpha-Actinin, a principal protein component of Z-disks, is removed from myofibrils by the calpains, and a 100-kDa polypeptide that comigrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the alpha-actinin subunit is released into the supernatant. Purified calpain does not degrade purified actin or purified alpha-actinin as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by N- and C-terminal amino acid analysis of calpain-treated and untreated alpha-actinin and actin. The 100-kDa polypeptide released from myofibrils by calpain elutes identically with native alpha-actinin off DEAE-cellulose and hydroxyapatite columns and, after purification, binds to pure F-actin in the same manner that untreated, native alpha-actinin binds. Calpain-released alpha-actinin also accelerates the rate of superprecipitation of reconstituted actomyosin, a sensitive property characteristic of native alpha-actinin. Consequently, the calpains release alpha-actinin from the Z-disk of myofibrils without degrading it or without altering its ability to bind to actin. These results indicate that alpha-actinin does not simply cross-link thin filaments across the Z-disk but that at least one additional protein (or perhaps an altered actin or alpha-actinin) is involved in the alpha-actinin/actin interaction in Z-disks.

Actinin↗
PubMed1990

Determination of the critical concentration required for desmin assembly.

The critical concentration required for filament assembly in vitro from highly purified desmin was determined by both turbidity and centrifugation assays. Assembly was done in the presence of 2 mM-Ca2+, 2 mM-Mg2+ or 150 mM-Na+ at 2, 22 and 37 degrees C. Similar values for critical concentration were obtained by both assays. As temperature increased, critical concentration decreased for each cation. The critical concentration was lowest in the presence of Ca2+ at 2, 22 and 37 degrees C, but was highest in the presence of 150 mM-Na+ at 2 degrees C. Negative staining showed that supernatants from the centrifugation assays contained protofilaments, protofibrils and short particles (less than 300 nm), but pellets contained long filaments (greater than 1 micron) with an average diameter of 10 nm. As the temperature increased, both the average diameter and average length of particles in the supernatant increased. Thermodynamic analysis indicated that hydrophobic interactions were dominant during desmin assembly, but that ionic interactions might also be involved. Our results demonstrated that the specific cation and temperature and temperature-cation interactions all are important in assembly of desmin intermediate filaments.

Actin Cytoskeleton↗
PubMed1989

Intermediate filaments.

It is likely that future studies involving a molecular biology approach, similar to those described in [7,27,28], will yield fruitful information regarding properties and cellular roles of IF. At this point, our knowledge of the properties and expression of IF remains in stark contrast to our lack of understanding of their biological functions. As pointed out by Franke et al. [29], several lines of investigation have suggested that IF do not serve a general cellular function (i.e. 'housekeeping duties'). Instead, the roles of IF and their constituent proteins are probably related to specific functions of the differentiated cell. Our search for these specific roles will be difficult, but exciting. In the meantime, it is rewarding that IF typing of cells and tumors has already yielded practical information useful in histology and cytology [30].

Animals↗
PubMed1988

Polarity and length of actin filaments at the fascia adherens of the cardiac intercalated disk.

Digestion of canine and bovine intercalated disks with a calcium-activated protease (CAF) removes the electron-dense material similar to that found at the Z-line and presumably consisting primarily of alpha-actinin. The major filaments exposed by CAF are actin, and the polarity is away from the intercalated disk, as was confirmed by decoration with heavy meromyosin. The length of actin filaments associated with the fascia adherens region at the concave region is 1.2- to 2.2-fold that of actin filaments (I-filaments) in the sarcomere and varies depending on the interdigitation of the membrane at the cell junction. Actin filaments at the intercalated disk seem to be attached (or very close) to the membrane in a direct, rather than looping, manner.

Actins↗
PubMed1987

Effect of cations and temperature on kinetics of desmin assembly.

Smooth-muscle desmin, which was isolated from avian gizzard, was purified and used to form reconstituted intermediate filaments. Filament assembly was done in the presence of physiological cations, Ca2+, Mg2+, Na+, and Na+ plus Mg2+, and with non-physiological cations Cu2+ and Ni2+. Assembly was done at 2 degrees, 22 degrees and 37 degrees C, and was monitored by absorbance and by electron microscopy. Absorbance increased most rapidly during the first 2-5 min and then increased at a slower rate with the physiological cations, but decreased after that time with the non-physiological cations. For each physiological cation, absorbance increased with increasing temperature. This was particularly evident with Ca2+, which produced the lowest absorbance at 2 degrees C and the highest at 37 degrees C. When ionic strength was comparable, filament-forming buffers that contained bivalent cations were associated with higher absorbance values. Filament diameters were significantly smaller 60 min after assembly initiation than after 5 min. Average filament diameters, when formed in the presence of Cu2+ or Ni2+, were 10% greater than in the presence of the physiological cations and did not show a consistent tendency to decrease as time increased. These results demonstrate the importance, not only of the pH and ionic composition of the filament-forming buffer, but also of the temperature and duration of dialysis for reconstitution of desmin filaments.

Animals↗
PubMed1987

Composition of intermediate filament subunit proteins in embryonic, neonatal and postnatal porcine skeletal muscle.

The intermediate (10-nm) filament subunit proteins (desmin and vimentin) in samples obtained from embryonic, neonatal, and postnatal porcine skeletal muscle were examined by two-dimensional electrophoresis (isoelectric focusing/sodium dodecylsulfate polyacrylamide gel electrophoresis). The skeletal muscle samples were taken from pig embryos at 45, 73 and 102 d of gestation; from neonatal pigs and from postnatal pigs at 1, 6 and 30 mo of age. Three fractions (namely, whole homogenized muscle, purified myofibrils and myofibrillar-protein-extracted residues) were prepared from each skeletal muscle sample for analysis. Vimentin was the major (approximately 75% vimentin: 25% desmin) 10-nm filament protein present in skeletal muscle samples obtained from the 45-d-old pig embryos. The relative proportion of vimentin decreased progressively during embryogenesis. At birth, the vimentin comprised approximately 15%, and desmin, 85%, of the 10-nm filament protein. The proportional amount of vimentin continued to decline postnatally, with the 10-nm filament protein of samples from the 30-mo-old animals consisting of less than approximately 5% vimentin and over 95% desmin. These results show a developmental stage-dependent pattern in the expression of vimentin and desmin intermediate filament subunit proteins in mammalian skeletal muscle. In the adult mammal, desmin is the significant 10-nm filament protein present.

Animals↗
PubMed1985

Fine structure of wide and narrow vertebrate muscle Z-lines. A proposed model and computer simulation of Z-line architecture.

A model of the structure of vertebrate Z-lines and Z-line analogs is introduced and supported by evidence from electron microscope studies of wide Z-lines (rat and feline soleus, and feline and canine cardiac muscles), narrow Z-lines (guppy, newt and frog skeletal muscles), and Z-rods (from a patient with nemaline myopathy and from cardiac muscles of aged dog). The model is based on a pair of Z-filaments (termed a Z-unit), which are linked near their centers at a 90 degrees angle and form bridges between neighboring antipolar thin (actin) filaments. A square lattice of four Z-filament pairs (the basic structure of the Z-line, termed a Z-line unit) defines the geometrical position of the I-square unit. In this native state of the Z-line, small square and large square net forms appear in cross-section. Other cross-sectional patterns of Z-lines, including basket-weave and diagonal-square net patterns, can be explained by detachment of the Z-filament from the Z-filament binding region within each Z-filament pair due to chemical or physical stress. Dissection of Z-lines and Z-line analogs with calcium-activated neutral protease provides evidence that the width of all wide Z-line structures is determined by the amount of overlap of antipolar thin filaments from adjacent sarcomeres. Longitudinal patterns of narrow and wide Z-lines are shown and described in relation to the model. To test the proposed model, the dynamics of the Z-line unit structure were computer-simulated. An attempt was made to correlate longitudinal (z direction) and cross-sectional (x and y directions) patterns and to determine the amount of movement of thin or Z-filaments that is required to explain the diversity observed in cross-sectional patterns of Z-lines. The computer simulations demonstrated that the structural transitions among the small square, and therefore large square net, as well as basket-weave and diagonal-square net forms seen in cross-sections could be caused by movements of thin filaments less than 10 nm in any direction (x, y or z).(ABSTRACT TRUNCATED AT 400 WORDS)

Actins↗