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Biomedical subjects

R Craig

Publications and source records attributed to R Craig.

At least 109 records · Page 6Linked to original sources
PubMed1987

Differential effects of chronic partial myelotomies on monoamine levels in cat spinal cord.

The concentrations of 5-hydroxytryptamine (5-HT), norepinephrine (NE) and dopamine (DA) were measured in samples of lumbar and cervical spinal cords from 6 cats with chronic (over 2 months) lesions of the thoracic spinal cord and from 7 unoperated cats. Lesions confined to the dorsal thoracic spinal cord significantly lowered lumbar concentrations of NE, but not 5-HT, compared with control lumbar or matched paired cervical samples. Both NE and 5-HT were significantly reduced by dorsal or ventral lesions that involved tissue ventral to the central canal. Only the largest lesion could be shown to reduce lumbar DA concentration.

Animals↗
PubMed1987

Structural changes accompanying phosphorylation of tarantula muscle myosin filaments.

Electron microscopy has been used to study the structural changes that occur in the myosin filaments of tarantula striated muscle when they are phosphorylated. Myosin filaments in muscle homogenates maintained in relaxing conditions (ATP, EGTA) are found to have nonphosphorylated regulatory light chains as shown by urea/glycerol gel electrophoresis and [32P]phosphate autoradiography. Negative staining reveals an ordered, helical arrangement of crossbridges in these filaments, in which the heads from axially neighboring myosin molecules appear to interact with each other. When the free Ca2+ concentration in a homogenate is raised to 10(-4) M, or when a Ca2+-insensitive myosin light chain kinase is added at low Ca2+ (10(-8) M), the regulatory light chains of myosin become rapidly phosphorylated. Phosphorylation is accompanied by potentiation of the actin activation of the myosin Mg-ATPase activity and by loss of order of the helical crossbridge arrangement characteristic of the relaxed filament. We suggest that in the relaxed state, when the regulatory light chains are not phosphorylated, the myosin heads are held down on the filament backbone by head-head interactions or by interactions of the heads with the filament backbone. Phosphorylation of the light chains may alter these interactions so that the crossbridges become more loosely associated with the filament backbone giving rise to the observed changes and facilitating crossbridge interaction with actin.

Actin Cytoskeleton↗
PubMed1987

Molecular structure and organization of filaments in single, skinned smooth muscle cells.

The contractile elements of single, skinned amphibian smooth muscle cells were resolved as fibrils containing F-actin and side-polar myosin filaments and adjacent cytoplasmic dense bodies, containing alpha-actinin and actin. The fibrils were found to be linked axially through actin filaments to the dense bodies, by electron microscopy. The arrangement of these two structures provides the essential features of a sarcomere to the contractile elements. The effect of contraction upon the movement of the dense bodies was followed dynamically by optical microscopy as a method of analyzing the organization of the contractile apparatus and resolving contractile units. Averaged over long time periods (30 to 50 seconds), the axial movements of dense bodies was uniform. The dense bodies were not simply displaced passively during contraction because the extent of radial movements averaged over the same (long) time periods were less than would be expected for the observed increases in cell diameter. Measured over short time intervals (3 to 6 seconds), the axial movement of some closely spaced dense bodies was not uniform: individual and groups of bodies moved significantly faster and slower than the mean rate of axial translocation. These shifts in coordinated and independent movements might reveal separate anisometric contractile units, or they could represent a random temporal pattern of localized activation and deactivation of small contractile elements.

Animals↗
PubMed1987

Platelet enhancement of tumor cell adhesion to subendothelial matrix: role of platelet cytoskeleton and platelet membrane.

Platelet involvement during tumor cell adhesion to subendothelial matrix was examined in vitro. Platelets were subjected to thrombin stimulation and mechanical lysis and examined for their effects on tumor cell adhesion. These treatments altered the platelet ultrastructure and cytoskeletal integrity. Untreated washed rat platelets (WRP) exhibited extensive adhesion to and spreading on substrates and substantially enhanced tumor cell adhesion to the same substrates (i.e., 250% greater than tumor cells without platelets). Thrombin prestimulation of platelets limited platelet adhesion and spreading and platelet facilitation of tumor cell adhesion. Complete mechanical lysis disrupted both the platelet membrane and the cytoskeleton and eliminated the ability of platelets to adhere or to enhance tumor cell adhesion. Partially lysed platelets resembled membrane ghosts and facilitated tumor cell adhesion by a mechanism independent of spreading and cytoskeletal rearrangement. Fractionation studies indicated that platelet cytoskeletal components played a role in the adhesion process. Pretreatment of WRP with cytochalasin A or B dose dependently inhibited microfilament-mediated platelet spreading and platelet-enhanced tumor cell adhesion. Colchicine and vinblastine induced microtubule depolymerization, but they had no observable effect on platelet spreading or platelet-enhanced tumor cell adhesion. It was concluded that platelet-enhanced tumor cell adhesion to subendothelial matrix depends on an intact platelet cytoskeleton and on a platelet membrane component(s) and is mediated by surface contact between platelets and tumor cells. Furthermore, platelet-mediated tumor cell adhesion to subendothelial matrix may involve two mechanisms: one dependent on, and one independent of, platelet spreading and cytoskeletal rearrangement.

Animals↗
PubMed1986

The ultrastructural location of C-protein, X-protein and H-protein in rabbit muscle.

Purified antibodies to the thick filament accessory proteins, C-protein, X-protein and H-protein, have been used to label fibres of three rabbit muscles, psoas (containing mainly fast white fibres), soleus (containing mainly slow red fibres) and plantaris (a muscle of mixed fibre type) and their location has been examined by electron microscopy. These accessory proteins are present on one or more of a set of eleven transverse stripes about 43 nm apart that have been observed previously in each half A-band. Each protein has a limited set of characteristic distributions. H-protein is present on stripe 3 (counting from the M-line) in the majority of psoas fibres but is absent in soleus and plantaris muscle. C-protein can occur on stripes 4-11 (the commonest pattern seen in psoas); on stripes 5-11 (in psoas and plantaris); on stripes 3 together with stripes 5-11 (in plantaris); or on none (in red fibres of all three muscles). X-protein can occur on stripes 3-11 in the red fibres of all three muscles; on stripe 4 only (in psoas and plantaris); on stripes 3 and 4 (in psoas and plantaris) or on none. Stripes labelled with anti-X are wider than those labelled with anti-C and consist of a doublet with an internal spacing of 16 nm. The patterns for the three accessory proteins, while overlapping, are in no case identical; this suggests the proteins do not simply substitute for one another. The precise axial positions of the anti-C labelled stripes differ from those of the anti-X stripes; the anti-X stripes lie about 8-9 nm further from the M-line than the corresponding anti-C stripes. This implies that the inner member of an X-protein doublet lies in a very similar position to a C-protein stripe. The anti-H labelled stripe seen in most psoas fibres lies 14 nm nearer the M-line than stripe 3 of the anti-X labelled array in psoas red fibres and is staggered from a continuation of the C-protein array by about 4 nm. The labelling patterns were constant within a fibre and suggest a very precise assembly mechanism. The number of classes of fibre, as defined by the accessory proteins present and their arrangement, exceeds the number of fibre types presently recognized.

Animals↗
PubMed1986

Trinitrotoluene: assessment of occupational absorption during manufacture of explosives.

Trinitrotoluene (TNT) absorption was assessed in groups of workers at two explosives factories by measuring the urinary concentrations of dinitroaminotoluene (DNAT) metabolites. DNAT was detected in most of the urine samples analysed and for postshift samples the mean (SD) concentration was 9.7 (7.9) mg/l (range 0.1-44 mg/l (n = 219)). Individual workers showed substantial day to day variations in DNAT concentrations in postshift urine samples, but on a group basis the concentrations remained fairly constant throughout the working week. Preshift urine samples taken at the beginning of a working week showed low concentrations of DNAT which initially suggested that the elimination of TNT metabolites is fairly rapid. A survey carried out of preshift and postshift urine samples collected from a group of workers for a full working week showed wide variations in the rate of clearance of TNT metabolites from the body and in some cases higher concentrations of metabolites were seen in the samples taken the morning after exposure. When urine samples were collected from the same group of workers after 17 days away from the workplace DNAT was still detectable in samples from eight of the nine subjects, indicating that a proportion of TNT or its metabolites is slowly excreted. When five subjects were monitored more intensively during two workshifts TNT was shown to be absorbed rapidly during the exposure period. In most cases the highest concentrations were seen in the postshift urine samples but significant proportions were still present in samples taken the morning after exposure. Atmospheric levels of TNT were found to be too low to account for the observed excretion of DNAT and dermal uptake rather than inhalation appears to be the major route of absorption.

Absorption↗
PubMed1985

Arrangement of the heads of myosin in relaxed thick filaments from tarantula muscle.

Thick filaments from leg muscle of tarantula, maintained under relaxing conditions (Mg-ATP and EGTA), were negatively stained and photographed with minimal electron dose. Particles were selected for three-dimensional image reconstruction by general visual appearance and by the strength and symmetry of their optical diffraction patterns, the best of which extend to spacings of 1/5 nm-1. The helical symmetry is such that, on a given layer-line, Bessel function contributions of different orders start to overlap at fairly low resolution and must therefore be separated computationally by combining data from different views. Independent reconstructions agree well and show more detail than previous reconstructions of thick filaments from Limulus and scallop. The strongest feature is a set of four long-pitch right-handed helical ridges (pitch 4 X 43.5 nm) formed by the elongated myosin heads. The long-pitch helices are modulated to give ridges with an axial spacing of 14.5 nm, lying in planes roughly normal to the filament axis and running circumferentially. We suggest that the latter may be formed by the stacking of a subfragment 1 (S1) head from one myosin molecule on an S1 from an axially neighbouring molecule. Internal features in the map indicate an approximate local twofold axis relating the putative heads within a molecule. The heads appear to point in opposite directions along the filament axis and are located very close to the filament backbone. Thus, for the first time, the two heads of the myosin molecule appear to have been visualized in a native thick filament under relaxing conditions.

Animals↗
PubMed1985

Arthrin: a new actin-like protein in insect flight muscle.

There are one or more proteins of 50,000 to 60,000 Mr in the thin filaments of insect flight muscle. A protein of 55,000 Mr has been isolated from insect fibrillar flight muscle and called arthrin. Despite its higher molecular weight, arthrin is in many ways like actin. The amino acid composition of arthrin was similar to that of actin. There were similarities in the peptides produced by digesting the denatured proteins and mild digestion of polymerized proteins cleaved similar-sized fragments from arthrin and actin. Polymerized arthrin activated the Mg2+ ATPase of myosin to the same extent as actin and the ATPase was regulated by rabbit or Lethocerus troponin and tropomyosin. Arthrin did not itself act as troponin-T. Electron microscopy of negatively stained specimens showed that arthrin and actin filaments were similar in structure and that arthrin could be decorated by rabbit subfragment-1 to form normal-looking arrowheads. Arthrin formed paracrystals at an optimum concentration of MgCl2 (25 mM) that was somewhat lower than the optimum for actin paracrystals. Optical diffraction showed that the structure of the paracrystals was similar to those formed from actin. The mass of arthrin and actin filaments relative to phage fd was measured by scanning transmission electron microscopy; the relative mass of arthrin and actin was 1.33, in agreement with molecular weight estimations. Therefore arthrin has the properties of a heavy form of actin. The proportion of actin, arthrin and troponin-T in Lethocerus myofibrils was six moles of actin to one mole of arthrin and one mole of troponin-T. The function of arthrin is not known.

Actins↗
PubMed1985

Dinitrotoluene: an assessment of occupational absorption during the manufacture of blasting explosives.

Two biological monitoring studies were carried out among workers in an explosives factory who were exposed to technical grade dinitrotoluene (DNT). In the first study urine samples from 28 workers were analysed for the metabolite 2,4-dinitrobenzoic acid (2,4-DNBA). Metabolite concentrations in urine were extremely low or non-detectable, prior to starting work at the beginning of the working week, but post-shift urine samples contained a mean 2,4-DNBA level of 17 mg/l. There were wide variations in the concentrations excreted in urine by different workers and by individual workers on consecutive days. Atmospheric levels of DNT (determined by personal monitoring) were found to be well below the recommended limit and therefore could not account for the observed excretion of 2,4-DNBA. It is suggested that skin may be the major route of absorption of DNT during this process. A second study was carried out to investigate the kinetics of absorption and excretion of DNT. Intensive urine sampling was carried out on five individuals over a 2-day period with additional samples over the subsequent 2 non-working days. Analysis for 2,4-DNBA showed that uptake of DNT is rapid and that the highest levels were normally seen in the end-of-shift specimens. Urine samples were analysed for other known metabolites of DNT which have been found in animal studies and it was shown that 2,4-DNBA is the major known metabolite which is excreted in human urine. Unchanged DNT was detected in blood samples taken during a single workshift at levels up to 250 ng/ml. It is concluded that there is a measurable uptake of DNT by the group of workers studied. The measurement of 2,4 DNBA in end-of-shift urine samples is an appropriate way of assessing the effectiveness of measures taken to reduce the absorption of DNT.

Absorption↗
PubMed1985

Structure of the actin-myosin complex in the presence of ATP.

The structure of the complex between actin and myosin subfragment 1 (S1), designated the acto-S1 complex, in the presence of ATP was examined by electron microscopy. This was accomplished by using negative staining to study a complex of S1 covalently crosslinked to actin by the zero-length crosslinker, 1-ethyl-3-[3-(dimethylamino)-propyl]carbodiimide. Two levels of S1 binding were studied, with a molar ratio of crosslinked S1 to total actin of either 20% or 50%. The lower percentage was used to observe individual S1 molecules attached to actin, while the higher percentage was used to look at the overall pattern of S1 decoration of the actin filament. In the absence of ATP, the appearances of both the 20% and 50% crosslinked filaments closely resembled the rigor appearances obtained with noncrosslinked proteins. The arrowheads observed had the conventional structure, and individual S1 molecules were elongated and curved and appeared to make an angle of 45 degrees with the thin filament. Addition of ATP to the crosslinked acto-S1 complex caused a radical change in the structure of the cross-bridges. At both 20 and 170 mM ionic strengths, individual S1 molecules appeared to be attached at variable angles which, in contrast to rigor, did not center on 45 degrees. In addition, the S1 molecules often appeared shorter and fatter than in rigor. The 50% crosslinked acto-S1 preparation no longer showed the arrowhead pattern of S1 decoration but instead appeared to be disordered with little obvious polarity. Control experiments with ADP suggest that these effects were not due simply to a weakening of the binding of S1 to actin in the presence of nucleotide but most likely were ATP-specific. The crosslinked acto-S1 complex, which hydrolyzes ATP at about the same rate as the maximal actin-activated ATPase of S1 (Vmax), is composed of a mixture of states A X M X ATP and A X M X ADP X Pi (in which A = actin and M = myosin), with more than 50% of the crosslinked S-1 occurring in state A X M X ATP. Therefore, it appears that both states A X M X ATP and A X M X ADP X Pi have a very different conformation from the classic arrowhead conformation of the A X M state.

Actins↗
PubMed1985

Structural changes that occur in scallop myosin filaments upon activation.

Myosin filaments isolated from scallop striated muscle have been activated by calcium-containing solutions, and their structure has been examined by electron microscopy after negative staining. The orderly helical arrangement of myosin projections characteristic of the relaxed state is largely lost upon activation. The oblique striping that arises from alignment of elongated projections along the long-pitched helical tracks is greatly weakened, although a 145 A axial periodicity is sometimes partially retained. The edges of the filaments become rough, and the myosin heads move outwards as their helical arrangement becomes disordered. Crossbridges at various angles appear to link thick and thin filaments after activation. The transition from order to disorder is reversible and occurs over a narrow range of free calcium concentration near pCa 5.7. Removal of nucleotide, as well as dissociation of regulatory light chains, also disrupts the ordered helical arrangement of projections. We suggest that the relaxed arrangement of the projections is probably maintained by intermolecular interactions between myosin molecules, which depend on the regulatory light chains. Calcium binding changes the interactions between light chains and the rest of the head, activating the myosin molecule. Intermolecular contacts between molecules may thus be altered and may propagate activation cooperatively throughout the thick filament.

Adenosine Triphosphate↗
PubMed1985

Potential effects of nutritional state on pulmonary function in cystic fibrosis.

There are a number of observations which suggest that malnutrition and decreasing pulmonary function are parallel phenomena in chronic lung disease. Causality for either has not been established. A potential link between the two may be the diminishing function of respiratory muscles which accompanies weight loss. Data from adult patients with chronic obstructive lung disease are presented which suggest that respiratory muscle fibers progressively atrophy, not hypertrophy, in the face of increasing airway obstruction. This lack of compensation is probably nonspecific since nonrespiratory muscles demonstrate the same trend. This observation may explain some of the impaired respiratory muscle function which has been documented in patients with chronic lung disease. There are no morphologic data available for older patients with cystic fibrosis, but the processes which impair muscle function may prove to be similar in both groups of patients. The effect of malnutrition on respiratory muscle fiber size remains to be determined.

Airway Obstruction↗
PubMed1985

Hypothalamic monoaminergic activity and pituitary function in male rats with estrogen-induced pituitary hyperplasia.

The mechanism by which estrogen enhances prolactin (PRL) secretion and induces hyperplasia of lactotrophs is not defined clearly. The objective of this study was to examine hypothalamic monoaminergic PRL regulatory systems and pituitary hormone secretion in the early and later stages of estrogen-induced hyperprolactinemia and pituitary hyperplasia. Dopamine (DA) and serotonin (5-HT) turnover were determined in microdissected brain regions 3 and 30 days after a single subcutaneous dose of estradiol (2 mg) to male ACI rats. Plasma samples were collected in animals with indwelling intra-atrial cannulae. 3 days after estrogen there was a significant increase in plasma PRL, pituitary PRL and growth hormone (GH), and DA turnover in the median eminence and arcuate nucleus. Plasma concentrations and pituitary content of PRL increased at 30 days. The responsiveness of PRL to thyrotropin-releasing hormone (TRH) was enhanced at both times. Concentrations of DA decreased considerably in the median eminence and arcuate nucleus by 30 days, and turnover decreased in the median eminence. 5-HT turnover was not affected in the early stages of hyperprolactinemia. Plasma GH increased and TSH was unchanged, even though pituitary content of both hormones decreased at 30 days. Estrogen had no effect on plasma corticosterone. These findings support the hypothesis that estrogen induces pituitary hyperplasia by antagonizing DA inhibition of PRL-secreting cells and by enhancing their responsiveness to TRH.

Animals↗
PubMed1985

Cysteamine effects on monoamines, dopamine-beta-hydroxylase and the hypothalamic-pituitary axis.

Cysteamine (beta-mercaptoethylamine, MEA) is a naturally occurring sulfhydryl compound that depletes pituitary PRL, causes a reduction in brain and gut somatostatin (SRIF), and suppresses norepinephrine (NE) and epinephrine (EPI) synthesis by inhibition of dopamine-beta-hydroxylase (DBH). SRIF inhibits GH and TSH secretion, whereas, NE and EPI facilitate their release. The objectives of this investigation were to: (1) determine the dose-response and time course of DBH inhibition by MEA in vivo and in vitro, and correlate these findings with MEA tissue levels and (2) assess the function of SRIF and NE/EPI in regulation of episodic GH and TSH secretion using MEA. Animals were administered MEA (75-300 mg/kg, s.c.) and hypothalamic levels of dopamine (DA), NE, EPI, serotonin (5-HT) and MEA were measured by high-performance liquid chromatography (HPLC) and electrochemical detection. DBH activity was measured in vitro after exposure to MEA +/- N-ethylmaleimide (NEMI). Chronically cannulated rats were administered MEA (100 or 300 mg/Kg) and serial blood samples were removed in undisturbed animals, and after 30 min swimming stress. Cannulated rats with bilateral lesions of the ventromedial/arcuate nuclei (VMN/ARC) were administered MEA (150 mg/kg). MEA caused a dose-related decrease in NE/EPI nd in increase in DA at doses greater than or equal to 150 mg/kg. Tissue MEA was highest at 4 h (679 +/- 64 pM/mg tissue), but still measureable after 24 h. MEA inhibited DBH in vitro (95% inhibition at 10(-3) M); NEMI blocked inhibition. Stress-induced GH supression and corticosterone release were partially blocked by a low dose of MEA (100 mg/kg).(ABSTRACT TRUNCATED AT 250 WORDS)

3,4-Dihydroxyphenylacetic Acid↗
PubMed1983

Signal sequences, secondary modification and the turnover of miscompartmentalized secretory proteins in Xenopus oocytes.

The cytoplasm of the Xenopus oocyte can be altered by the microinjection of proteins and the regulatory responses to such perturbations can then be studied. We have investigated proteolytic systems within the oocyte which may be involved in the maintenance of the integrity of the different subcellular compartments. Thus primary translation products, made in the wheat germ system under the direction of frog liver, chicken oviduct, rat liver rapidly sedimenting endoplasmic reticulum, rat seminal vesicle, guinea pig mammary gland or honey been venom gland RNA, were injected into oocytes. Their stability in the frog cell cytosol was in general low compared to that of their processed counterparts. The latter were usually obtained by collecting the heterologous proteins exported by RNA-injected oocytes. Electrophoretic analysis of oocytes injected with particular primary and processed polypeptides permitted measurement of the stabilities of proteins differing only by the presence or absence of a detachable signal sequence, or by the presence of a specific secondary modification. The effect of the latter on protein stability appears slight. However, the presence of a detachable signal sequence destabilizes those miscompartmentalized secretory proteins which are otherwise stable. Indeed all other results are consistent with this concept for they show that primary translation products are in general much less and are never more stable than their processed counterparts. Thus we provide evidence that errors of compartmentation can be corrected in living cells and that this process is often facilitated by the properties conferred on a protein by a detachable signal sequence.

Animals↗