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Biomedical subjects

D Stafford

Publications and source records attributed to D Stafford.

36 records · Page 2Linked to original sources
PubMed1996

Non-erythroid alpha-spectrin breakdown by calpain and interleukin 1 beta-converting-enzyme-like protease(s) in apoptotic cells: contributory roles of both protease families in neuronal apoptosis.

The cytoskeletal protein non-erythroid alpha-spectrin is well documented as an endogenous calpain substrate, especially under pathophysiological conditions. In cell necrosis (e.g. maitotoxin-treated neuroblastoma SH-SY5Y cells), alpha-spectrin breakdown products (SBDPs) of 150 kDa and 145 kDa were produced by cellular calpains. In contrast, in neuronal cells undergoing apoptosis (cerebellar granule neurons subjected to low potassium and SH-SY5Y cells treated with staurosporine), an additional SBDP of 120 kDa was also observed. The formation of the 120 kDa SBDP was insensitive to calpain inhibitors but was completely blocked by an interleukin 1 beta-converting-enzyme (ICE)-like protease inhibitor, Z-Asp-CH2OC(O)-2,6-dichlorobenzene. Autolytic activation of both calpain and the ICE homologue CPP32 was also observed in apoptotic cells. alpha-Spectrin can also be cleaved in vitro by purified calpains to produce the SBDP doublet of 150/145 kDa and by ICE and ICE homologues [ICH-1, ICH-2 and CPP32(beta)] to produce a 150 kDa SBDP. In addition, CPP32 and ICE also produced a 120 kDa SBDP. Furthermore inhibition of either ICE-like protease(s) or calpain protects both granule neurons and SH-SY5Y cells against apoptosis. Our results suggest that both protease families participate in the expression of neuronal apoptosis.

Amino Acid Sequence↗
PubMed1994

Eye dominance in the small-eared bushbaby, Otolemur garnettii.

Eye-preference has been measured in the small-eared bushbaby, Otolemur garnettii, using two testing conditions, one requiring the subject to look through a grid and the other involving trained looking through a small hole. Monocular eye use was scored for viewing a variety of stimuli. The six subjects (four adult females and two babies) tested using the grid showed left-eye dominance for viewing the tester. Five subjects were tested for viewing food and all were similarly left-eye preferent. That is, there was indication of an eye preference at the group level. The eye preference did not correlate with handedness for food reaching or holding in the same individuals. When three of the subjects were tested viewing the more arousing stimulus of their babies held in the tester's hand, the eye-preference changed; there was either no preference or a weaker left-eye preference. One subject was tested with novel stimuli (a toy monkey, the tester wearing a mask and a rubber snake) and showed a significant shift to a right-eye preference for viewing two of these stimuli. Increased arousal, or fear, was apparent in the latter tests. Comparison is made to eye dominance data for humans.

Animals↗
PubMed1994

In vitro mutagenesis study of two critical glutamic acids in the calcium binding loop of the factor IX heavy chain.

We investigated the structural and functional significance of calcium binding in the factor IXa heavy chain by introducing point mutations into the probable calcium binding site (residues 235 and 245). According to factor IXa computer modelling based on trypsin x-ray structure, side chains of two glutamic acid residues, 235 and 245, together with backbone carbonyl groups of residues 237 and 240, bind a calcium ion. Factor IX clotting activity decreased approximately 25 percent on substitution of glutamic acid 235 with lysine. Activity decreased more than 90 percent on substitution of glutamic acid 245 with lysine. Activity also decreased more than 90 percent on substitution of both glutamic acids by lysines. Factor XIa cleavage of factor IXGlu235Lys and factor IXGlu245Lys appeared normal by polyacrylamide gel analysis. (Factor IXGlu235Lys: Factor IX with Lysine substituted for Glutamic acid at residue 235. Factor IXGlu245Lys: Factor IX with Lysine substituted for Glutamic acid at residue 245. Factor IXGlu235&245Lys: Factor IX with Lysine substituted for Glutamic acid at residues 235 and 245.) Activated factor IXGlu235Lys bound the fluorescent active site probe, p-aminobenzamidine, normally, while factor XIa cleaved factor IXGlu245Lys and factor IXGlu235&245Lys failed to bind p-aminobenzamidine. Plasma purified factor IX titrated with terbium showed an increase in luminescence; however, factor IXGlu235Lys and factor IXGlu245Lys had no effect on terbium luminescence. Radioimmunoassays indicate that in calcium's absence, factor IXGlu245Lys adopts a conformation similar to normal factor IX in the presence of calcium. By contrast, factor IXGlu245Lys's conformation in the presence of calcium is similar to that of plasma purified factor IX in the absence of calcium.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigens↗
PubMed1987

Carrier testing in hemophilia B with an immunoassay that distinguishes a prevalent factor IX dimorphism.

Immunoassays with a monoclonal antibody (A-1) detect a prevalent dimorphism in plasma coagulation factor IX. The antibody was shown to react with a dimorphic segment of the normal factor IX sequence as follows. First, A-1 bound to isolated activation peptide (residues 146 through 180) prepared from activated factor IX from a normal plasma pool. Second, binding of recombinant factor IXs with A-1 or factor IX from normal individuals was strong only when they had Threonine (Thr) at position 148; factor IXs with the Alanine (Ala) allele at that position were far less reactive. Third, immunoblot reactivity of Escherichia coli fusion proteins containing known segments of the factor IX sequence restricted the epitope to residues 147 through 153. In 120 hemophilia B pedigrees, the Ala immunoassay allele frequency was 0.19 and did not differ from the Ala frequency in normal males. In 22 of 49 families, immunoassay testing was informative for classification of obligate or possible carriers. In one large family, 4 obligate carriers were heterozygous for the dimorphism and 3 of their 7 daughters were classified as carriers. In other families, when the affected member had less than 1 nmol/L factor IX antigen, heterozygosity for Thr/Ala alleles excluded the carrier state even when DNA studies were not informative. Strong linkage disequilibrium of Thr/Ala alleles with the common TaqI DNA polymorphism was found. Nineteen of 75 normal and hemophilic factor IX genes had the 1.3-kilobase (kb) fragment and coded for the Ala allele; the rest had the 1.8-kb fragment and coded for Thr. In selected families, the A-1 immunoassay is an inexpensive and rapid method to confirm and supplement restriction fragment length polymorphism analyses of DNA for carrier testing.

Alleles↗
PubMed1986

Antigenic determinant in human coagulation factor IX: immunological screening and DNA sequence analysis of recombinant phage map a monoclonal antibody to residues 111 through 132 of the zymogen.

As an approach to the study of structure-function relationships in the normal and defective forms of human coagulation factor IX, we have begun to develop a series of monoclonal antibodies against specific sites on the protein. Zymogen and activated forms of normal factor IX were used initially as antigen for the preparation of monoclonal antibodies. Recombinant phage were prepared by cloning small (50- to 500-nucleotide) random DNA fragments from the coding region of a factor IX cDNA clone into the expression vector lambda gt11. Immunological screening of these recombinants with mixtures of monoclonal antibodies identified several immunoreactive phage. Further analysis showed that the monoclonal antibody designated IX-30 was generating the positive signals at a frequency of approximately 1/2,500 recombinants. Subcloning and sequence analysis of the inserted DNA in the immunoreactive phage revealed overlapping in-frame insertions, from which it could be inferred that the site in factor IX recognized by IX-30 is confined to residues 111 through 132 in the light chain. Similar mapping with other monoclonal antibodies should provide additional probes for the protein structure of human factor IX.

Amino Acid Sequence↗
PubMed1985

Murine monoclonal anti-DNA autoantibodies bind to endogenous bacteria.

Several bacterial species (including Streptococcus faecalis, Bacillus cereus, Staphylococcus aureus, and Escherichia coli) were tested for their ability to react with monoclonal anti-DNA antibodies that were derived from MRL-lpr/lpr mice. S. faecalis reacted with 8/15 of such antibodies. The binding was unaffected by DNase, but it was competitively inhibited by DNA. F(ab')2 fragments of the monoclonal antibodies reacted with the bacteria, but Fc fragments did not. Phospholipids extracted from the bacterial cells were able to bind to three representative anti-DNA antibodies that also bound to whole bacteria. The results suggest that bacterial phospholipids might provide an immunogenic stimulus for the production of antibodies that cross-react with DNA. We propose that some anti-DNA auto-antibodies and anti-bacterial antibodies evolve from a restricted group of antibodies with high avidity for the phosphodiester groups that occur in DNA and bacterial cells walls.

Animals↗
PubMed1981

Muscle fiber recruitment and blood pressure response to isometric exercise.

Blood pressure was recorded during fatiguing and nonfatiguing isometric contractions of a slow-twitch muscle (the soleus) and a mixed muscle (the medial gastrocnemius) of the cat. Four tensions were examined in each muscle; 10, 25, 40, and 70% of the muscle's initial strength (tetanic tension of the unfatigued muscle). All experiments were also repeated at two muscle temperatures, 28 and 38 degrees C. For the soleus muscle, there was no change in the blood pressure during isometric contractions. For the medial gastrocnemius muscle, both the systolic and diastolic blood pressure increased markedly when either all or just the fast-twitch motor units were stimulated; however, when only the slow-twitch motor units were stimulated, a lower pressor response was observed. Venous blood samples were drawn before, during, and after fatiguing and nonfatiguing contractions of both muscles to determine the K+ concentration in the venous blood. The mean increase in the K+ concentration during contractions was 0.6 meq/l for the slow-twitch motor units of the soleus and 5.1 meq/l for the motor units in the medial gastrocnemius.

Animals↗
PubMed1981

Blood flow and metabolism during isometric contractions in cat skeletal muscle.

The muscle blood flow, oxygen uptake, carbon dioxide production, muscle and blood lactate, muscle ATP, creatinine phosphate, glycogen, and venous pH were measured in the soleus (a slow-twitch muscle) and the medial gastrocnemius (a fast-twitch muscle) of the cat during fatiguing isometric exercise. Five tensions were examined: 10, 25, 50, 75, and 100% of the initial strength of the muscles (tetanic tension of the unfatigued muscle). Contractions were either sustained to fatigue or, for tensions of 10 and 25% initial strength of the soleus muscle, were sustained for 3 min. Analysis of the blood flow and metabolites from these muscles showed that the soleus was heavily dependent on its blood supply, using aerobic metabolism as the predominant pathway, whereas the medial gastrocnemius muscle seemed to use anaerobic metabolism even at low isometric tensions.

Adenosine Triphosphate↗
PubMed1977

Platelet Autoimmunity.

Autoimmunity to platelets was looked for by complement fixation, antiglobulin consumption, transformation and migration-inhibition techniques. Only the migration-inhibition method gave consistently positive results. The test was positive in nine out of 14 patients with idiopathic thrombocytopenic purpura and all five patients with glandular fever.

Autoantibodies↗
PubMed1974

Isolation and some properties of the genes coding for histone proteins.

The DNA coding for histone proteins, together with the intervening spacer DNA, has been isolated from the sea urchin, Psammechinus miliaris, by repeated centrifugation on CsCl-asctinomycin gradients. The biophysical data show that a major portion of the spacer is 37% G+C and very divergent in sequence, while the DNA segments coding for histone proteins are high in G+C and have diverged to a lesser extent within the species.

Animals↗