Comparison of islet autoantibodies in 'pre-diabetes' and recommendations for screening.
Explore the source record for details and available documents.
Autoantibodies↗
Biomedical subjects
M A Atkinson
Publications and source records attributed to M A Atkinson.
Insulitis and diabetes in NOD mice reduced by prophylactic insulin therapy.
Intensive insulin therapy in patients with recently diagnosed insulin-dependent diabetes mellitus (IDDM) has been reported to result in a prolonged increase in endogenous insulin-secreting capacity. Because the clinical onset of IDDM occurs only after most insulin-secreting beta-cells have been destroyed, we tested whether prophylactic insulin therapy might prevent IDDM in nonobese diabetic (NOD) mice. One hundred fourteen NOD mice were randomized at weaning into a protamine zinc pork insulin-treated (I) group or a placebo-treated (P) group given insulin diluent. All insulin treatments were adjusted to the maximum tolerable dosages and continued until 180 days of age. The cumulative IDDM frequency within the female I group was significantly less (3 of 34, 8%) than in female P controls (17 of 26, 65%; P less than 0.0001). This beneficial effect was limited to females, however, because the frequency of IDDM in male I mice (3 of 32, 9%) was not significantly different from the frequency in male P controls (1 of 22, 5%; P less than 0.5). Pancreatic histological examinations of nondiabetic animals revealed that insulin treatment resulted in significant reductions in islet cell inflammation and damage and improvements in insulin content. In summary, NOD mice given insulin therapy from weaning until 180 days of age had significantly lower frequencies of diabetes and pancreatic insulitis than sex-matched control littermates treated with insulin diluent. These results suggest that prophylactic insulin therapy to prevent IDDM in humans should be considered for clinical trials.
Animals↗
Cooperative dependence of the actin-activated Mg2+-ATPase activity of Acanthamoeba myosin II on the extent of filament phosphorylation.
The actin-activated Mg2+-ATPase of myosin II from Acanthamoeba castellanii is regulated by phosphorylation of 3 serine residues at the tip of the tail of each of its two heavy chains; only dephosphorylated myosin II is active, whereas the phosphorylated and dephosphorylated forms have identical Ca2+-ATPase activities and Mg2+-ATPase activities in the absence of F-actin. We have now chemically modified phosphorylated and dephosphorylated myosin II with N-ethylmaleimide (NEM). The modification occurred principally at a single site within the NH2-terminal 73,000 Da of the globular head of the heavy chain. NEM-myosin II bound to F-actin and formed filaments normally, but the Ca2+- and Mg2+-ATPase activities of phosphorylated and dephosphorylated myosin II and the actin-activated Mg2+-ATPase activity of NEM-dephosphorylated myosin II were inhibited. Only filamentous myosin II has actin-activated Mg2+-ATPase activity. Native phosphorylated myosin II acquired actin-activated Mg2+-ATPase activity when it was co-polymerized with NEM-inactivated dephosphorylated myosin II, and the increase in its activity was cooperatively dependent on the fraction of NEM-dephosphorylated myosin II in the filaments. From this result, we conclude that the specific activity of each molecule within a filament is independent of its own state of phosphorylation, but is highly cooperatively dependent upon the state of phosphorylation of the filament as a whole. This enables the actin-activated Mg2+-ATPase activity of myosin II filaments to respond rapidly and extensively to small changes in the level of their phosphorylation.
Acanthamoeba↗
The effect of external fixation stiffness on early healing of transverse osteotomies.
The effect of bending stiffness of external fixators on the early healing of transverse tibial osteotomies was studied in a canine model to determine whether there is a particular range of fixation stiffness where healing is poor. Five unilateral fixators with different stiffnesses were developed and employed along with a dynamic compression plate to fix experimental osteotomies of dog tibias and the contralateral intact tibias served as controls. The six experimental fixation devices were: plate (P), stainless steel sidebar fixator (SS), stiff aluminum sidebar fixator (SA), intermediate aluminum sidebar fixator (IA), flexible aluminum sidebar fixator (FA), and Delrin sidebar fixator (D). Compared with human tibial fixation methods, the fixators ranged from a rigidity greater than a plate on a human tibia to a flexibility similar to a plaster cast on a human lower leg. Sixty-four adult male mongrel dogs (29 +/- 3 kg) underwent surgery and 52 completed the 9-week study, leaving 7 to 10 dogs in each experimental group (P = 8, SS = 7, SA = 9, IA = 9, FA = 10, D = 9). After the 9-week survival period the experimental tibias were radiographed and callus area was digitized. Both the experimental and control tibias were then tested to failure in four-point bending, and failure moment, stiffness, and energy to failure were determined. Of the 52 osteotomies, only four developed nonunions: 3 of 10 in the FA group and 1 of 7 in the SS group. Using analysis of variance, no statistical differences were detected in failure force, stiffness, or energy to failure as a function of fixator groups.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Sequence similarities between chicken intestinal 110-kDa ATPase and myosin I-like enzymes.
We report the partial amino acid sequence of chicken intestinal microvillar 110-kDa protein that, as a complex with calmodulin, has previously been shown to exhibit myosin-like ATPase and actin-binding activities. The sequence shows a high degree of similarity to the sequence of a novel vertebrate myosin I-like heavy chain encoded by a cDNA isolated from bovine intestine. This confirms that the bovine and chicken proteins are the first examples of Acanthamoeba myosin I-like proteins from higher eukaryotes. Comparison of available structural and functional data leads us to postulate that the myosin I family of proteins result from the fusion of a conserved myosin headlike motor domain, with variable COOH-terminal domains responsible for binding to specific intracellular structures.
Adenosine Triphosphatases↗
Enzymatic activity and filament assembly of Acanthamoeba myosin II are regulated by adjacent domains at the end of the tail.
Polyclonal antibodies raised against a synthetic peptide consisting of the last 19 amino acids at the end of the coiled-coil region of the heavy chains inhibited the actin-activated Mg2+-ATPase activity of myosin II and its ability to form filaments. Antibodies against a synthetic peptide corresponding to the 21 adjacent amino acids at the beginning of the non-helical tailpiece, which include the three regulatory phosphorylatable serines, had no effect on either activity.
Acanthamoeba↗
Structure-function studies on Acanthamoeba myosins IA, IB, and II.
Myosins IA and IB are globular proteins with only a single, short (for myosins) heavy chain (140,000 and 125,000 daltons for IA and IB, respectively) and are unable to form bipolar filaments. The amino acid sequence of IB heavy chain shows 55% similarity to muscle myosins in the N-terminal 670 residues, which contain the active sites, and a unique 500-residue C-terminus highly enriched in proline, glycine, and alanine. The C-terminal region contains a second actin-binding site which allows myosins IA and IB to cross-link actin filaments and support contractile activity. Myosins IA and IB are regulated solely by phosphorylation of one serine on the heavy chain positioned between the catalytic site and the actin-binding site that activates ATPase. Myosin II is a more conventional myosin in composition (two heavy chains and two pairs of light chains), heavy chain sequence (globular head 45% identical to muscle myosins and a coiled-coil helical tail), and structure (bipolar filaments). The tail of myosin II is much shorter than that of other conventional myosins, and it contains a 25 amino acid sequence in which helical structure is predicted to be weak or absent. The position of this sequence corresponds to the position of a bend in the monomer. Myosin II heavy chains also have a 29-residue nonhelical tailpiece which contains three regulatory, phosphorylatable serines. Phosphorylation at the tip of the tail regulates ATPase activity in the globular head apparently through an effect on filament structure.
Acanthamoeba↗
Insulin autoantibodies in pre-diabetes.
Explore the source record for details and available documents.
Adolescent↗
Autoantibodies in nonobese diabetic mice immunoprecipitate 64,000-Mr islet antigen.
In insulin-dependent diabetes mellitus (IDDM) in humans and BB rats, islet cell autoimmunities associated with autoantibodies to a beta-cell protein of 64,000 Mr (64K) have been described. We report that sera from newly diagnosed nonobese diabetic (NOD) mice similarly contain an autoantibody that immunoprecipitates 64K autoantigen from detergent lysates of [35S]methionine-labeled murine islet cells. The autoantibody was detectable by weaning; it disappeared within weeks after diabetes onset and was absent in older nondiabetic NOD mice as well as all of three non-diabetes-prone control strains tested. The 64K beta-cell autoantigen may be a critical target in the immunopathogenesis of IDDM.
Animals↗
Dietary protein restriction reduces the frequency and delays the onset of insulin dependent diabetes in BB rats.
Environmental agents have been implicated in the pathogenesis of insulin dependent diabetes (IDD). These studies were designed to learn if dietary protein influences the development of IDD in the BB rat. Specifically, analysis involved the effects of substituting a modified, semi-synthetic diet (AIN-76) containing soy protein as the sole protein source for the standard chow containing a mixture of animal and non-animal protein. IDD was less frequent (73% vs. 38%, P less than or equal to 0.01), and the onset of diabetes was retarded (110 +/- 11.0 vs. 92 +/- 15.5 days, P less than or equal to 0.01) in rats fed the study diet versus standard chow, respectively. The frequency of thyroid collodal autoantibodies was also significantly decreased in rats fed the study diet (56% vs. 23%, P less than or equal to 0.04), whereas frequencies of smooth muscle and gastric parietal cell autoantibodies were less frequent, but not significantly so. Lymphocyte counts and subsets were unaffected. In non-diabetic rats at greater than 180 days of age, insulitis was less severe in the experimental group. These findings suggested that dietary protein may influence the development of IDD in the BB rat.
Animals↗
A model for the polymerization of Acanthamoeba myosin II and the regulation of its actin-activated Mg2+-ATPase activity.
Explore the source record for details and available documents.
Acanthamoeba↗
Electric birefringence study of the solution structure of chymotrypsin-cleaved Acanthamoeba myosin II.
After removal of the 66 COOH-terminal amino acids from each of its two heavy chains by chymotrypsin digestion, Acanthamoeba myosin II forms only parallel dimers under conditions in which native myosin II forms bipolar filaments (Kuznicki, J., Cote, G. P., Bowers, B., and Korn, E. D. (1985) J. Biol. Chem. 260, 1967-1972). We have studied the solution structure of the chymotrypsin-cleaved myosin II by electric birefringence. Only two species, known to be monomer and parallel dimer from previous studies, were detected. The contribution to the birefringence decay from dimer increased from about 10 to 70% as the KCl concentration was lowered from 100 mM to 0 in 50% glycerol. At all ionic strengths, the monomer had a relaxation time corrected to water at 20 degrees C of 8.2 microseconds, whereas a relaxation time of 10.3 microseconds was expected for monomers with straight rigid rods. This strongly indicates that the myosin rod in solution is bent. On the assumption that there is a single bend 26 nm from the tip of the tail, as suggested by electron microscopy, it was calculated that the average bend angle would be 110 degrees, in solution, if as seems most likely, the average angle between the two globular heads were 180 degrees. The observed relaxation time of the dimer corrected to water at 20 degrees C was 25 microseconds, independent of ionic strength, which, if the motion of the heads were unrestricted, is consistent with a structure for a parallel dimer in which either the two monomer subunits have straight rigid rods and are staggered by about 28 nm or only one is bent and the stagger is 30 nm. As described in the accompanying Appendix, either of these dimers can be assembled into a bipolar filament compatible with the apparent structure of filaments of native myosin II (Pollard, T.D. (1982) J. Cell Biol. 95, 816-825).
Acanthamoeba↗
Bovine albumin antibodies in NOD mice.
Sera from NOD mice were studied for the presence of bovine serum albumin (BSA) antibodies using an ELISA assay. NOD mice had higher levels of BSA binding and higher frequencies of BSA antibodies than did control mice. Furthermore, diabetic NOD mice had higher BSA binding, and higher frequencies of BSA Ab than did nondiabetic NOD mice. These findings are indicative of an exaggerated immune response in NOD mice to BSA that may be associated with diabetes.
Animals↗
The purification and characterization of a globular subfragment of Acanthamoeba myosin II that is fully active when cross-linked to F-actin.
Acanthamoeba myosin II contains two heavy chains of Mr 185,000 and two pairs of light chains of Mr 17,500 and 17,000. We now report the purification of a globular proteolytic 103-kDa subfragment of myosin II which contained a 68-kDa NH2-terminal segment of the heavy chain and one pair of intact light chains. The myosin II head fragment expressed full Ca2+-ATPase activity but its actin-activated Mg2+-ATPase activity had a Vmax of only 0.07 s-1 compared to 1.9 s-1 (per head) for filaments of native unphosphorylated myosin II. The head fragment had a similar KATPase to that of filaments (5 versus 4 microM) and about 75% of the head fraction could bind to F-actin in the presence of ATP with a Kbinding of 5.6 microM. The Kbinding of the head fragment may be similar to that of individual heads in the native myosin II filaments although the experimentally determined apparent Kbinding for filaments is much lower, 0.3 microM. The head fragment was covalently cross-linked to F-actin in the absence of nucleotide using the zero length cross-linker 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. The cross-linked actin-myosin head complex hydrolyzed MgATP at a rate equivalent to Vmax for the active dephosphorylated native myosin II. These data indicate that the isolated head fragment had intact catalytic and actin-binding domains but that it bound to F-actin in the presence of ATP in a relatively inactive conformation. When covalently cross-linked to F-actin the head fragment was apparently locked into a catalytically fully active conformation.
Actins↗
Amino acid sequence of the active site of Acanthamoeba myosin II.
We have used the substrate [5,6-3H]UTP for direct photoaffinity labeling of the active site of the heavy chain of myosin II from Acanthamoeba castellanii. The only labeled peptide in a total tryptic digest had the sequence of Thr-Glu-Asn-Thr-Me2Lys-Lys (where Me2Lys represents dimethyllysine) with the substrate covalently bound to the Glu residue. This sequence differs at only one position from the sequence of residues 184-189 of nematode myosin heavy chain (Me2Lys----Lys), a post-translational modification, and at two additional positions from residues 185-190 of rabbit skeletal muscle myosin (Glu----Val and Lys----Arg). The partial sequence of a larger labeled peptide derived from total chymotryptic digestion was compatible with and extended this sequence. A 20-residue sequence that contains the active site, tryptic hexapeptide is otherwise identical in Acanthamoeba and rabbit skeletal muscle myosins and has only one more difference in nematode myosin. Because UTP is a substrate for myosin II and a "zero-length" probe, we believe that it identifies amino acid residues that are very close to the substrate during the catalytic cycle.
Affinity Labels↗
Are insulin autoantibodies markers for insulin-dependent diabetes mellitus?
Recent studies have shown that insulin autoantibodies occur in patients with newly diagnosed insulin-dependent diabetes mellitus (IDDM) before exogenous insulin treatment. Our study was designed to test the hypothesis that insulin autoantibodies, like cytoplasmic islet cell antibodies (ICAs), can identify individuals with ongoing autoimmune beta-cell destruction and increased risk of IDDM development. Insulin autoantibodies detected by use of a radioligand-binding assay were found in 1.4% of normal controls, 4% of first-degree relatives of IDDM patients, and in 37% of newly diagnosed IDDM patients. A strong positive correlation between insulin autoantibodies and ICAs was observed. HLA typing of insulin-autoantibody-positive first-degree relatives of IDDM patients, as well as in the general population, revealed a strong association with HLA-DR3 and/or-DR4, suggesting that insulin autoantibodies are restricted to persons genetically susceptible to IDDM. In an ongoing study of beta-cell function in ICA-positive nondiabetic individuals, the additional presence of insulin autoantibodies significantly increased the likelihood of beta-cell dysfunction. After intravenous glucose stimulation, insulinopenia was present in 70% of ICA and insulin-autoantibody-positive individuals in contrast to only 23% of ICA-positive, insulin-autoantibody-negative persons. These data document a significant association between insulin autoantibodies and ICAs and support the contention that insulin autoantibodies, like ICAs, are markers of ongoing beta-cell destruction.
Autoantibodies↗
Effects of limited tryptic cleavage on the physical and enzymatic properties of myosin II from Acanthamoeba castellanii.
Limited digestion of Acanthamoeba myosin II by trypsin selectively cleaved the 185,000-Da heavy chains into a 73,000-Da peptide containing the catalytic and actin-binding sites and a 112,000-Da peptide containing the regulatory phosphorylatable sites. The light chains were unaffected. The proteolytic products remained associated and formed bipolar filaments that were very similar in appearance to filaments of native myosin by negative staining electron microscopy. Filaments of trypsin-cleaved, dephosphorylated myosin, however, had a smaller sedimentation coefficient than filaments of native dephosphorylated myosin. Trypsin-cleaved dephosphorylated myosin retained complete Ca2+-ATPase activity but had no actin-activated ATPase activity under conditions that are optimal for native, dephosphorylated myosin (pH 7.0, 4 mM MgCl2, 30 degrees C or pH 6.4, 1 mM MgCl2, 30 degrees C). Trypsin-cleaved dephosphorylated myosin had higher actin-activated ATPase activity at pH 6.0 and 1 mM MgCl2 than undigested dephosphorylated myosin which is appreciably inhibited under these conditions. Trypsin-cleaved, dephosphorylated myosin inhibited the actin-activated ATPase activity of native, dephosphorylated myosin when both were present in the same co-polymers, when enzymatic activity was assayed at pH 7.0, 4 mM MgCl2, and 30 degrees C, but this inhibition was overcome by raising the MgCl2 to 6 mM. These results provide additional evidence that regulation of acanthamoeba myosin II occurs at the filament level and that, under most conditions of assay, the heavy chains must be intact and the regulatory serines unphosphorylated for actin-activated ATPase activity to be maximally expressed.
Adenosine Triphosphatases↗