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H Kaplan

Publications and source records attributed to H Kaplan.

At least 109 records · Page 6Linked to original sources
PubMed1990

Unusual proteolysis of the protoxin and toxin from Bacillus thuringiensis. Structural implications.

Trypsin is shown to generate an insecticidal toxin from the 130-kDa protoxin of Bacillus thuringiensis subsp. kurstaki HD-73 by an unusual proteolytic process. Seven specific cleavages are shown to occur in an ordered sequence starting at the C-terminus of the protoxin and proceeding toward the N-terminal region. At each step, C-terminal fragments of approximately 10 kDa are produced and rapidly proteolyzed to small peptides. The sequential proteolysis ends with a 67-kDa toxin which is resistant to further proteolysis. However, the toxin could be specifically split into two fragments by proteinases as it unfolded under denaturing conditions. Papain cleaved the toxin at glycine 327 to give a 34.5-kDa N-terminal fragment and a 32.3-kDa C-terminal fragment. Similar fragments could be generated by elastase and trypsin. The N-terminal fragment corresponds to the conserved N-terminal domain predicted from the gene-deduced sequence analysis of toxins from various subspecies of B. thuringiensis, and the C-terminal fragment is the predicted hypervariable sequence domain. A double-peaked transition was observed for the toxin by differential scanning calorimetry, consistent with two or more independent folding domains. It is concluded that the N- and C-terminal regions of the protoxin are two multidomain regions which give unique structural and biological properties to the molecule.

Amino Acid Sequence↗
PubMed1990

Characterization of the cysteine residues and disulphide linkages in the protein crystal of Bacillus thuringiensis.

Bacillus thuringiensis produces a 130-140 kDa insecticidal protein in the form of a bipyramidal crystal. The protein in the crystals from the subspecies kurstaki HD-1 and entomocidus was found to contain 16-18 cysteine residues per molecule, present primarily in the disulphide form as cystine. Evidence that all the cysteine residues form symmetrical interchain disulphide linkages in the protein crystal was obtained from the following results: (i) the disulphide diagonal procedure [Brown & Hartley (1966) Biochem. J. 101, 214-228] gave only unpaired cysteic acid peptides in diagonal maps; (ii) the disulphide bridges were shown to be labile in dilute alkali and the crystal protein could be released quantitatively with 1 mM-2-mercaptoethanol; (iii) the thiol groups of the released crystal protein were shown by competitive labelling [Kaplan, Stevenson & Hartley (1971) Biochem. J. 124, 289-299] to have the same chemical properties as exposed groups on the surface of the protein; (iv) the thiol groups in the released crystal protein reacted quantitatively with iodoacetate or iodoacetamide. The finding that all the disulphide linkages in the protein crystal are interchain and symmetrical accounts for its alkali-lability and for the high degree of conservation in the primary structure of the cystine-containing regions of the protein from various subspecies.

Amino Acids↗
PubMed1990

Low-dose methotrexate compared with auranofin in adult rheumatoid arthritis. A thirty-six-week, double-blind trial.

Weekly treatment with low-dose oral methotrexate (MTX) was compared with daily auranofin (AUR) treatment in a 36-week double-blind, randomized, multicenter study of 281 patients with active, adult-onset rheumatoid arthritis. Both treatment groups showed significant improvement by the usual measures of clinical efficacy. The response with MTX occurred earlier and was consistently greater than that with AUR. An intent-to-treat analysis showed significantly greater improvement (P less than 0.01) with MTX for painful and swollen joint counts and physician and patient global assessments of disease activity. Adverse reactions were reported more frequently in the AUR group, and more AUR-treated patients were withdrawn from the study because of toxicity. MTX was thus more effective and better tolerated than AUR in this study.

Administration, Oral↗
PubMed1990

Secondary structure of the entomocidal toxin from Bacillus thuringiensis subsp. kurstaki HD-73.

The secondary structure of the toxin from Bacillus thuringiensis subsp. kurstaki (Btk) HD-73 was estimated by Raman, infrared, and circular dichroism spectroscopy, and by predictive methods. Circular dichroism and infrared spectroscopy gave an estimate of 33-40% alpha-helix, whereas Raman and predictive methods gave approximately 20%. Raman and circular dichroism spectra, as well as predictive methods, indicated that the toxin contains 32-40% beta-sheet structure, whereas infrared spectroscopy gave a slightly lower estimate. Thus, all of these approaches are in agreement that the native conformation of Btk HD-73 toxin is highly folded and contains considerable amounts of both alpha-helical and beta-sheet structures. No significant differences were detected in the secondary structure of the toxin either in solution or as a hydrated pellet.

Bacillus thuringiensis↗
PubMed1990

Binding of glucagon to lipid bilayers.

At physiological pH and temperature, glucagon binds to liposomes composed of egg phosphatidylcholine and cholesterol (2:1 mol/mol) in a highly specific manner. The chemical reactivities of the functional groups were determined over the concentration range of 1.0 X 10(-6)-3.0 X 10(-8) M by the method of competitive labelling with 1-fluoro-2,4-dinitrobenzene as the labelling reagent. At concentrations above 3 X 10(-7) M, the amino terminal histidine and the two tyrosine residues showed a marked decrease in reactivity in the presence of liposomes, but the reactivity of the Lys-12 N epsilon-amino group was unaltered. At lower concentrations the Lys-12 reactivity also decreased markedly, owing to a change in the environment of this group. These results indicated that two different forms of glucagon existed over the concentration range studied. Both in the absence and presence of liposomes the Lys-12 N epsilon-amino groups showed a transition in reactivity at 1.8 X 10(-7) M. In the presence of liposomes the other functional groups also showed a transition in reactivity at 2 X 10(-7) M but the change was much smaller. The pattern of reactivities were consistent with the X-ray crystallographic structure of the type 2 glucagon trimer being the predominant species at 10(-6) M, with free monomeric glucagon occurring at 3 X 10(-8) M. A trimerization constant of 4 X 10(13) M-2 at pH 7.5 and 37 degrees C was determined.(ABSTRACT TRUNCATED AT 250 WORDS)

Dipeptides↗
PubMed1990

Multifactorial facial pain--differential diagnosis: a case report.

A multidisciplinary algology team was formed to facilitate the diagnosis and treatment of complex head and neck pain disorders. The standard patient evaluation includes a history and physical, surface electromyography, Minnesota Multi-phasic Personality Inventory (MMPI), brief psychiatric interview, dental/occlusal analysis, a postural/musculoskeletal examination; and necessary diagnostic imaging. Clinicians meet in conference after each clinic session. Organic and psychiatric findings are compiled and a differential diagnosis is made. Treatment recommendations are outlined and a review of the evaluation and the therapeutic plan are forwarded to the referring doctor. A typical conference discussion is presented here.

Adult↗
PubMed1990

Octapeptide segments from the amino terminus of glycophorin A contain the antigenic determinants of the M and N blood groups systems.

Human red blood cells with phenotype N/N and M/M were tested in an agglutination assay with anti-N and anti-M antibodies, respectively. After incubation of the synthesized octapeptide (Leu-Ser-Thr-Thr-Glu-Val-Ala-Met) from the N-amino terminus of glycophorin A, with anti-N antibody, there was significant inhibition of the agglutination of the N-positive cells. There was also inhibition of the agglutination of the M-positive cells with anti-M antibody by the synthesized octapeptide (Ser-Ser-Thr-Thr-Gly-Val-Ala-Met) from the M-amino terminus of glycophorin A. There was no inhibition, however, of the agglutination of M-positive cells with anti-M antibodies by the N-amino terminal octapeptide. Likewise, the M-amino terminal octapeptide did not inhibit agglutination of N-positive cells with anti-N. Because the synthesized octapeptides contained no carbohydrate, the anti-N and anti-M specificity appears to be determined principally by the peptides themselves. Further studies with the use of chimeric peptides indicate that the amino terminal amino acid leucine of N-glycophorin A is a primary determinant of the N antigen, whereas the amino terminal serine of M-glycophorin A is a primary determinant for the M antigen.

Amino Acid Sequence↗
PubMed1989

Chemical properties of the histidine residue of secretin: evidence for a specific intramolecular interaction.

Secretin has a single histidine residue located at the amino terminus which plays a crucial role in its biological activity. The chemical properties, viz. pK and reactivity, of the alpha-amino and imidazole groups of this residue were determined at a secretin concentration of 10(-6) M in 0.1 M KCl at 37 degrees C. Competitive labelling using tritiated 1-fluoro-2,4-dinitrobenzene (DNP-F) as the labelling reagent was the experimental approach employed. The alpha-amino group was found to have a pK value of 8.83 and a reactivity 5-times that of the alpha-amino group in the model compound, histidylglycine. For the imidazole function a pK value of 8.24 and a reactivity 26-times that of the imidazole function in histidylglycine was found. Both these groups in secretin had pK values which were shifted one pK unit higher than in histidylglycine, but like the model compound the reactivity of the imidazole function was still linked to the state of ionization of the alpha-amino group. These observations are interpreted as evidence for the existence of a major conformational state in dilute aqueous solution in which the amino-terminal histidine of secretion is interacting with a negatively charged carboxyl group.

Binding, Competitive↗
PubMed1989

Facile preparation and characterization of the toxin from Bacillus thuringiensis var. kurstaki.

We report a simple three-step method of generating a homogeneous toxic fragment (toxin) in high yield from B. thuringiensis var. kurstaki. Purified crystals were digested with trypsin at pH 10.5, followed by (NH4)2SO4 precipitation and dialysis. For the HD73 strain the preparation is toxic to eastern-spruce-budworm (Choristoneura fuminiferana) larvae. It gives a single 66 kDa band on polyacrylamide-gel electrophoresis and a single band with an isoelectric point of 5.5 on an isoelectric-focusing gel. A single isoleucine N-terminus was detected, and the first 20 amino acids were found to be identical with those predicted from the gene nucleotide sequence. A single lysine C-terminus was detected, and the amino acid composition was in excellent agreement with tryptic cleavages at arginine-28 and lysine-623 of the protoxin. Raman spectroscopic analysis gave values of 20% alpha-helix, 35% beta-sheet and 45% unordered structure. The resistance of the toxin to most proteinases and its susceptibility to proteolysis by papain and Pronases indicates a compact multidomain structure.

Bacillus thuringiensis↗
PubMed1989

Euthyroid hyperthyroxinaemia due to endogenous antibodies to thyroxine and tri-iodothyronine. A case report.

A case of euthyroid hyperthyroxinaemia caused by auto-antibodies to thyroxine and tri-iodothyronine is presented. Gel filtration chromatography of the patient's serum showed increased binding of radio-labelled thyroxine analogue to a macromolecular component, which migrated in the gammaglobulin region on electrophoresis. Precipitation by protein A confirmed that this was an immunoglobulin. The importance of recognising this condition so that inappropriate therapy can be avoided is stressed.

Autoantibodies↗
PubMed1989

Isolation of carboxyl-terminal peptides from proteins by diagonal electrophoresis: application to the entomocidal toxin from Bacillus thuringiensis.

A procedure for the selective isolation of the C-terminal peptides from enzymatic digests of proteins is described. The methodology is based on the diagonal electrophoretic procedure described by R. G. Duggleby and H. Kaplan (1975) Anal. Biochem. 65, 346-354). The carboxyl groups in the protein are amidated with [14C]-methylamine followed by enzymatic digestion. Since only the C-terminal peptides lack a free carboxyl group, these peptides will lie on a diagonal line of a two-dimensional electrophoretogram run at pH 2.1 and 4.4. The diagonal line is delineated by autoradiography using [14C]taurine (net charge = 0 at pH 2.1 and 4.4) and [14C]choline (net charge = +1 at pH 2.1 and 4.4). Radioactive C-terminal peptides lie between these markers and can be directly excised for analysis. This procedure permits the detection and selective isolation of C-terminal peptides with minimal losses. The procedure was applied to the test proteins alpha-chymotrypsin and ribonuclease A. It was used to determine the C-terminus of the Bacillus thuringiensis toxin generated by tryptic cleavage of the protoxin.

Amino Acid Sequence↗
PubMed1989

Neck contracture as a rare complication of cervical soft tissue expansion.

An unusual complication using an inflatable silicon implant for removing postburn scarred tissue of the neck and face is described. The appearance of a fibrotic band along the inflating tube caused neck contracture. The purpose of this article is to present this uncommon complication and to emphasize the technical error when using the tissue expander in the neck region.

Adult↗
PubMed1989

[Angioblastoma (hypertrophic hemangioma). Preliminary report].

We are presenting herewith the case of a rare vascular tumor called angioblastoma which histopathological and clinical aspects may lead into errors of diagnosis and therapy. Its fast development, tendency to ulceration and bleeding and dark colour, are signs that may be confused with those of hemangiosarcomas and, inclusive, melanomas, because, from a clinical point of view, its pleomorphism and cellular atypical nature, due to immaturity, may induce diagnostic and histological doubts. In order to simplify diagnosis and therapy of an angioblastoma, we have treated it within the complex subject of vascular hyperplasias in general. Thereby, we have made three synopses of interest for such a purpose. Out of the consulted bibliography, its limited frequency is stated, as relative cases don't amount to a hundred worldwide and none of them was found in our country. We have reported the most accurate data for its correct clinical and histopathological diagnosis to avoid extremely aggressive therapies, since it is a lesion of benign biological behaviour.

Child↗
PubMed1989

[Early microsurgical reconstruction of extremities with free microvascular flaps].

The key to successful reconstruction of the extremities after complex trauma is often adequate soft tissue cover of the injured area. In the past, when surgical debridement of wounds was too extensive, or it exposed vital structures such as blood vessels, nerves, tendons or bones, the limb either had to be amputated or reconstructed in multiple surgical procedures. The use of free microvascular flaps in the immediate postinjury period allows single stage reconstruction of wounds including exposed vital tissues. It promotes limb salvage and shortens recovery time after severe trauma. 4 patients with severe complex trauma of the extremities underwent reconstruction with immediate free microvascular flaps, which included latissimus dorsi and rectus abdominis muscle and free scapular flaps. The surgical protocol included debridement of wounds on arrival with primary fixation of fractures and additional debridement procedures in the first week until the wound was stable. Then final fixation of fractures was performed and the wound was covered by a free microvascular flap. With this technique, single stage reconstruction is possible in most cases, with improvement in function and cosmetic outcome, multiple operations are avoided and the recovery period is shortened.

Adult↗
PubMed1988

[Serratia marcescens. Cutaneous involvement. Preliminary report].

Three cases of skin infections are reported. The biological examination revealed the presence of gram-negative bacillus Serratus marcescens whose chemical characteristics enabled its identification. Treatment was based on the respective antibiograms, cure being obtained in two patients. The third patient was treated with an antibiotic to which he was not sensible, with the consequent failure. Perusal of the literature at our disposal failed to reveal skin infections due to Serratus marcescens. Thus, in such patients who do not respond to the usual therapy, it is suggested that the Serratus marcescens bacillus be systematically looked for an reported in order to confirm this preliminary publication.

Adult↗