Biomedical subjects

A Holm

Publications and source records attributed to A Holm.

At least 73 records · Page 4Linked to original sources

PubMed1996

Characterization of two conformational epitopes of the Chlamydia trachomatis serovar L2 DnaK immunogen.

Chlamydia trachomatis DnaK is an important immunogen in chlamydial infections. DnaK is composed of a conserved N-terminal ATP-binding domain and a variable C-terminal peptide-binding domain. To locate the immunogenic part of C. trachomatis Dnak, we generated monoclonal antibodies (MAbs) against this protein. By use of recombinant DNA techniques, we located the epitopes for two MAbs in the C-terminal variable part. Although the antibodies reacted in an immunoblot assay, it was not possible to map the epitopes completely by use of 16-mer synthetic peptides displaced by one amino acid corresponding to the C-terminal part of C. trachomatis DnaK. To determine the limits of the epitopes, C. trachomatis DnaK and glutatione S-transferase fusion proteins were constructed and affinity purified. The purified DnaK fusion proteins were used for a fluid-phase inhibition enzyme-linked immunosorbent assay with the two antibodies. The epitopes were found not to overlap. To obtain DnaK fragments recognized by the antibodies with the same affinity as native C. trachomatis DnaK, it was necessary to express, respectively, regions of 127 and 77 amino acids. The MAbs described in this study thus recognized conformational epitopes of C. trachomatis DnaK.

Adenosine Triphosphatases↗

PubMed1996

The 18-kilodalton Chlamydia trachomatis histone H1-like protein (Hc1) contains a potential N-terminal dimerization site and a C-terminal nucleic acid-binding domain.

The Chlamydia trachomatis histone H1-like protein (Hc1) is a DNA-binding protein specific for the metabolically inactive chlamydial developmental form, the elementary body. Hc1 induces DNA condensation in Escherichia coli and is a strong inhibitor of transcription and translation. These effects may, in part, be due to Hc1-mediated alterations of DNA topology. To locate putative functional domains within Hc1, polypeptides Hc1(2-57) and Hc1(53-125), corresponding to the N- and C-terminal parts of Hc1, respectively, were generated. By chemical cross-linking with ethylene glycol-bis (succinic acid N-hydroxysuccinimide ester), purified recombinant Hc1 was found to form dimers. The dimerization site was located in the N-terminal part of Hc1 (Hc1(2-57)). Moreover, circular dichroism measurements indicated an overall alpha-helical structure of this region. By using limited proteolysis, Southwestern blotting, and gel retardation assays, Hc1(53-125) was shown to contain a domain capable of binding both DNA and RNA. Under the same conditions, Hc1(2-57) had no nucleic acid-binding activity. Electron microscopy of Hc1-DNA and Hc1(53-125)-DNA complexes revealed differences suggesting that the N-terminal part of Hc1 may affect the DNA-binding properties of Hc1.

Amino Acid Sequence↗

PubMed1995

Novel avidin and streptavidin binding sequences found in synthetic peptide libraries.

Synthetic resin-bound peptide libraries made of protein L-amino acids have been synthesized. During screening of the libraries, peptides that bind to avidin have been identified containing a novel motif with two histidines separated by one residue. A sub-library was synthesized and screened, and new critical residues appeared surrounding the two histidines. Additionally peptide libraries made of D-amino acids have been screened with avidin and streptavidin and novel motifs have been found.

Amino Acid Sequence↗

PubMed1995

1H, 13C and 15N random coil NMR chemical shifts of the common amino acids. I. Investigations of nearest-neighbor effects.

In this study we report on the 1H, 13C and 15N NMR chemical shifts for the random coil state and nearest-neighbor sequence effects measured from the protected linear hexapeptide Gly-Gly-X-Y-Gly-Gly (where X and Y are any of the 20 common amino acids). We present data for a set of 40 peptides (of the possible 400) including Gly-Gly-X-Ala-Gly-Gly and Gly-Gly-X-Pro-Gly-Gly, measured under identical aqueous conditions. Because all spectra were collected under identical experimental conditions, the data from the Gly-Gly-X-Ala-Gly-Gly series provide a complete and internally consistent set of 1H, 13C and 15N random coil chemical shifts for all 20 common amino acids. In addition, studies were also conducted into nearest-neighbor effects on the random coil shift arising from a variety of X and Y positional substitutions. Comparisons between the chemical shift measurements obtained from Gly-Gly-X-Ala-Gly-Gly and Gly-Gly-X-Pro-Gly-Gly reveal significant systematic shift differences arising from the presence of proline in the peptide sequence. Similarly, measurements of the chemical shift changes occurring for both alanine and proline (i.e., the residues in the Y position) are found to depend strongly on the type of amino acid substituted into the X position. These data lend support to the hypothesis that sequence effects play a significant role in determining peptide and protein chemical shifts.

Amino Acid Sequence↗

PubMed1995

Neuropsychological deficit in newly diagnosed patients with schizophrenia or schizophreniform disorder.

Forty-six patients with schizophrenia or schizophreniform disorder admitted to hospital for the first time were compared with 21 healthy volunteers on neuropsychological tests reflecting prefrontal and left respectively right hemisphere function. The patients with schizophrenia or schizophreniform disorder had a poorer performance on neuropsychological tests (such as Wisconsin Card Sorting) compared with healthy volunteers. Both left and right hemisphere seemed to be involved. Especially poor performance was found on somewhat complicated tests requiring ability of analysis, abstraction and memory, thus indicating dysfunction of prefrontal and temporohippocampal regions. Signs of sulcal enlargement and size of lateral ventricles on computed tomographic scan correlated with poor test performance on some tests both in patients and in healthy volunteers. No correlations were found between performance on neuropsychological test and negative symptoms.

Adolescent↗

PubMed1995

Mapping of the delayed-type hypersensitivity-inducing epitope of secreted protein MPT64 from Mycobacterium tuberculosis.

The gene encoding the immunogenic protein MPT64 found in culture filtrates of Mycobacterium tuberculosis H37Rv was expressed in Escherichia coli K-12 and purified as a recombinant protein. The purified recombinant MPT64 elicited delayed-type hypersensitivity (DTH) in outbred guinea pigs sensitized with Mycobacterium bovis BCG Tokyo. The skin reactions were comparable to those obtained with native MPT64. No skin reactions were observed when either recombinant MPT64 or native MPT64 was used in guinea pigs sensitized with M. bovis BCG Danish 1331. Amino- and carboxy-terminal deletion mutants of MPT64 were purified as fusion proteins for the mapping of DTH-inducing epitopes on recombinant MPT64 by use of the guinea pig skin test model. The part of the molecule responsible for the biological activity was located at the carboxy-terminal end. Further studies with overlapping synthetic peptides have pinpointed the biological activity at a single DTH-inducing epitope consisting of 15 residues between amino acids Gly-173 and Ala-187. Screening by PCR of 56 clinical isolates of M. tuberculosis from Danish and Tanzanian patients demonstrated the presence of mpt64 in all of the strains. These results point to MPT64 as a possible candidate for a skin test reagent specific for diagnosis of human tuberculosis.

Amino Acid Sequence↗

PubMed1994

A cleaved form of the receptor for urokinase-type plasminogen activator in invasive transplanted human and murine tumors.

It was recently found that urokinase-type plasminogen activator (uPA) is involved in the cleavage of its receptor (uPAR) on cultured cells, thereby releasing one of the receptor's 3 domains (the ligand binding domain I) and leaving the 2 others [uPAR(2 + 3)] anchored to the cell surface. With monoclonal antibodies (MAbs) we have now identified human uPAR(2 + 3) in lysates of invasive human MDA-MB-231 mammary carcinomas xenografted into nude mice. The production of peptide antibodies recognizing different domains of murine uPAR made it possible to identify a similar cleaved form of uPAR, murine uPAR(2 + 3), in extracts of primary Lewis lung carcinomas. Cleavage of uPAR also occurs in cultured MDA-MB-231 cells and Lewis lung carcinoma cells. This cleavage is inhibited by anticatalytic antibodies to either human or murine uPA, respectively, indicating that it is catalyzed by either uPA or plasmin generated by uPA. The amount of uPAR(2 + 3) may therefore be directly related to the activity of the uPA system and it is possible that the level of uPAR(2 + 3) in cancer tissue may prove to be a stronger prognostic parameter than the levels of either full-length uPAR or UPA.

Amino Acid Sequence↗

PubMed1994

Synthesis and characterization of Desulfovibrio gigas rubredoxin and rubredoxin fragments.

The 52-residue Desulfovibrio gigas rubredoxin peptide chain has been synthesized and a procedure for chain folding around iron(II) developed. The folded, stable synthetic rubredoxin can be subjected to purification, and reversibly oxidized and reduced. Ultraviolet/visible absorption and CD spectra of both forms show all the same features as native D. gigas rubredoxin, and the symmetric and asymmetric Fe-S stretching bands in the resonance Raman spectrum can be identified. In addition, the matrix-assisted laser desorption mass spectrum of a peptide sample exposed to trace amounts of iron is dominated by a peak at 5735Da very close to the value for the calculated molecular mass. Details in the ultraviolet/visible bandshape and mass spectrum, however, indicate remaining impurities. In comparison, a previously synthesized 25-residue rubredoxin fragment with the non-conserved positions 13-35 and 51-52 omitted and Val5-Glu50 anchored via glycine folds gives the correct molecular mass and ultraviolet/visible spectrum, but is much more labile than the 52-residue protein. This shows that non-conserved residues are crucial in protein folding and that chemical metalloprotein synthesis offers alternative prospects to microbiological protein engineering.

Circular Dichroism↗

PubMed1994

A quantitative assay to measure the interaction between immunogenic peptides and purified class I major histocompatibility complex molecules.

A direct and sensitive biochemical assay to measure the interaction in solution between peptides and affinity-purified major histocompatibility complex (MHC) class I molecules has been generated. Specific binding reflecting the known class I restriction of cytotoxic T cell responses was obtained. Adding an excess of beta 2-microglobulin (beta 2m) significantly increased the rate of peptide association, but it did not affect the rate of dissociation. Binding was complicated by a rapid and apparently irreversible loss of functional MHC class I at 37 degrees C which might limit the life span of empty MHC class I thereby preventing the inadvertent exchange of peptides at the target cell surface. All class I molecules tested bound peptides of the canonical octa- to nona-meric length. However, one class I molecule, Kk, also bound peptides, which were much longer suggesting that the preference of class I molecules for short epitopes is not absolute and may be caused by factors other than the peptide-MHC class I binding event itself.

Amino Acid Sequence↗

PubMed1994

Incomplete Fmoc deprotection in solid-phase synthesis of peptides.

During solid-phase peptide synthesis of homo-oligopeptides containing leucine or alanine using the Fmoc strategy, we have observed ineffective N-alpha-deprotection with piperidine in a sequence-dependent manner. Incomplete deprotection was found to be associated with subsequent slow or incomplete amino acid coupling. Optimization of the deprotection step was carried out by varying the experimental conditions e.g. deprotection time, temperature, solvents and addition of chaotropes. Coupling and deprotection steps have been investigated using color monitoring, as well as FAB MS and HPLC for product analysis. The phenomena of difficult coupling and deprotection steps in the investigated systems have been demonstrated to have the same physical chemical origins, beta-sheet formation.

Acylation↗

PubMed1994

Characterization of a linear epitope on Chlamydia trachomatis serovar L2 DnaK-like protein.

A cytoplasmic 75-kDa immunogen from Chlamydia trachomatis serovar L2 has previously been characterized as being similar to the Escherichia coli heat shock protein DnaK. We have localized a linear epitope for one monoclonal antibody specific for C. trachomatis DnaK. By use of a recombinant DNA technique, the epitope was limited to 14 amino acids. With synthetic peptides, the epitope was further limited to eight amino acids. Six of these amino acids are conserved in bovine HSP70, which has a known three-dimensional structure. The amino acid sequence homologous to the epitope is located in a linear part of the HSP70 molecule known as connect II.

Amino Acid Sequence↗

PubMed1993

Peptide dot immunoassay and immunoblotting: electroblotting from aluminum thin-layer chromatography plates and isoelectric focusing gels to activated nitrocellulose.

Nitrocellulose membrane was preactivated with divinyl sulfone, and a spacer of 1,6-diaminohexane was coupled to the membrane which was functionalized by glutaraldehyde, leaving a reactive carbonyl group. The peptides were coupled to the carbonyl by the side chain and terminal amino groups. The octapeptide angiotensin II (sequence: DRVYIHPF) and peptide analogs containing 6-10 amino acid residues were dotted directly onto the matrix at 45 degrees C for 15 min and detected by specific antisera, which were raised in rabbits against angiotensin I and II, respectively. They were visualized by peroxidase-coupled anti-rabbit IgG antibodies. The detection limit for synthetic angiotensin II was 500 fg per cm2 (= 500 amol per cm2) and for the decapeptide angiotensin I (sequence: DRVYIHPFHL) it was 500 pg per cm2 (= 400 fmol per cm2). Separation of synthetic angiotensin analogs by high performance thin-layer chromatography on silica coated aluminum plates was followed by electroblotting onto activated nitrocellulose and detection with specific antibodies, showing a sensitivity of 100 fg and 1 pg for angiotensin II and angiotensin I, respectively. Isoelectric focusing in agarose using Ampholine carrier ampholytes and immunoblotting with specific antisera displayed a lower sensitivity for angiotensin II and angiotensin I of 2 ng and 20 ng, respectively. The isoelectric focusing and immunoblotting technique was applied for separation of angiotensin I and II and related peptides in serum, where synthetic angiotensin I was degraded in the presence of 1 mM phenylmethylsulfonyl fluoride and 10 mM ethylenediaminetetraacetic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Acquired Immunodeficiency Syndrome↗

PubMed1993

Chemical activation of nitrocellulose membranes for peptide antigen-antibody binding studies: direct substitution of the nitrate group with diaminoalkane.

A method to covalently link peptide and proteins, through a diaminoalkane spacer to nitrocellulose membrane was developed for immunochemical applications. Initially the nitrocellulose membrane was modified by covalent incorporation of diaminoalkane spacers without any prior activation. The incorporation was shown primarily to involve alpha-elimination of the nitrate groups, and an imine was formed between the carbonyl group on the membrane and the diaminoalkane. The rate of incorporation increased exponentially with the length of the diaminoalkane as determined by a ninhydrin colorimetric reaction, which was developed for the study. More than 200 nmole diamine per mg nitrocellulose could be incorporated, but less than 11 nmol/mg (63 nmol/cm2) was chosen in order to retain the strength of the membrane. The primary amino groups of the modified membrane was glutaraldehyde activated and the octapeptide, angiotensin II, was covalently bound. A dot immunoassay was performed where specific anti-angiotensin II antibodies reacted with the peptide and was visualized by peroxidase coupled secondary antibodies. The results were quantified by video densitometry above 0.005 microgram AII per cm2. The immunoassay showed improved detection of the peptide on the activated as compared to unactivated membrane as well as increased retention of radiolabeled [125I]angiotensin II.

Angiotensin II↗

PubMed1993

Solid-phase peptide synthesis on proteins.

A new method for solid-phase peptide synthesis in which a protein is used as the solid support has been developed. Two aspects of the method have been demonstrated. The peptides H-Phe-Leu-Glu-Glu-Val-OH (1) and H-Leu-Leu-Ala-Gly-Val-OH (2), respectively, were synthesized on the amino groups of BSA via a cleavable linker, using the Fmoc group protecting scheme. The free peptides were obtained by cleavage from the protein with 95% TFA, precipitation in diethyl ether and additional work-up by either dialysis or centrifugation through a membrane followed by gel filtration. The identity of the products was determined by amino acid analysis and HPLC. The peptide-protein conjugates, H-Ser-Met-Asp-Thr-Ser-Asn-Lys-Glu-Glu-Lys-BSA (3) and H-Thr-Val-Leu-BTG (4), were obtained in the same manner, omitting the cleavable linker group. It was found that 35-50 peptide chains were conjugated per molecule BSA and BTG, respectively. Immunization of rabbits with conjugate 3 gave rise to peptide specific antibodies. This method will be useful for generation of sequence specific antibodies, since the peptide is conjugated to the carrier protein exclusively via its C-terminus, and will allow synthesis of highly specific peptide-protein conjugates.

Amino Acid Sequence↗

PubMed1993

Multiple column peptide synthesis, Part 2 (1, 2).

A manually operated apparatus for parallel multiple column soli-phase peptide synthesis is described. It employs Fmoc-amino acid-O-Dhbt or -Pfp esters in the continuous flow version of the polyamide method on small packed columns of kieselguhr supported resin in a reaction block of Teflon. The solvents and deprotecting reagents are dispensed from two washers in a parallel fashion and reagent consumption is low. Activated and protected amino acids are transferred from a dispenser tray as solutions, eight at a time. The use of the method is demonstrated by the synthesis of overlapping peptides from a protein structure and of analogous protease substrates. The products have been characterized by HPLC, FAB mass spectroscopy and amino acid analysis.

Amino Acid Sequence↗

PubMed1993

Killing of Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus by human polymorphonuclear leukocytes in serum and saliva.

The ability of polymorphonuclear leukocytes from human peripheral blood to kill Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus was examined with fresh isolates and laboratory strains from each species (5 strains within each group) under different conditions. Bacterial cells were mixed with a polymorphonuclear leukocyte suspension in the presence of either active serum or heat-inactivated serum or active serum together with sterile-filtered saliva. Surviving bacteria were determined by counting the number of bacterial colony-forming units in the mixtures after a 60-min incubation at 37 degrees C. Mixtures without polymorphonuclear leukocytes served as controls for the evaluation of the degree of killing of the bacteria. In general, A. actinomycetemcomitans resisted phagocytic killing to a greater extent than H. aphrophilus, and the killing of the former species mainly depended on the presence of heat-labile serum components, probably complement factors. Laboratory strains of A. actinomycetemcomitans were more easily killed than fresh isolates. The presence of saliva in the reaction mixtures decreased the degree of killing. However, strain-dependent variations in the killing were found under either condition. The leukotoxic activity of A. actinomycetemcomitans strains, determined by a [51Cr]-release assay, was not correlated with the resistance of these strains to the phagocytic killing. The results point out a strain-dependent difference in the ability of A. actinomycetemcomitans to evade the inflammatory response associated with polymorphonuclear leukocytes. This difference may constitute a potential virulence factor for this periodontopathogen. Furthermore, the leukotoxicity of the strains is not the main determinant that modifies the interaction of A. actinomycetemcomitans with human neutrophils.

Aggregatibacter actinomycetemcomitans↗

PubMed1993

Polymorphonuclear leukocyte chemiluminescence induced by Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus in serum and saliva.

The ability of different strains of Actinobacillus actinomycetemcomitans (A.a.) and Haemophilus aphrophilus (H.a.) to trigger activation of an oxidative burst in human polymorphonuclear leukocytes (PMNL) was examined by measuring the luminol-amplified light emission--chemiluminescence (CL)--from these cells. Bacterial cells were incubated with PMNL from one healthy subject, in the presence of either active serum, heat-inactivated serum, saliva, or saliva and active serum. In the presence of active serum, all five H.a. strains and two out of five A.a. strains triggered a CL response. The CL induced in the presence of heat-inactivated serum was considerably less than that achieved with fresh serum. In the presence of only saliva, all strains induced considerably weaker CL responses than those induced in the presence of saliva with active serum. In the presence of serum, intracellular reactions appeared to be the main source of CL, while addition of saliva and active serum increased the extracellular CL. The results indicate that strain-dependent differences exist among A.a. strains in their ability to trigger the oxygen-dependent bactericidal mechanisms of human PNML. In contrast, the CL patterns of H.a. strains were equivalent. Various factors in the environment, such as activated complement and salivary compounds, affect the interaction of these species with neutrophils.

Aggregatibacter actinomycetemcomitans↗

PubMed1993

Adherence and surface properties of buoyant density subpopulations of group B streptococci, type III.

From clinical isolates of group B streptococci, buoyant density subpopulations with variable amounts of type-specific polysaccharide can be derived. The ability of these subpopulations to adhere to vaginal epithelial cells and to glass was assessed by using [3H]-labeled bacteria. Subpopulations with no or minute amounts of capsule showed good adherence capacity and a hydrophobic cell surface. In contrast, a large capsule made the bacteria less adherent and rendered their surface hydrophilic. A negative cell surface charge, judged by determination of the z-potential, was directly related to the amount of capsule. Proteinase K treatment of less encapsulated variants significantly decreased the cell hydrophobicity. The ability of a GBS strain to express different surface characteristics may be a mechanism for adaptation to environmental changes, and may thereby contribute to the pathogenicity of GBS.

Bacterial Adhesion↗