Biomedical subjects

A Holm

Publications and source records attributed to A Holm.

At least 19 recordsLinked to original sources

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

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

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

Identification of murine T-cell epitopes on the S4 subunit of pertussis toxin.

The aim of the present study was to identify murine T-cell epitopes on pertussis toxin subunit S4. Six mouse strains with five different haplotypes at the H-2 locus were immunized with the pertussis toxin B oligomer. Lymph node lymphocytes were isolated and stimulated in an in vitro proliferation assay with pertussis toxin components and 11 overlapping synthetic peptides synthesized on the basis of the primary sequence of S4. In vitro proliferative responses to the synthetic peptides revealed the presence of four distinct murine T-cell epitopes on subunit S4. The recognition of the peptides was major histocompatibility complex restricted. Immunizing four of the six mouse strains with the synthetic peptides showed that the peptides which were demonstrated to contain T-cell epitopes following immunization with the B oligomer were able to induce proliferative responses to detoxified pertussis toxin and pertussis toxin components containing subunit S4. One of the identified murine T-cell epitopes corresponded to one of the major human T-cell epitopes previously identified on subunit S4. It is hoped that this murine model system will facilitate the development of a synthetic immunogen mimicking the protective properties of pertussis toxin.

Animals↗

PubMed1993

Identification of B-cell epitopes on the S4 subunit of pertussis toxin.

The main purpose of the present study was to identify B-cell epitopes on the S4 subunit of pertussis toxin (PT) by the synthetic peptide approach. Two strategies were followed: (i) screening of two series of overlapping peptides (12- and 25-residue peptides) covering the entire S4 sequence by a panel of murine monoclonal anti-PT antibodies and various polyclonal anti-PT antisera in an enzyme-linked immunosorbent assay (ELISA), and (ii) analysis of the S4 amino acid sequence by a predictive algorithm followed by synthesis and immunization of mice with the predicted peptides coupled to diphtheria toxoid. The anti-peptide conjugate antisera were tested in an ELISA for cross-reactivity with native PT, B oligomer, and S4. Screening of the free peptides in an ELISA by the PT antisera indicated the presence of six B-cell epitope-containing domains covered by residues 18 to 32, 33 to 46, 39 to 52, 51 to 65, 71 to 84, and 91 to 106. None of the peptides, however, were recognized by the monoclonal anti-PT antibodies in an ELISA. Immunization with six computer-predicted peptides (B1 to B6) and three potential T-cell epitopes (T1 to T3) gave rise to very high antibody responses towards the homologous conjugates. With the exception of the anti-T1/diphtheria toxoid antisera, all anti-peptide conjugate antisera cross-reacted with PT in an ELISA at different levels. None of these anti-peptide conjugate antisera, however, showed any PT-neutralizing effect as measured by the Chinese hamster ovary cell assay and the leukocytosis-promoting activity test. The results of the present study suggest that discontinuous epitopes are predominant in the S4 subunit of native PT.

Amino Acid Sequence↗

PubMed1992

Synthesis and characterization of a 25-residue rubredoxin(II)-like metalloprotein and its valine-leucine mutant.

An iron-sulfur metalloprotein containing the 5-12 and 35-50 residues of Desulfovibrio gigas rubredoxin has been synthesized by Fmoc solid phase peptide synthesis and subsequent peptide folding. A Gly links the two residue chains between Val-5 and Glu-50. Sybyl Tripos structure optimization indicates only minor structural changes of the folded synthetic protein compared to the similar residue positions in the native protein. The UV-VIS spectrum of the reduced synthetic protein is very similar to that of native D. gigas rubredoxin and the molecular mass determined by laser mass spectrometry has the expected value (+/- 2D). No metal is transferred to the gas phase by the laser beam merely by mixing the peptide and iron(II), substantiating that the folding procedure is a necessary pre-requisite for protein formation. The Val-->Leu41 chemical mutant has also been synthesized and behaves in a closely similar fashion.

Amino Acid Sequence↗

PubMed1992

Induction of polyclonal antibodies to the S1 subunit of pertussis toxin by synthetic peptides coupled to PPD: effect of conjugation method, adjuvant, priming and animal species.

Two peptides, designated L and K, covering a sequence near the NH-terminal end of the S1 subunit of pertussis toxin (PT) were conjugated to the PPD (purified protein derivative) of M. tuberculosis by either glutaraldehyde (GLUT) or succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) and N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) and injected into groups of mice and guinea pigs. Initially, the effect of priming the animals with BCG vaccine and the use of aluminium hydroxide as adjuvant for the anti-peptide antibody response was studied. The group of BCG-primed mice immunized with adsorbed peptide conjugates showed the highest anti-peptide conjugate antibody response. Based on this finding, groups of BCG-primed mice were immunized four times with either adsorbed peptide L-GLUT, peptide L-SMCC/SPDP or peptide K-SMCC/SPDP conjugates and the fine peptide specificity as well as the PT and S1 cross-reactivity was investigated in ELISA. Mice immunized with peptide L-GLUT showed a significant antibody response to the homologous conjugate, only, whereas the group injected with the peptide L-SMCC/SPDP conjugate gave a significant response to both peptide K and L conjugated by the SMCC-SPDP method. Likewise, mice immunized with the peptide K-SMCC/SPDP conjugate reacted with the homologous and peptide L-SMCC/SPDP conjugate, although only the response to the former conjugate was significantly greater than the response to PPD. All groups showed a strong anti-PPD response. The anti-PT/S1 cross-reactivity of the antisera varied considerably within each group but was found to be highest in the peptide L-GLUT-immunized animals. The results of the present study not only stress the importance of BCG priming and use of aluminium hydroxide adjuvants for the immunogenicity of the peptides in question but also point to the specificity of the conjugation methods employed as low cross-reactivity between the anti-peptide L-GLUT and L-SMCC/SPDP antisera was noted. Moreover, it appeared that the choice of conjugation method may have an effect on the ability of the peptide conjugates to induce an antibody response cross-reacting with the native protein.

Adjuvants, Immunologic↗

PubMed1992

Identification of human T-cell epitopes on the S4 subunit of pertussis toxin.

Ten adult humans were vaccinated with the Japanese acellular pertussis vaccine JNIH-3, containing detoxified pertussis toxin (PT), formaldehyde, and filamentous hemagglutinin. The vaccination induced a specific antibody response to PT and filamentous hemagglutinin, and a Western blot (immunoblot) analysis of the antibody response to PT revealed antibodies to PT subunits S1, S2, S3, S4 and S5. The response of peripheral lymphocytes to PT was assessed in an in vitro proliferation assay. A proliferative response to detoxified PT and PT dimers S2-S4 and S3-S4 was found, and it was further demonstrated that the proliferative response to detoxified PT and dimer S2-S4 was mediated by T cells of the CD4+ phenotype. The specificity of the proliferative response to subunit S4 was analyzed with a range of synthetic peptides synthesized on the basis of the primary sequence of subunit S4. The proliferative response to the peptides revealed two major and one minor T-cell epitope located in the NH2-terminal end of subunit S4.

Adult↗

PubMed1992

Antibodies to a recombinant glutamate-rich Plasmodium falciparum protein: evidence for protection of individuals living in a holoendemic area of Liberia.

A Plasmodium falciparum antigen gene coding for a 220-kD glutamate-rich protein (GLURP) has been cloned, and the 783 C-terminal amino acids of this protein (GLURP489-1271) have been expressed as a beta-galactosidase fusion protein in Escherichia coli. The encoded 783 amino acid residues contain two areas of repeated amino acid sequences. Antibodies against recombinant GLURP489-1271, as well as against a synthetic peptide corresponding to GLURP899-916, and against a synthetic peptide representing the major glutamate rich repeat sequence from the P. falciparum ring erythrocyte surface antigen (Pf155/RESA) (EENV)6 were examined in 423 individuals (age range 30 days-78 years) living in a malaria holoendemic area of Liberia. In the 5-9-year-old age group, subjects with anti-GLURP489-1271 antibody concentrations greater than the mean value of the group had lower parasite densities than those with low antibody concentrations (P = 0.0151). High levels of anti-GLURP899-916 antibodies did not correlate with low parasite densities. However, high levels of anti-(EENV)6 antibodies were associated with significantly lower parasite densities in the 2-4-year-old age group (P = 0.0189). There was no correlation between the anti-GLURP489-1271 and anti-(EENV)6 antibody responses. The data provide indirect evidence for a protective role of antibodies reacting with recombinant GLURP489-1271 as well as with the synthetic peptide (EENV)6 from the Pf155/RESA.

Adolescent↗

PubMed1991

On the mechanism of kidney stone disintegration by acoustic shock waves.

In the present study we have, by theoretical and experimental investigations, especially concentrated on the importance of acoustic streaming, transient cavitation and microstreaming in the fluid close to the stone. Artificial stones, round stones 20 mm in diameter made of Plaster of Paris or rectangular flat stones, 25 x 25 x 15 mm, were suspended in a water bath at the focus of an EDAP LT01 lithotripter. "Unprotected" stones were disintegrated while stones surrounded by a layer of silicone showed no or very small disintegration. For successful destruction of the calculus, it seems to be essential that the stones are surrounded by a liquid, i.e., water.

Humans↗

PubMed1991

Measurements of ultrasonic fields with optical diffraction tomography.

This article describes a light diffraction technique that allows a measurement of ultrasonic pressure and phase in the cross-section of an ultrasonic beam. The light diffraction measurements are based on the Raman-Nath model of ultrasonic continuous-wave light diffraction. The diffracted light intensity can be expressed as an integration of sound pressure along the light path. This integration is analogous to the integration of the absorption coefficient in x-ray tomography. Tomography algorithms can be employed on the light measurement data to calculate the pressure and phase in a measuring plane perpendicular to the ultrasound propagation vector. The reconstruction of the measurement plane is done with an implementation of an algebraic reconstruction technique (ART). Satisfactory results are obtained with 10 projections. The measurement method is applicable up to Raman-Nath parameter values of 2.3. The experimental results are compared to miniature PVDF hydrophone measurements.

Algorithms↗

PubMed1991

Cell surface hydrophobicity and electrokinetic potential of Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus.

Laboratory strains and fresh isolates of Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus were examined for cell surface hydrophobicity and for electrokinetic properties under different experimental conditions. Fresh isolates of A. actinomycetemcomitans and H. aphrophilus were hydrophobic. Laboratory strains of A. actinomycetemcomitans were 20-30% less hydrophobic than fresh isolates. No difference was observed between laboratory and fresh isolates of H. aphrophilus. The pH of the suspending buffer, growth phase or incubation atmosphere did not significantly affect the hydrophobicity of the 2 species, whereas agar cultures of H. aphrophilus were less hydrophobic than broth cultures. All A. actinomycetemcomitans strains treated with sterile filtered saliva showed a concentration-dependent decrease in hydrophobicity of at most 30%. H. aphrophilus strains were not affected by the same treatment. Laboratory strains of H. aphrophilus were more negatively charged than A. actinomycetemcomitans, whereas fresh isolates of the 2 species exhibited similar surface charge. In the presence of saliva the mean cell surface charge of laboratory strains decreased by 56% for A. actinomycetemcomitans and by 73% for H. aphrophilus. The results indicate that the 2 species differ in expression of cell structures accounting for hydrophobicity and surface charge and that environmental factors might differently influence the physical properties of the two species analyzed.

Aggregatibacter actinomycetemcomitans↗

PubMed1991

Digitizing 35 mm colour slides for computerized general image handling in ophthalmology.

Standard personal computers with appropriate graphic user interfaces can now be configured for work with near photographic quality colour image handling. Advantages and limitations of digitized colour image work, including cost considerations, are discussed in relation to some common ophthalmic photography situations. Practical methods are briefly described. Future developments are outlined, particularly methods for direct acquisition of high quality colour images and image compression/reduction.

Computer Systems↗

PubMed1991

Peroxide chemistry of triaryl-substituted imidazoles. Fenflumizole, a non-steroidal, anti-inflammatory agent.

Fenflumizole, [2-(2,4-difluorophenyl)4,5-bis(4-methoxyphenyl)imidazole] is a nonsteroidal, anti-inflammatory analgesic. It reacts quantitatively with 1O2 forming 2-(2,4-difluorophenyl)-4-hydroperoxy-4,5-bis(4-methoxyphenyl)imidazole in a reversible reaction. In ethanol solution at ambient temperatures, the peroxide regenerates parent fenflumizole and 1O2 together with minor quantities of other products. The structures of those products point to the intermediacy of a 1,3-endoperoxide and a dioxetane. These observations may be relevant to the biological activity of fenflumizole.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed1990

The role of fragment X polymers in the fibrin enhancement of tissue plasminogen activator-catalyzed plasmin formation.

When thrombin-mediated fibrin formation and tissue plasminogen activator (t-PA)-mediated fibrinolysis proceed in dynamic interaction, desA-(desB beta 1-42)-fragment X polymers are shown to be the predominant fibrin derivatives present during the rapid second phase of Glu1- and Lys78-plasminogen activation. To further investigate the effect of this intermediate, a method was developed for the production and purification of fibrinogen-derived desA-(desB beta 1-42)-fragment X, deprived of both COOH-terminal A alpha-chains, but still capable of thrombin-mediated polymerization. DesA-(desB beta 1-42)-fragment X polymer was compared to intact fibrin with regard to its stimulatory effect on Glu1-, Lys78-, and Val443-plasminogen activation, and its binding of Glu1- and Lys78-plasminogen. Pure fragment X polymer gave rise to a biphasic activation pattern like that of fibrin, demonstrating similar kinetics of rapid phase activation. The dissociation constant for the binding of plasminogen to the effector decreases by a factor of 14, and the stoichiometry increases by a factor of 2 upon plasmin-catalyzed cleavage of both native Glu1- to Lys78-plasminogen, and fibrin to fragment X polymer. We conclude that desA-fibrin protofibril formation is sufficient to initiate fibrin enhancement of t-PA-catalyzed plasminogen activation, and that optimal stimulation depends on further plasmin-mediated modification of the fibrin effector to desA-fragment X-related moieties. Optimal stimulation is dependent on the presence of the kringle 1-4 domains of plasminogen and probably results from altered and increased binding of both plasminogen and t-PA to the modified effector.

Amino Acid Sequence↗

PubMed1990

Dot immunobinding and immunoblotting of picogram and nanogram quantities of small peptides on activated nitrocellulose.

Nitrocellulose was activated with divinyl sulfone, a spacer of ethylenediamine, and glutaraldehyde. The aldehyde groups on the activated nitrocellulose, Nit-CHO, were stable through one month at 4 degrees C. Peptides were attached to the membrane by reaction of the amino group with the free carbonyl, forming peptide bonds. The decapeptide angiotensin I (AI), the octapeptide angiotensin II (AII), angiotensin analogues, Met- and Leu-enkephalin (Met-E and Leu-E) were tested on the membranes with specific rabbit antibodies (sRaAb) against the peptides, and visualized by horseradish peroxidase conjugated anti-rabbit antibody (HRP-anti-RaAb). With this technique AII could be detected with a sensitivity of 20 pg/cm2 and AI by 500 pg/cm2. Substitution of Ala7 for Pro7 in AI and AII caused a marked reduced binding of anti-AI and antid-AII antisera, respectively, and it completely abolished crossreactivity of anti-AI with Ala7-AII as well as anti-AII with Ala7-AI. Peptides from the gp41 and gp36 antigens corresponding to the sequence aa596-618 of the human immunodeficiency viruses type 1 and 2, HIV-1 and HIV-2, were tested on Nit-CHO with two human sera from infected patients. The serological reactions were specific for both the HIV-1 and HIV-2 peptide, respectively. This indicated that the technique could be exploited for serological testing of humans. Separation of peptides by high performance thin layer chromatography (HPTLC) and identification by immunoblotting was demonstrated with angiotensin analogues. After separation by HPTLC on silica aluminium plates the peptides were electrotransfered by semidry electroblotting on Nit-CHO, followed by specific antibody overlays and developed as for the dot immunobinding technique. This combined method enabled us to differentiate between closely related peptide analogues and it improved the sensitivity of peptide detection 100-1000 fold as compared to visualization by quenched fluorescence on chromatography plates.

Angiotensin I↗

PubMed1990

Covalently linked peptides for enzyme-linked immunosorbent assay.

A general method is described, by which synthetic peptides are covalently linked via their carboxyl group to microtiter plates (CovaLink) for enzyme-linked immunosorbent assay (ELISA). Plates were prepared by this method with an angiotensin II peptide and with an HIV-2 peptide and attachment detected by rabbit anti-angiotensin serum and with a positive serum from an HIV-2-infected patient, respectively, using the common ELISA procedure in the last steps. The method is simple to perform, it constitutes an alternative to the common ELISA method, and eliminates the risk of inadvertent loss of peptide during the procedure. The method is highly reproducible and has a high sensitivity. It may be used for either antigen or antibody detection.

Angiotensin II↗