Biomedical subjects
Publications and source records attributed to D Stoll.
A modular mannanase (Man26A) from the bacterium Cellulomonas fimi contains a mannan-binding module (Man26Abm) that binds to soluble but not to insoluble mannans. Man26Abm does not bind to cellulose, chitin or xylan. The K(d) for binding of Man26Abm to locust bean gum (LBG) is approximately 0.2 microM. Man26A is the first mannanase reported to contain a mannan-binding module.
Yersinia heat-shock protein 60 (Ye-hsp60) has recently been found to be a dominant CD4 and CD8 T cell Ag in Yersinia-triggered reactive arthritis. The nature of this response with respect to the epitopes recognized and functional characteristics of the T cells is largely unknown. CD4+ T cell clones specific for Ye-hsp60 were raised from synovial fluid mononuclear cells from a patient with Yersinia-triggered reactive arthritis. and their specificity was determined using three recombinant Ye-hsp60 fragments, overlapping 18-mer synthetic peptides as well as truncated peptides. Functional characteristics were assessed by cytokine secretion analysis in culture supernatants after specific antigenic stimulation. Amino acid positions relevant for T cell activation were detected by single alanine substitutions within the epitopes. Fragment II comprising amino acid sequence 182-371 was recognized by the majority of clones. All these clones were specific for peptide 319-342. Th1 clones and IL-10-secreting clones occurred in parallel, sometimes with the same fine specificity. The 12-mer core epitope 322-333 is a degenerate MHC binder and is presented to some T cell clones in a "promiscuous" manner. This epitope is almost identical with a B27-restricted CTL epitope of Ye-hsp60. Cross-reactivity of Ye-hsp60-specific T cell clones with self-hsp60 was not observed. In conclusion, an interesting Ye-hsp60 T cell epitope has been identified and characterized. It remains to be determined whether this epitope is also relevant in other reactive arthritis patients.
The genes man26a and man2A from Cellulomonas fimi encode mannanase 26A (Man26A) and beta-mannosidase 2A (Man2A), respectively. Mature Man26A is a secreted, modular protein of 951 amino acids, comprising a catalytic module in family 26 of glycosyl hydrolases, an S-layer homology module, and two modules of unknown function. Exposure of Man26A produced by Escherichia coli to C. fimi protease generates active fragments of the enzyme that correspond to polypeptides with mannanase activity produced by C. fimi during growth on mannans, indicating that it may be the only mannanase produced by the organism. A significant fraction of the Man26A produced by C. fimi remains cell associated. Man2A is an intracellular enzyme comprising a catalytic module in a subfamily of family 2 of the glycosyl hydrolases that at present contains only mammalian beta-mannosidases.
Bovine viral diarrhea virus (BVDV) isolates can either be cytopathogenic (cp) or noncytopathogenic (noncp). While both biotypes express the nonstructural protein NS2-3, generation of NS3 strictly correlates with the cp phenotype. The production of NS3 is usually caused by cp specific genome alterations, which were found to be due to RNA recombination. Molecular analyses of the cp BVDV strain Oregon revealed that it does not possess such genome alterations but nevertheless is able to generate NS3 via processing of NS2-3. The NS3 serine protease is not involved in this cleavage, which, according to protein sequencing, occurs between amino acids 1589 and 1590 of the BVDV Oregon polyprotein. Transient-expression studies indicated that important information for the cleavage of NS2-3 is located within NS2. This was verified by expression of chimeric constructs containing cDNA fragments derived from BVDV Oregon and a noncp BVDV. It could be shown that the C-terminal part of NS2 plays a crucial role in NS2-3 cleavage. These data, together with results obtained by site-specific exchanges in this region, revealed a new mechanism for NS2-3 processing which is based on point mutations within NS2.
Pestiviruses represent the first RNA viruses for which recombination with cellular protein-coding sequences has been reported. As a result of such recombinations cytopathogenic (cp) pestiviruses can develop from noncytopathogenic (noncp) viruses. In the case of bovine viral diarrhea virus (BVDV), the generation of cp mutants is linked to the induction of the lethal syndrome mucosal disease (MD) in cattle. The cp BVDV JaCP was isolated from an animal which had come down with MD. The genome of JaCP contains a novel kind of cellular insertion (LC3*) which is flanked by duplicated pestivirus sequences. Neither insertion nor duplication is present in the genome of the accompanying noncp virus JaNCP. As part of the viral polyprotein, the insertion in the JaCP genome is translated into a polypeptide almost identical to a fragment of light chain 3, a subunit of the microtubule-associated proteins 1A and 1B from the rat. Transient-expression studies revealed that the LC3* sequence is able to induce an additional cleavage of the viral polyprotein. The respective cleavage occurs directly downstream of the LC3*-encoded sequence and is not dependent on the NS3 serine protease. Insertion of LC3* into an infectious noncp pestivirus cDNA clone without duplicated viral sequences resulted in recovery of a defective cp virus able to replicate only in the presence of a noncp helper virus. In contrast, introduction of both insertion and duplication led to an autonomously replicating cp virus.
In a review of the literature, the authors have studied the biochemical aspects of the sensory physiology of the nasal mucosa. In a previous publication, the abundant presence of neuropeptides in the afferent sensory nerves of the pituitary gland was studied. In this work, the authors have sought to determine the physiological role of these molecules in transmitting messages of aggression, and their implication in the defense mechanisms.
A retrospective study of the records of 392 consecutive patients prospectively operated for non-vasomotor nasal obstruction, has allowed us to identify a syndrome that we would call morphological syndrome. This is caused by anatomical deformities of the nasal septum, inferior turbinate hypertrophy frequently found being the most often only a compensation for the septal deviation. This endonasal abnormality, which can be congenital or post-traumatic, is most frequently of developmental origin and therefore most often found in Caucasian men, whose facial and septal growth is considerable. This morphological syndrome comprises unilateral or bilateral nasal obstruction, noisy nocturnal breathing (20% of patients), a posterior nasal drip (38% of patients), frontal headaches (49% of patients) and recurrent viral or bacterial rhinosinusitis (36% of patients). Other associated symptoms include epistaxis, olfactory dysfunction (hyposmia) and the manifestations of eustachian tube dysfunction. That this syndrome exists is confirmed by the effectiveness of septoplastic operation, performed in our hospital according to the method of Cottle, but without any surgical associated intervention on the inferior turbinates. This operation is an effective treatment both in a global fashion (95% of good results) and in relation to each of the symptoms (except postnasal drip) and is stable in the longterm and well tolerated (minor associated morbidity). This study allow us to re-establish the almost exclusive role of the nasal septum in the genesis of morphological nasal obstruction.
Failure of inactivation is the typical response of voltage-gated Na+ channels to the cytosolic presence of proteolytic enzymes, protein reagents such as N-bromoacetamide (NBA) or iodate, and antibodies directed against the linker between domains III and IV of the alpha-subunit. The present patch clamp experiments with cardiac Na+ channels aimed to test the hypothesis that these interventions may provoke the occurrence of non-inactivating Na+ channels with distinct kinetic properties. A site-directed polyclonal antibody (anti-SLP2, target sequence 1481-1496 of the cardiac Na+ channel alpha-subunit) eliminated fast Na+ inactivation to induce burst activity which was accompanied by the occurrence of two open states. A deactivation process terminated channel activity during membrane depolarization proceeding with time constants of close to 40 ms (at -40 mV). NBA-modified and iodatemodified Na+ channels were kinetically indistinguishable from the anti-SLP2-modified type since they likewise deactivate and, thus, attain an only moderate Po of close to 20%. This is fundamentally different from the behaviour of enzymatically-modified Na+ channels: after cytosolic proteolysis with alpha-chymotrypsin, trypsin or pronase, mean Po during membrane depolarization amounted to approximately 40% because deactivation operated extremely slowly and less efficiently (time constants 100-200 ms at -40 mV, as a minimum) or was virtually non-operating. Invitro cleavage of the synthetic linker sequence 1481-1496 confirmed that this part of the alpha-subunit provides a substrate for these peptidases or reactants for NBA but cannot be chemically modified by iodate. This iodate resistance indicates that iodate-modified Na+ channels are based on a structural alteration of still another region which is also involved in Na+ inactivation, besides the linker between domains III and IV of the alpha-subunit. Endogenous peptidases such as calpain did not affect Na+ inactivation. This stresses the stochastic nature of a kinetic peculiarity of cardiac Na+ channels, mode-switching to a non-inactivating mode.
The single-stranded genomic RNA of pestiviruses is of positive polarity and encompasses one large open reading frame of about 4,000 codons. The resulting polyprotein is processed co- and posttranslationally by virus-encoded and host cell proteases to give rise to the mature viral proteins. A serine protease residing in the nonstructural (NS) protein NS3 (p80) has been shown to be essential for the release of the NS proteins located downstream of NS3. In this report the NS3 serine protease-dependent cleavage sites for bovine viral diarrhea virus (BVDV) strain CP7 are described. Proteins used for analysis were generated in Escherichia coli or in eukaryotic cells by the use of the T7 vaccinia virus system. The N termini of NS4A, NS4B, NS5A, and NS5B were determined by protein sequencing. Analysis of the data obtained showed that leucine at P1 is the only position conserved for all cleavage sites. At P1' alanine is found at the NS4A-NS4B site, whereas serine resides at this position at the NS3-NS4A, NS4B-NS5A, and NS5A-NS5B cleavage sites. For all cleavage sites the amino acids found at P1 and P1' are conserved for different genotypes of pestiviruses, despite the high degree of sequence variation found between these viruses. It is therefore assumed that the cleavage sites determined for BVDV CP7 are representative of those for all pestiviruses.
Isolated inflammatory sphenoid sinusitis, associated with complications are rare. Authors report 23 cases in 8 years. The left sphenoid is the most often involved, in 50 years or more old females. Neurological complications are the most frequent (75% of the cases): isolated headache, neuro-ophtalmologic paralysis, meningitis, before respiratory complications (20%). A bacteriological etiology was found in 60% of the cases, and a fungal etiology in 25%. The treatment is based on a large endoscopic marsupialization of the sphenoid. Recovery is the rule, except for neuro-ophtalmologic paralysis which recovery is inconstant.
The authors report their experience with 31 hypoglosso-facial anastomoses mainly carried out after removal of tumours of the cerebello-pontine angle, 26 were available for evaluation: at 18 months there were 5 of grade II, 19 of grade IV and 2 of grade V. Only one patient was dissatisfied with the result. 24 of the patients had received their hypoglosso-facial anastomosis using the clinical technique of Korte. In 3 cases the operation was by the technique of May. The authors suggest a variation of May's technique: it was carried out on the last 4 patients. It consists of a hemi-hypoglosso-facial anastomosis with rerouting of the mastoid portion of the facial nerve, without using a nerve graft. The aim of this technique is to reduce the sequelae of hemi-lingual atrophy and paralysis (which gives trouble with articulation, mastication and deglutition). The authors' experience confirms that these sequelae are greatly reduced, if not abolished. The functional results in terms of facial movements were satisfactory and consistant: 43.7% were quantified--with 3 of grade II and one of grade IV on the House Brackmann scale.
Major histocompatibility complex (MHC) class II molecules present peptide antigens to CD(4+)-T cells. These heterogeneous peptides are derived from internalized exogenous proteins or from endogenous membrane proteins that are processed by the antigen-presenting cell. Peptides are bound to the MHC class II molecules in an extended conformation and extend out of the binding groove. The aim of this study was to estimate the influence of every amino acid in all the possible undecapeptide amides (2.048 x 10(14) individuals) on the binding to human MHC-DRB1*0101 molecules (HLA-DR1) and to identify new peptide ligands. 220 undecapeptide sublibraries, O/X10, each composed of ten degenerate positions and one defined position, were screened for binding to isolated HLA-DR1. Competition of the sublibraries with a fluorescence-labeled peptide ligand allowed definition of the amino acids favourable or unfavourable for DR1-binding at every sequence position. From the activity pattern of the undecapeptide library, 54 individual peptides were deduced (27 potential hits and 27 potential falls) and prepared by chemical synthesis. As anticipated, 27 positive and 27 negative results were obtained from the competition experiments. The 27 peptides that bind obey the rules for the HLA-DR1-binding motif. The synthetic peptide library approach proved to be valuable for the design of synthetic MHC class II ligands and thus can be considered as a basis for drug design in immunotherapy.
The rate of racemization during solid-phase peptide synthesis was studied using capillary electrophoresis and 18-crown-6 tetracarboxylic acid as chiral selector. For this purpose, the tripeptide D-Tyr-L-Lys-L-Trp as a model compound was synthesized by solid-phase peptide synthesis. A separation method based on capillary electrophoresis was developed which allowed all eight optical isomers of the tripeptide to be separated in a single run. The separation method was validated and was found to be well suited for purity analysis, with a limit of detection of 0.05% of the major compound. The method was revealed to be highly sensitive even to small variations in the buffer pH. Capillary electrophoresis was also employed to prove the enantiomeric purity of the Fmoc-protected amino acids used for peptide synthesis. A separation method based on micellar electrokinetic chromatography and gamma-cyclodextrin was developed for this purpose. The formation of optical isomers during peptide synthesis was investigated in the final product without hydrolyzing the tripeptide. This strategy allowed the rate of racemization to be determined by activation of amino acids in coupling cycles and cleavage of the peptides from the resin and from side-chain protecting groups. The formation of stereoisomers could be verified and was 0.4% or less per synthesis cycle. The experimental data agreed well with theoretical considerations, showing that racemization takes place mainly at the carboxy-activated amino acid during coupling.
The pestivirus genome encodes a single polyprotein which is subject to co- and posttranslational processing by cellular and viral proteases. The map positions of all virus-encoded proteins are known with the exception of a hypothetical peptide (p?) which interlinks the glycoprotein E2 and the nonstructural protein NS2-3 approximately between amino acid positions 1060 and 1130. Expression studies with recombinant vaccinia viruses bearing a set of C-terminally truncated E2-p?-NS2-encoding sequences derived from a bovine viral diarrhea virus (BVDV) strain led to the identification of a minor fraction of E2 which had an increased molecular mass due to a C-terminal extension. This larger form of E2 (E2p7) was specifically recognized by an antiserum raised against the amino acid sequence from 1065 to 1125. In addition, the antibodies revealed a BVDV-encoded 7-kDa protein (p7) in infected cells. By radiosequencing it was determined that Val-1067 was the N-terminal amino acid of in vitro-synthesized p7. Analyses of BVDV and classical swine fever virus virions suggest that neither p7 nor E2p7 is a major structural constituent.
Following a review of the literature, the authors focused their interest on the sensitive physiology of the biochemistry of the nasal mucosa. After a general presentation of the neurotransmitter molecules studied (the tachykinins), the authors have determined their presence in the sensitive afferent nerves of the pituitary gland, evidencing their co-existence with various other neuropeptides. The distribution of these molecules was studied, given their capacity of acting on peripheral tissular receptors. The widespread distribution and the considerable potential of such neuropeptides reveal the important they play in nasal physiology.
The authors, on the basis of 140 cases, analyze the bacterial flora of essentially chronic rhino-sinusitis observed in hospital consultations at the Pellegrin Hospital, the referral centre of the Aquitaine region. Samples taken in the operating theatre from 100 cases, by aspiration and mucosal biopsy, were studied in aerobic and anaerobic media. The results marked by the scarcity of sterile samples and of anaerobic germs, evidence the predominance of an aerobic flora, with in particular Haemophilus influenzae, Staphylococcus aureus and beta-hemolytic Streptococci. No significant difference was recorded between the bacteriology of acute sinusitis as against chronic sinusitis. The flora of sinusitis appear to depend more on the age factor than on the pathogenic type. Branhamella catharralis is present only in patients under 30. The enterobacteria appear in the adult 30-50 year-old age group and, as from the age of 50, are combined by Pseudomonas aeruginosa, which account for 18% of the strains. These two types of germs then appear to be predominant over the Staphylococci and the Haemophilus.