Efficient assembly of recombinant major histocompatibility complex class I molecules with preformed disulfide bonds.

The expression of major histocompatibility class I (MHC-I) crucially depends upon the binding of appropriate peptides. MHC-I from natural sources are therefore always preoccupied with peptides complicating their purification and analysis. Here, we present an efficient solution to this problem. Recombinant MHC-I heavy chains were produced in Escherichia coli and subsequently purified under denaturing conditions. In contrast to common practice, the molecules were not reduced during the purification. The oxidized MHC-I heavy chain isoforms were highly active with respect to peptide binding. This suggests that de novo folding of denatured MHC-I molecules proceed efficiently if directed by preformed disulfide bond(s). Importantly, these molecules express serological epitopes and stain specific T cells; and they bind peptides specifically. Several denatured MHC-I heavy chains were analyzed and shown to be of a quality, which allowed quantitative analysis of peptide binding. The analysis of the specificity of the several hundred human MHC haplotypes, should benefit considerably from the availability of pre-oxidized recombinant MHC-I.

Cholesterol-dependent interaction of syncollin with the membrane of the pancreatic zymogen granule.

Syncollin is a protein of the pancreatic zymogen granule that was isolated through its ability to bind to syntaxin. Despite this in vitro interaction, it is now clear that syncollin is present on the luminal side of the zymogen granule membrane. Here we show that there are two pools of syncollin within the zymogen granule: one free in the lumen and the other tightly associated with the granule membrane. When unheated or cross-linked samples of membrane-derived syncollin are analysed by SDS/PAGE, higher-order forms are seen in addition to the monomer, which has an apparent molecular mass of 16 kDa. Extraction of cholesterol from the granule membrane by treatment with methyl-beta-cyclodextrin causes the detachment of syncollin, and this effect is enhanced at a high salt concentration. Purified syncollin is able to bind to brain liposomes at pH 5.0, but not at pH 11.0, a condition that also causes its extraction from granule membranes. Syncollin binds only poorly to dioleoyl phosphatidylcholine liposomes, but binding is dramatically enhanced by the inclusion of cholesterol. Finally, cholesterol can be co-immunoprecipitated with syncollin. We conclude that syncollin is able to interact directly with membrane lipids, and to insert into the granule membrane in a cholesterol-dependent manner. Membrane-associated syncollin apparently exists as a homo-oligomer, possibly consisting of six subunits, and its association with the membrane may be stabilized by electrostatic interactions with either other proteins or phospholipids.

Adaptation of wild-type measles virus to CD46 receptor usage.

Vaccine strains of measles virus (MV) use CD46 as receptor and downregulate CD46 from the surface of infected cells. MVs isolated and passaged on B-lymphoid cells (wild-type MVs) seem to use another receptor and do not downregulate CD46. In the present study, we found that isolation of MV on human or marmoset B-lymphoid cells did not alter the MV haemagglutinin (H) protein relative to that in the patient. The wild-type isolates were adapted to the human epithelial HEp-2 cell line or the monkey fibroblast Vero cell line. All HEp-2 cell adapted viruses and 1 out of 4 Vero cell adapted viruses acquired the capacity to use CD46 as receptor, as measured by their ability to infect murine cells expressing human CD46. Adaptation to CD46 receptor usage was coupled to substitution of amino acid 481 of the MV H protein from asparagine to tyrosine but not to CD46 downregulation. The present study demonstrates that CD46 receptor usage can be induced by adaptation of wild-type MV to cells that do not express a wild-type receptor and suggests that a similar mechanism acted on the progenitor viruses of the present MV vaccine strains during their isolation and attenuation.

Analysis of the association of syncollin with the membrane of the pancreatic zymogen granule.

Syncollin is a pancreatic zymogen granule protein that was isolated through its ability to bind to syntaxin. Here we show that syncollin has a cleavable signal sequence and can be removed from granule membranes by washing with sodium carbonate. When membranes were subjected to Triton X-114 partitioning, syncollin was found predominantly in the aqueous phase, indicating that it is not sufficiently hydrophobic to be embedded in the membrane. Syncollin has intramolecular disulfide bonds and was accessible to water-soluble cross-linking and biotinylating reagents only when granules were lysed by sonication. These results indicate that syncollin is tightly bound to the luminal surface of the granule membrane. In situ, syncollin was resistant to proteases such as trypsin. When granule membranes were solubilized in ionic detergents such as deoxycholate, this trypsin resistance was maintained, and syncollin migrated on sucrose density gradients as a large (150 kDa) protein. In contrast, in non-ionic detergents such as Triton X-100, syncollin became partially sensitive to trypsin and behaved as a monomer. Syncollin in alkaline extracts of granule membranes was also monomeric. However, reduction of the pH regenerated the oligomeric form, which was insoluble. We conclude that syncollin exists as a homo-oligomer and that its ability to self-associate can be reversibly modulated via changes in pH. In light of our findings, we reassess the likely role of syncollin in the pancreatic acinar cell.

A single-chain fusion molecule consisting of peptide, major histocompatibility gene complex class I heavy chain and beta2-microglobulin can fold partially correctly, but binds peptide inefficiently.

The function of major histocompatibility complex class I (MHC-I) molecules is to sample peptides from the intracellular environment and present these peptides to CD8+ cytotoxic T lymphocytes (CTL). We have attempted to develop a general approach to produce large amounts of pure and active recombinant MHC-I molecules. A convenient source of MHC-I molecules would be a valuable tool in structural and biochemical analysis of MHC-I, and in experiments using MHC-I molecules to enable specific manipulations of experimental and physiological CTL responses. Here we describe the generation of a recombinant murine MHC-I molecule, which could be produced in large amounts in bacteria. The recombinant MHC-I protein was expressed as a single molecule (PepSc) consisting of the antigenic peptide linked to the MHC-I heavy chain and further linked to human beta2-microglobulin (hbeta2m). The PepSc molecule was denatured, extracted, purified and folded using a recently developed in vitro reiterative refolding strategy. This led to the formation of soluble, recombinant MHC-I molecules, which migrated as monomers of the expected size when submitted to non-reducing sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Serological analysis revealed the presence of some, but not all, MHC-I-specific epitopes. Biochemically, PepSc could bind peptide, however, rather ineffectively. We suggest that a partially correctly refolded MHC-I has been obtained.

Identification of SNAREs involved in regulated exocytosis in the pancreatic acinar cell.

The molecular basis of exocytotic membrane fusion in the pancreatic acinar cell was investigated using an in vitro assay that measures both zymogen granule-plasma membrane fusion and granule-granule fusion. These two fusion events were differentially sensitive to Ca(2+), suggesting that they are controlled by different Ca(2+)-sensing mechanisms. Botulinum neurotoxin C (BoNT/C) treatment of the plasma membranes caused cleavage of syntaxin 2, the apical isoform of this Q-SNARE, but did not affect syntaxin 4, the basolateral isoform. BoNT/C also cleaved syntaxin 3, the zymogen granule isoform. BoNT/C treatment of plasma membranes abolished granule-plasma membrane fusion, whereas toxin treatment of the granules reduced granule-plasma membrane fusion and abolished granule-granule fusion. Tetanus toxin cleaved granule-associated synaptobrevin 2 but caused only a small reduction in both granule-plasma membrane fusion and granule-granule fusion. Our results indicate that syntaxin 2 is the isoform that mediates fusion between zymogen granules and the apical plasma membrane of the acinar cell. Syntaxin 3 mediates granule-granule fusion, which might be involved in compound exocytosis. In contrast, the major R-SNARE on the zymogen granule remains to be identified.

Prion diseases (transmissible spongiform encephalopathies): a review.

Bovine spongiform encephalopathy (BSE), or "mad cow disease", as well as Creutzfeldt-Jakob-Disease in humans, have recently been attracting public attention. The European Society of Gastrointestinal Endoscopy (ESGE) has issued guidelines on the topic (Endoscopy 1997; 29: 203-4), which were accompanied by a review article by T. Ponchon ("Transmission of hepatitis C and prion diseases through digestive endoscopy: evaluation of risk and recommended practices", Endoscopy 1997; 29: 199-202) dealing with the potential transmission of BSE and hepatitis C through gastrointestinal endoscopy. The following article on prion diseases in general provides a more in-depth overview of these disorders and their epidemiology and pathophysiology.

Progressive multifocal leucoencephalopathy.

Progressive multifocal leucoencephalopathy (PML) is a rarely occurring demyelinating disease of the central nervous system caused by a neurotropic papovavirus named JC virus (JCV). The most frequently affected affected regions are the cerebral hemispheres, especially the parietooccipital region, followed by the cerebellum and brain stem. The disease occurs predominantly in individuals with an immunocompromised state and impaired cellular mediated immunity (CMI) due to other underlying illness. More extensive use of irradiation and immunosuppressive therapy in relation to increased transplantational activities as well as treatment of autoimmune diseases and malignancies, in addition to the appearance of the acquired immunodeficiency syndrome (AIDS) as a consequence of infection with the human immunodeficiency virus (HIV), has caused a considerable increase in the occurrence of PML. The course of the disease is still most often rapidly progressive and fatal, but several cases with prolonged survival and even remission have been reported, and various antiviral treatments have been tried. The only drug that until now has shown favourable results is cytosine arabinoside. In HIV-infected PML-patients immunomodulation with AZT/zidovudine may alleviate the course and improve the prognosis in some patients. Suspicion of PML should lead to an extensive immunological investigation before considering of brain biopsy, which is still the only specific test. On the basis of the increased frequency of PML in relation to HIV-infection, it is likely that our knowledge of the pathogenetic aspects will increase, which, hopefully, may lead to an effective therapeutic strategy.

A model phage display subtraction method with potential for analysis of differential gene expression.

In order to establish a subtractive procedure that makes it possible to enrich selectively phage displayed antibodies directed against proteins constituting a difference between two populations of cells, a competitive selection strategy utilising two solid phases was developed and tested. Antibodies recognising a defined difference between two otherwise identical protein mixtures were isolated and their specificity confirmed. To test further the efficacy of selection inhibition during the competitive selections, selections towards a total cell extract were performed with and without competition from the same extract. An analysis of the resulting phage antibodies confirmed the subtractive nature of the system described.

[Human spongiform encephalopathies. Diseases caused by prions]. / Humane spongiforme encefalopatier Sygdomme forårsaget af prioner.

Spongiform encephalopathies or prion diseases are common denominators for a group of diseases, all fatal, which show characteristic neuropathological changes. In man the group includes four diseases: kuru, Creutzfeldt-Jakob's disease (CJD), Gerstmann-Sträussler-Scheinker's disease (GSS) and fatal familial insomnia (FFI). In animals it comprises the following six: scrapie (sheep and goats), transmissible mink encephalopathy (mink), chronic wasting disease (mule/elk), exotic ungulate encephalopathy (antelopes) and feline and bovine spongiform encephalopathy (cats and cattle). The diseases can be transmitted to other animal species, including mice, hamsters, rats, monkeys and chimpanzees. The diseases show common histopathological changes in both animals and man, which are restricted to the CNS, in none of them are there signs of an inflammatory process or fever, and the cellcount in the CSF is normal. Disease symptoms are characterized by loss of higher levels of brain functions resulting in dementia and ataxia as the most pronounced symptoms. All prion diseases are associated with accumulation of an abnormal, partially proteinase-resistant isoform of a host-coded protein, the prion-protein (PrP), which is a cell surface sialoglycoprotein of unknown significance. PrP is highly conserved among mammals and is expressed in most tissues. Diseases caused by prions are exceptional as they can occur sporadically and are transmissible both genetically and infectiously. This review attempts to elucidate the present knowledge of the natural history of these diseases in man.

A fast, economical and efficient method for DNA purification by use of a homemade bead column.

A fast, efficient and economical method for purification of DNA from various sources by the use of silica particles packed in homemade bead columns is described. The method is a further development of the method devised by Carter and Milton (1), improved by the introduction of a column operated with a vacuum system. The method is highly suitable for multiple sample handling in the preparation of DNA for sequencing and other purposes. Protocols are devised for plasmid minipreparation, gel elution and purification of DNA from solutions e.g. enzyme reactions.

[Progressive multifocal leukoencephalopathy]. / Progressiv multifokal leukoencefalopati.

Progressive multifocal leucoencephalopathy (PML) is a rarely occurring demyelinating disease of the central nervous system caused by a neurotropic papovavirus named JC virus (JCV). The most frequently affected areas are the cerebral hemispheres, especially the parieto-occipital region, followed by the cerebellum and brain stem. The disease occurs predominantly in individuals with an immunocompromised state and impaired cellular mediated immunity (CMI) due to other underlying illness. More extensive use of irradiation and immunosuppressive therapy in relation to increased transplantational activities as well as treatment of autoimmune diseases and malignancies, in addition to the appearance of the acquired immunodeficiency syndrome (AIDS) as a consequence of infection with the human immunodeficiency virus (HIV), has caused a considerable increase in the occurrence of PML. The course of the disease is still most often rapidly progressive and fatal, but several cases with prolonged survival and even remission have been reported, and various antiviral treatments have been tried. The only drug that until now has shown favourable results is cytosine arabinoside. In HIV-infected PML-patients immunomodulation with AZT/zidovudine may alleviate the course and improve the prognosis in some patients. Suspicion of PML should lead to an extensive immunological investigation before considering of brain biopsy, which is still the only specific test. On the basis of the increased frequency of PML in relation to HIV-infection, it is likely that our knowledge of the pathogenetic aspects will increase, which, hopefully, may lead to an effective therapeutic strategy. A review of this disease, based upon studies of the literature, is presented.

Prediction of body composition in premenopausal females from dual-energy X-ray absorptiometry.

Four methods for predicting body composition were compared in premenopausal females (n = 100), 28-39 yr old, by using underwater weighing (UWW) as the criterion method. The four methods were dual energy X-ray absorptiometry (DEXA), skinfolds, bioelectrical impedance, and body mass index. The sample had a mean percent fat (%fat) of 29.7 +/- 6.8% (SD) by DEXA and 29.9 +/- 5.8% measured by UWW. DEXA yielded a standard error of estimate (SE) of 2.4% (r = 0.91) for the prediction of %fat from UWW. When %fat was estimated from other methods, larger SEs were obtained: 3.0% for skin-folds, 3.3% for body mass index, and 2.9% for bioelectrical impedance (height2/resistance) plus weight. Individual body density values derived from UWW were corrected for bone mineral variation. DEXA predicted the corrected body density with a lower SE (0.0040 vs. 0.0053 g/ml) than the original density values. We conclude that DEXA was a precise method and correlated highly with fat-free body weight and %fat from UWW in this homogeneous female sample.

[Neurological complications of Mycoplasma pneumoniae infections]. / Neurologiske komplikationer ved Mycoplasma pneumoniae infektioner.

Mycoplasma pneumoniae (Mp) infections are well known for the classical clinical picture of primary atypical pneumonia. The infection shows a predilection for young age groups. Every fourth-fifth year Mp epidemics are seen, lasting several months particularly in autumn/wintertime. The last Mp epidemic in Denmark was seen autumn/winter 1991-1992. The central nervous system (CNS) is involved in less than 0.1% of all Mp infections, but among patients treated in hospital, CNS involvement occurs in up to 7%. Among patients with acute, febrile, nonbacterial CNS affection the incidence of Mp infections is shown to be 5%, with a maximum of 10% during Mp epidemics. In up to 20% the CNS complications are seen without preceding pulmonary symptoms. The pathogenesis is unknown, but probably involves several mechanisms. The spectrum of clinical findings is wide, ranging from mild meningeal signs to severe neurological symptoms and a poor outcome. Mp encephalitis has a particularly high morbidity and mortality. The effect of antibiotic treatment is doubtful, but the treatment is often instituted late. It may be debated, whether early antibiotic treatment can reduce the frequency of the CNS complications and their sequelae. Mp infection should be remembered as a differential diagnosis in any patient with fever and neurological symptoms. It can be recommended to add Mp diagnostic measures to the screening investigations, especially in patients with recent respiratory symptoms and during Mp epidemics. It is important to attempt to detect Mp by culture or polymerase chain reaction (PCR) from throat, respiratory tract and cerebrospinal fluid (CSF). Mp serology from blood and CSF should be performed early in cases where Mp infection is suspected.

Neurocysticercosis: a short review and presentation of a Scandinavian case.

Cysticercosis is a disease entity caused by the larval form (Cysticercus cellulosae) of the pork tapeworm (Taenia solium). When man becomes the intermediate host, cysticercal cysts can develop in various organs. Neurocysticercosis, i.e. cysticercosis of the central nervous system, can lead to a broad range of neurological disturbances. The disease is usually confined to geographical regions where sanitation is poor but can occur among immigrants or travellers from such regions. Due to increased travel and immigration the disease may appear in non-endemic areas. We describe a recent case of neurocysticercosis in a 28-year-old Danish woman, who had been travelling in the Far East. She was successfully treated with praziquantel. A short review of the literature is given as the knowledge of the diagnosis and treatment of the disease has increased greatly in the last decade.

[Cerebral cysticercosis]. / Cerebral cysticerkose.

A Danish woman aged 28 years who had travelled in the Far East developed cerebral symptoms with headache and visual disturbances. Migraine was suspected. Subsequent CT scanning revealed multiple processes and metastases were suspected. As the patient had travelled in the Far East 1 1/2 years previously, she was examined for neurocysticercosis. This diagnosis was established and the patient was successfully treated with praziquantel. On account of increased travelling activity, the possibility of neurocysticercosis should be borne in mind when dealing with patients with cerebral symptoms and relevant travelling histories.