Three novel human VMD2-like genes are members of the evolutionary highly conserved RFP-TM family

The RFP-TM protein family was first described in Caenorhabditis elegans as hypothetical transmembrane proteins containing a conserved 350-400 amino acid domain including the invariant peptide motif RFP. The VMD2 gene underlying Best disease was shown to represent the first human member of the RFP-TM protein family. More than 97% of the disease-causing mutations are located in the N-terminal RFP-TM domain implying important functional properties. Here, we have identified three novel VMD2-related human genes (VMD2L1, VMD2L2 and VMD2L3) demonstrating a high degree of conservation in their respective RFP-TM domains. Each of the VMD2-like proteins has a unique C-terminus that lack similarity to other proteins or motifs. By FISH analysis, VMD2L1 was localised to chromosome 19p13.2-p13.12, VMD2L2 to 1p32.3-p33 and VMD2L3 to 12q14.2-q15. RT-PCR analyses revealed tissue-restricted expression of the three genes with both VMD2L1 and VMD2L2 abundantly transcribed in colon. VMD2L1 is present in the retinal pigment epithelium while VMD2L3 shows predominant expression in skeletal muscle.     

Generalized AA-amyloidosis in a 58-year-old Caucasian woman with an 18-month history of gastrointestinal tuberculosis

 We report on a 58-year-old Caucasian woman who went to a general practitioner about recurrent abdominal pain, night sweats and weight loss of a few weeks’ duration. Once gynaecological disease had been ruled out, the patient was admitted to hospital with severe abdominal pain and intestinal obstruction and a right-sided hemicolectomy was performed. Following the investigation of osteolytic lumbar vertebrae, 18 months after visiting the general practitioner the patient was finally found to be suffering from generalized AA-amyloidosis secondary to gastrointestinal tuberculosis. This had been misinterpreted as Crohn’s disease. Re-examination of the specimens from the right-sided hemicolectomy demonstrated that scanty deposits of AA-amyloid were present 9 months after the first presentation. AA-amyloid can thus be present in serious inflammatory disease even during the first 9 months after the initial clinical presentation. Received: 23 June 1998 / Accepted: 19 August 1998     

Detection of a trigger zone of bradykinin-induced fast calcium waves in PC12 neurites

 Bradykinin and caffeine were used as two different agonists to study inositol 1,4,5-trisphosphate (IP₃)-sensitive and caffeine/ryanodine-sensitive intracellular Ca²⁺ release in the outgrowing neurites of nerve-growth-factor (NGF)-treated rat phaeochromocytoma cells (PC12). Changes in neuritic intracellular free Ca²⁺ ([Ca²⁺]_i) in single cells were measured after loading with a 1:1 mixture of the acetoxymethyl (AM) ester of the Ca²⁺-sensitive dyes Fura-red and Fluo-3, in combination with confocal microscopy. Bradykinin-induced Ca²⁺ release was blocked by U73211, a specific phospholipase C inhibitor. Caffeine-induced Ca²⁺ release was very low in neurites at rest. It increased after the cells were preloaded with Ca²⁺. The Ca²⁺ signal evoked at high concentrations of bradykinin (>500 nM) arose from a trigger zone in the proximal part of the neurite, as a bi-directional wave towards the growth cone and cell body. The speed of neuritic Ca²⁺ waves was reduced in cells loaded with the Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-tetraacetic acid/AM. Preloading of Ca²⁺ stores led to increased bradykinin-induced Ca²⁺ release, as seen for caffeine, and faster Ca²⁺ wave speeds. Caffeine evoked a simultaneous [Ca²⁺]_i rise along the neurites of Ca²⁺ preloaded cells. Higher Ca²⁺ signal amplitudes and faster Ca²⁺ wave speeds, but no longer-lasting IP₃-induced [Ca²⁺]_i signals, correlated with increased caffeine-induced Ca²⁺ release in the neurites. At low concentrations of bradykinin (<1.0 nM), the Ca²⁺ signals ceased to propagate as complete Ca²⁺ waves. Instead, repetitive stochastic Ca²⁺ release events (neuritic Ca²⁺ puffs) were observed. Neuritic Ca²⁺ puffs spread across only a few microns, at a slower speed than neuritic Ca²⁺ waves. These Ca²⁺ puffs represent elementary Ca²⁺ release units, whereby the released Ca²⁺ ions form these elementary events into the shape of a Ca²⁺ wave. Received: 16 April 1996 / Received after revision and accepted: 13 May 1996     

Divergent regulation of vascular endothelial growth factor and of erythropoietin gene expression in vivo

There is accumulating evidence from in vitro experiments that the gene expression of the vascular endothelial growth factor (VEGF) is, like that of the erythropoietin (EPO) gene, regulated by the oxygen tension and by divalent cations such as cobalt. Since the information about the regulation of VEGF gene expression in vivo is rather scarce, this study aimed to examine the influence of hypoxia and of cobalt on VEGF gene expression in different rat organs and to compare it with that on EPO gene expression. To this end male Sprague-Dawley rats were exposed to carbon monoxide (0.1% CO), hypoxia (8% O₂ ) or to cobalt chloride (12 and 60 mg/kg s.c.) for 6 h. mRNA levels for VEGF- 188, -164, and -120 amino acid isoforms in lungs, hearts, kidneys and livers were semiquantitated by RNase protection. For these organs we found a rank order of VEGF mRNA abundance of lung >> heart > kidney = liver. EPO mRNA levels were semiquantitated in kidneys and livers. Hypoxia, CO and cobalt increased EPO mRNA levels 60-fold, 140-fold and 5-fold, respectively, in the kidneys, and 11-fold, 11-fold and 3-fold, respectively, in the livers. None of these manoeuvres caused significant changes of VEGF mRNA in lung, heart or kidneys. Only in the livers did hypoxia lead to a significant (50%) increase of VEGF mRNA. These findings suggest that, in contrast to the in vitro situation, the expression of the VEGF gene in normal rat tissues is rather insensitive to hypoxia. In consequence, the in vivo regulation of the VEGF and the EPO genes appear to differ substantially, suggesting that the regulation of the VEGF and EPO genes may not follow the same essential mechanisms in vivo. Received: 31 July 1995/Received after revision: 20 November 1995/Accepted: 27 November 1995     

The effect of crosslinking in elastomers investigated by NMR analysis of 13C edited transverse 1H NMR relaxation

The transverse nuclear ¹H magnetization decay in poly(styrene-co-butadiene) (SBR) is investigated by editing ¹³C NMR spectra. This technique allows for the assignment of localized ¹H dynamical information by discriminating the chemical sites based on their chemical shift in the ¹³C dimension. Here, the homo- and heteronuclear dipolar couplings contribute to the ¹H NMR relaxation giving additional information to a homonuclear experiment. In this heteronuclear 2D experiment two prominent peaks are observed in the ¹³C dimension, which correspond to CH and CH₂ groups, respectively. The decay rate in the ¹H dimension is found for both groups to scale with the crosslink density. An additional ultra-fast magnetization decay is reported. The effect of the carbon black filler is investigated for this component. The analysis of the ¹³C NMR edited transverse ¹H magnetization relaxation is a useful tool in combining high resolution NMR spectra with information on molecular dynamics, providing insight into crosslink density and filler effects.     

Dominant negative mutations in the C-propeptide of COL2A1 cause platyspondylic lethal skeletal dysplasia, torrance type, and define a novel subfamily within the type 2 collagenopathies

Platyspondylic lethal skeletal dysplasia (PLSD) Torrance type (PLSD-T) is a rare skeletal dysplasia characterized by platyspondyly, brachydactyly, and metaphyseal changes. Generally a perinatally lethal disease, a few long-term survivors have been reported. Recently, mutations in the carboxy-propeptide of type II collagen have been identified in two patients with PLSD-T, indicating that PLSD-T is a type 2 collagen-associated disorder. We studied eight additional cases of PLSD-T and found that all had mutations in the C-propeptide domain of COL2A1. The mutational spectrum includes missense, stop codon and frameshift mutations. All non-sense mutations were located in the last exon, where they would escape non-sense-mediated RNA-decay. We conclude that PLSD-T is caused by mutations in the C-propeptide domain of COL2A1, which lead to biosynthesis of an altered collagen chain (as opposed to a null allele). Similar mutations have recently been found to be the cause of spondyloperipheral dysplasia, a non-lethal dominant disorder whose clinical and radiographical features overlap those of the rare long-term survivors with PLSD-T. Thus, spondyloperipheral dysplasia and PLSD-T constitute a novel subfamily within the type II collagenopathies, associated with specific mutations in the C-propeptide domain and characterized by distinctive radiological features including metaphyseal changes and brachydactyly that set them apart from other type 2 collagenopathies associated with mutations in the triple-helical domain of COL2A1. The specific phenotype of C-propeptide mutations could result from a combination of diminished collagen fibril formation, toxic effects through the accumulation of unfolded collagen chains inside the chondrocytes, and alteration of a putative signaling function of the carboxy-propeptide of type 2 collagen.     

Detection of perforin and granzyme A mRNA in infiltrating cells during infection of mice with lymphocytic choriomeningitis virus

The analysis of gene expression in cytotoxic T cells by in situ hybridization of serial liver and brain sections from mice infected with lymphocytic choriomeningitis virus (LCMV) and immunostaining with T cell marker- and virus-specific antibodies revealed a close histological association of infiltrating lymphocytes expressing the perforin and granzyme A genes with virally infected cells. Maximal frequency of perforin and granzyme A mRNA-containing cells on liver sections preceded by about 2 days maximal LCMV-specific cytotoxicity of the lymphoid liver infiltrating cells. These results are most consistent with an involvement of perforin and granzyme A in cell-mediated cytotoxicity in vivo.     

Anaphylaxis to wheat beer.

BACKGROUND: Despite its worldwide and abundant consumption, beer has rarely been found to cause anaphylaxis. Barley malt contained in lager beers seems to be an important elicitor. OBJECTIVE: To report the unusual case of severe anaphylaxis following the ingestion of wheat beer. METHODS: A 59-year-old man experienced angioedema, generalized urticaria, and unconsciousness after ingestion of wheat beer. He tolerated lager beer well. For diagnostic evaluation, skin prick tests, oral challenge tests, and identification of specific IgE antibodies were performed. RESULTS: Skin prick test results with standard series of common aeroallergens and food allergens were negative with the exception of a 1 + reaction to wheat flour. The results of skin prick tests with native materials were positive for 2 brands of wheat beer and wheat malt shred but negative for baker's yeast, hops, and a brand of lager beer. Oral challenges with wheat beer or wheat flour elicited urticaria. By CAP-FEIA, specific IgE antibodies to wheat and barley flour but not to hops or baker's yeast were found in serum. Immunoblot analysis revealed that patient's IgE was bound to a protein of approximately 35 kDa in wheat extract. CONCLUSIONS: This is the first report, to our knowledge, on anaphylaxis to beer attributable to wheat allergy.