Novel and recurrent TRPV4 mutations and their association with distinct phenotypes within the TRPV4 dysplasia family.

BACKGROUND: Mutations in TRPV4, a gene that encodes a Ca(2+) permeable non-selective cation channel, have recently been found in a spectrum of skeletal dysplasias that includes brachyolmia, spondylometaphyseal dysplasia, Kozlowski type (SMDK) and metatropic dysplasia (MD). Only a total of seven missense mutations were detected, however. The full spectrum of TRPV4 mutations and their phenotypes remained unclear. OBJECTIVES AND METHODS: To examine TRPV4 mutation spectrum and phenotype-genotype association, we searched for TRPV4 mutations by PCR-direct sequencing from genomic DNA in 22 MD and 20 SMDK probands. RESULTS: TRPV4 mutations were found in all but one MD subject. In total, 19 different heterozygous mutations were identified in 41 subjects; two were recurrent and 17 were novel. In MD, a recurrent P799L mutation was identified in nine subjects, as well as 10 novel mutations including F471del, the first deletion mutation of TRPV4. In SMDK, a recurrent R594H mutation was identified in 12 subjects and seven novel mutations. An association between the position of mutations and the disease phenotype was also observed. Thus, P799 in exon 15 is a hot codon for MD mutations, as four different amino acid substitutions have been observed at this codon; while R594 in exon 11 is a hotspot for SMDK mutations. CONCLUSION: The TRPV4 mutation spectrum in MD and SMDK, which showed genotype-phenotype correlation and potential functional significance of mutations that are non-randomly distributed over the gene, was presented in this study. The results would help diagnostic laboratories establish efficient screening strategies for genetic diagnosis of the TRPV4 dysplasia family diseases.

A novel recombinant bispecific single-chain antibody, bscWue-1 x CD3, induces T-cell-mediated cytotoxicity towards human multiple myeloma cells.

The development of antibody-based strategies for the treatment of multiple myeloma (MM) has been hampered so far by the fact that suitable plasma cell-specific surface antigens have been missing. However, recently a novel monoclonal antibody, designated Wue-1, has been generated that specifically recognizes normal and malignant human plasma cells. Therefore, Wue-1 is an interesting and promising candidate to develop novel immunotherapeutic strategies for the treatment of MM. One variant for an antibody-based strategy is the bispecific antibody approach. Recombinant bispecific single-chain (bsc) antibodies are especially interesting candidates because they show exceptional biological properties. We have generated a novel MM-directed recombinant bsc antibody, bscWue-1 x CD3, and analyzed the biological properties of this antibody using the MM cell line NCI-H929 and primary cells from the bone marrow of patients with MM. We were able to show that bscWue-1 x CD3 induces efficient and selective T-cell-mediated cell death of NCI-H929 cells and primary myeloma cells in nine out of 11 cases. The bscWue-1 x CD3 Ab is efficacious even at low E:T ratios, and with or without additional T-cell pre- or costimulation. Target cell lyses were specific for Wue-1 antigen-positive cells and could be blocked by the Wue-1 monoclonal antibody.

Protection against hydrogen peroxide cytotoxicity in rat-1 fibroblasts provided by the oncoprotein Bcl-2: maintenance of calcium homoeostasis is secondary to the effect of Bcl-2 on cellular glutathione.

The oncoprotein Bcl-2 protects cells against apoptosis, but the exact molecular mechanism that underlies this function has not yet been identified. Studying H2O2-induced cell injury in Rat-1 fibroblast cells, we observed that Bcl-2 had a protective effect against the increase in cytosolic calcium concentration and subsequent cell death. Furthermore, overexpression of Bcl-2 resulted in an alteration of cellular glutathione status: the total amount of cellular glutathione was increased by about 60% and the redox potential of the cellular glutathione pool was maintained in a more reduced state during H2O2 exposure compared with non-Bcl-2-expressing controls. In our cytotoxicity model, disruption of cellular glutathione homoeostasis closely correlated with the pathological elevation of cytosolic calcium concentration. Stabilization of the glutathione pool by Bcl-2, N-acetylcysteine or glucose delayed the cytosolic calcium increase and subsequent cell death, whereas depletion of glutathione by dl-buthionine-(S, R)-sulphoximine, sensitized Bcl-2-transfected cells towards cytosolic calcium increase and cell death. We therefore suggest that the protection exerted by Bcl-2 against H2O2-induced cytosolic calcium elevation and subsequent cell death is secondary to its effect on the cellular glutathione metabolism.

[Examination of antigen preparations from the virus of enzootic bovine leukosis with regard to suitability for immunoblotting]. / Prüfung von Antigenpräparationen des Virus der enzootischen Rinderleukose auf Eignung im Immunblot.

8 different leukosis antigen preparations for immunodiffusion from 6 European countries were examined regarding their suitability for immunoblotting. Emphasis was made on demonstration of immunoreactions at 51 kDa beside reactions at 24 kDa. The whole protein content of the different preparations varied very much. gp 51 could be detected only by monoclonal antibodies at a molecular weight of 53 kDa, in one preparation at 26 kDa. With sera from field or with the European reference serum E4 none or only weak reactions could be observed at these molecular weights. All antigen preparations contained bovine IgG that is divided in L- and H-chains by SDS-electrophoresis under reducing conditions. These single chains reacted also with anti-bovine-IgG-biotin conjugate und disturbed the detection of the leukosis specific reactions. The bovine IgG could be removed by a Protein-G-column. One of all tested leukosis antigens, which are prepared for immunodiffusion, was suitable for immunoblotting. This antigen preparation contained only few bovine IgG and had strong reactions at 24 kDa. A special antigen preparation for detection of other reliable immunoreactions in the immunoblot has to be developed.

Cerebral hemodynamics and cerebral metabolism during cold and warm stress.

The purpose of this study was to examine if local thermo-applications affect central nervous reactions. In a crossover study, six normal, healthy volunteers at first received cold packs (Cryogel, 8-12 degrees C; Pino GmbH, Hamburg, Germany) and afterwards hot packs (Parafango, 50-60 degrees C; Pino GmbH), and another six volunteers started with the hot packs and had the cold packs later; both groups administered the hot and cold packs to their thighs. Before, during, and after treatment, cerebral blood flow velocity (CBFV) in the middle cerebri-artery (MCA) was measured continuously by transcranial Doppler sonography, whereas cerebral respiratory chain enzyme cytochrome aa3 (cCytaa3) and cerebral oxygen saturation (cHbO2) were measured by transcranial near infrared spectroscopy in frontal brain tissue. Furthermore, CO2 end-tidal and arterial blood pressure (noninvasive) were also measured. Six other volunteers received only one treatment; therefore, 15 measurements with cold and 15 measurements with hot packs were performed. During application of cold packs, a decrease of cHbO2 of 10.5% (P < 0.001) and cCytaa3 of 6.7% (P < 0.001) was found, whereas the CBFV(MCA) increased significantly (3.9%; P < 0.001) between preliminary and post-stimulus periods. When cold packs were removed, a significant increase of the cHbO2 (16.9%; P < 0.001) and cCytaa3 (9.7%; P < 0.001) was measured. With these values, cHbO2 and cCytaa3 showed an overshooting counterreaction beyond the initial level. When applying the hot packs, a contrary course of the parameters was found. cCytaa3 showed a significant increase of 9.3% (P < 0.001) at the end of the stimulus phase and a decrease of 1.9% (P = 0.02) during the post-stimulus period. The correlating increase of cHbO2 was significant at 13.7% (P < 0.005). At the end of the post-stimulus phase, a significant decrease of cHbO2 at 1.9% (P = 0.004) was recorded. With Parafango applications, a significant decrease of CBFV(MCA) at 6.9% (P < 0.001) was measured at the end of the stimulus in comparison with the preliminary phase. Crossover analysis showed no significant period effects and intraindividual changes between period and treatment. Therefore, both treatments can be compared within the individual using paired t test. Local cold and warm stimuli influence the cerebral hemodynamics and cerebral metabolism. Cerebral hemodynamics (CBFV(MCA) in comparison with cerebral metabolism (cCytaa3, cHbO2) show opposite reactions under thermo-stress. Of special interest is the overshooting counter-regulation of cerebral metabolism after cold stimulation. These effects may open new thermotherapeutic aspects in central nervous system diseases.

[Electrophoretic analysis on the proteolytic decomposition of of FSH preparations]. / Elektrophoretische Untersuchungen zum proteolytischen Abbau von FSH-Präparaten.

We investigated the influence of some proteolytic enzymes being present in the digestive canal (pepsin, trypsin, chymotrypsin) on the digestion of porcine FSH-samples by electrophoresis. Trypsin and Chymotrypsin digested the 18-kDa-protein much faster than pepsin, whereas a 67-kDa-protein was much faster digested by Pepsin than by Trypsin or Chymotrypsin. The overall proteolytic effect of Chymotrypsin is small compared with Pepsin and Trypsin. A subsequent digestion with Pepsin, Trypsin and Chymotrypsin led to a complete proteolysis of all proteins being present in the sample.

[Detection of antibodies against the virus of enzootic bovine leukosis in serum and milk samples using an immunoblot]. / Nachweis von Antikörpern gegen das Virus der enzootischen bovinen Leukose aus Serum-und Milchproben mit Hilfe eines Immunblots.

A method for detection of antibodies against the enzootic bovine leukemia virus in single serum and milk samples, as well as in pooled milk samples, is described. After the electrophoretical separation of the antigen proteins with SDS-PAGE, a transfer onto an immobilizing membrane is done, followed by immunodetection based on the reaction of the leukemia virus antibodies with antigen proteins. The antibody-antigen complex is visualized by a second, biotinylated antibody and peroxidase-avidin. This system is developed for verification of ELISA-results in surveillance of official admitted herds free of leukosis. The threshold for positive samples is tested by the European reference serum E-4, Copenhagen, the dilution 1:25,000 is recognized as positive. The detected antigen-antibody reactions are determined by scanning and comparison with the MW-markers.

The impact of gelatin-resorcinol glue on aortic tissue: a histomorphologic evaluation.

PURPOSE: Although gelatin-resorcinol-formaldehyde glue has been used to treat acute aortic dissections for some time, concerns about formaldehyde's mutagenicity and carcinogenicity made it imperative to develop a new glue compound. Gelatin-dialdehyde glue was produced by omitting the formaldehyde component and replacing it with two less toxic aldehydes, glutaraldehyde and glyoxal. This study evaluated the histomorphologic effects of the new glue through in vivo use on the aortic tissue of domestic pigs. METHODS: Each animal's infrarenal aorta was glued around an implanted prosthesis. Histomorphologic evaluation was performed after operation after 1 and 4 weeks. RESULTS: The results demonstrated that the clinically observed tanning effect can be attributed primarily to the disintegration of the fiber texture, specifically collagenous, as well as smooth muscle fibers, and to the reciprocal alterations of the proteoglycan interstitial substance in the aortic wall. Macroscopic, microscopic, and electron microscopic analysis of the gluing process revealed an adequate healing process without any morphologically significant difference between formaldehyde and formaldehyde-free gelatin-resorcinol glue. CONCLUSIONS: Gelatin-dialdehyde glue is able to produce the same effects in the area of the aortic wall as the substantially more toxic gelatin-resorcinol-formaldehyde glue and thus could be recommended for clinical trials for treating acute aortic dissections thus far yielding excellent initial results.

Quantitation of electrophoretically separated proteins in the submicrogram range by dye elution.

A new method for the fast, reliable and reproducible submicrogram quantitation of proteins separated by different electrophoretic techniques is presented. The method is based on a modified sensitive staining technique using Coomassie Brilliant Blue G-250 in colloidal solution combined with an optimized elution procedure of the bound dye in a 3% w/v solution of sodium dodecyl sulfate followed by photometric determination of dye concentration in the eluate. In addition a new method is provided for background correction, even suitable for gels showing strong background staining. The staining procedure allows the detection of 20 ng depending on the nature of the protein and the separation technique used. Quantitation is linear at least in the range from 50 ng to 10 micrograms and highly reproducible even under non-optimized conditions. The presented method can be applied to sodium dodecyl sulfate-electrophoresis, isoelectric focusing and two-dimensional electrophoresis.

Formaldehyde-free collagen glue in experimental lung gluing.

Because of the well-known limitations of the adhesive strength of fibrin glue, it is imperative to develop a stronger glue with acceptable biocompatibility. This was accomplished by removing the formaldehyde component from gelatin-resorcinol-formaldehyde glue and replacing it by two less toxic aldehydes--pentanedial and ethanedial. To evaluate the adhesive strength of this new glue, GR-DIAL, lung incisions in rabbit hybrids were glued together. Each group (n = 5) was examined histologically after 2 days and 1, 2, and 4 weeks. The glue disintegrated gradually with good bioresorption when the incision was closed with a thin layer of glue. The healing process was favorable, indicating good biocompatibility. Therefore, GR-DIAL glue is capable of enhancing the use of surgical glues in the field of thoracic surgery by enabling surgeons to close larger parenchymal lesions than with fibrin glue.

Studies on the mutagenicity of a peptoplast adhesive in Salmonella typhimurium.

A new class of adhesives--peptoplasts--was tested for their mutagenic potential in Salmonella typhimurium. The tested peptoplasts revealed no mutagenic properties under the present test conditions.

A new histological embedding method by low-temperature polymerisation of methyl methacrylate allowing immuno- and enzyme histochemical studies on semi-thin sections of undecalcified bone marrow biopsies.

A new procedure of embedding in methyl methacrylate (MMA) is introduced, which enables immunostaining by preservation of cellular epitopes. This could be achieved by reduction of polymerisation temperature from ca. 60 degrees C to 22 degrees C within the core of tissue blocks. Reduction of the polymerisation temperature is due to destabilisation of acrylate monomer, reduction of catalyst, exclusion of molecular oxygen, chemical initiation and reduction of environmental temperature. This results in good preservation of antigens and enzymes in the haematopoietic and lymphatic tissue of bone marrow as well as lymphoid, epithelial and mesenchymal markers in other tissues, comparable to paraffin embedding. Results are demonstrated by application of monoclonal and polyclonal antibodies and by demonstration of enzyme activity conventionally used in haematology.

Quantitation of proteins separated in N, N'-1,2-dihydroxyethylenebisacrylamide-crosslinked polyacrylamide gels.

A simple and rapid method for the quantitation of proteins separated either by sodium dodecyl sulfate-electrophoresis or by isoelectric focusing in slab gels is presented. The method is based on the solubility of polyacrylamide gels crosslinked with N, N'-1, 2-dihydroxyethylenebisacrylamide (DHEBA) in periodic acid. After electrophoretic separation proteins are stained with Coomassie brilliant blue G-250. DHEBA gels show considerable swelling during the staining and destaining process but can be shrunk to their normal size in a 10% (w/v) solution of ammonium sulfate. Stained bands are cut from the gel and solubilized in periodic acid. During dissolution the dye decolorizes. Protein concentration in the solution is determined by a modified Coomassie dye-binding assay. Quantitation is linear in the range of 100 ng to 5 micrograms and not disturbed by dissolved gel. Separations in N, N'-1, 2-dihydroxyethylenebisacrylamide-crosslinked gels show qualities similar to those in normal crosslinked gels.

Preclinical safety testing of plastic products intended for use in man.

Plastic material is used for medical devices including biomaterial for numerous applications in man. These applications range from disposable plastic syringes to hip prostheses. In order to assume safety and to comply with legal requirements, preclinical tests are necessary. The plastic material employed should in most cases be tested or evaluated at three levels: (1) toxicity tests with the various ingredients used to manufacture the basic plastic, (2) toxicity tests with the final plastic and (3) toxicity tests with the final device. However, individual testing protocols are required due to the different toxicological profiles of the plastics and the heterogenicity of their application-sites in man. This paper evaluates the studies which may be needed to fulfill these requirements.

Changes of the surface proteolytic activity in synchronized Ehrlich ascites tumor cells grown in vivo.

Changes of the cell surface proteolytic activity during the cell cycle in vitro were reported. Using an easy assay, with casein as a substrate, the proteolytic activity on the surface of Ehrlich ascites tumor (EAT) cells grown in vivo was determined. The cleavage of casein incubated with EAT cells increased linearly for 20 min and permitted reproducible enzyme activity determinations. If the proliferation of exponentially growing EAT cells was partially synchronized by an intraperitoneal bleomycin injection, a significant increase of the surface enzymatic activity was observed in cells with an increased DNA content. This finding supports the results obtained with transformed cells in vitro, indicating that elevated proteolytic surface activity occurs in the late synthesis phase and prior to mitosis. However, the observed effect may also be due to changes of gene expression caused by bleomycin.

Experimental glueing of injured spleen by collagen glue.

This paper discusses the suitability of collagen adhesive for the treatment of a ruptured spleen, and presents a preliminary study on 20 Sprague Dawley rats. Results indicate that this system should be further investigated for clinical applications.

Toxicological aspects of synthetic tissue adhesives.

Generally, in order to be able to conduct clinical trials or to obtain a product licence for adhesives, toxicological studies have to be conducted in such a way as to provide sufficient data to be able to assess the toxicity, i.e. the risk. This paper discusses the types of studies which may be needed to fulfill these requirements.

Growth acceleration of Ehrlich ascites tumor cells treated by proteinase in vitro.

Since the cytotoxic effect of ionizing radiation increases in faster proliferating cell populations, the effects of a mild trypsin and bromelain treatment on the growth rate of Ehrlich ascites tumor (EAT) cells cultured in vitro were studied. A continuous exposure to proteinase started at the time of cell plating caused a temporary block of DNA synthesis that was followed by an accelerated growth rate 48 h later (approximately 1.5-fold). EAT cells exposed to bromelain and trypsin after completed adaptation to the substratum demonstrated a similar increase of growth 2 days after the beginning of the enzyme treatment. The acceleration of growth was also observed when exposure to proteinase was interrupted after 24 h but the stimulation effect was reversible and continued only 2 days. It is concluded that bromelain and trypsin are able to modify reversibly the growth rate of EAT cell population cultured in vitro.

Comparison of hydroxypropyl methylcellulose 2% (Adatocel) and hyaluronic acid 1% (Healon).

Hydroxypropyl methylcellulose solutions (2% HPMC) were examined for impurities. There was no evidence of any particulate matter in two solutions from different sources: one prepared by a hospital pharmacy, the other industrially prepared (Adatocel). In a controlled clinical study, Adatocel was compared with Healon (1% hyaluronic acid solution). The postoperative irritation (cellular response) in the anterior chamber was the same in both groups.

[2-Chloro-1-methylpyridinium iodide. A suitable reagent for peptide synthesis]. / 2-Chlor-1-methylpyridiniumjodid. Ein geeignetes Kupplungsreagenz für Peptidsynthesen.

2-Chloro-1-methylpyridinium iodide is a coupling reagent for peptide synthesis. By its use different peptides were synthesized with protected di- and trifunctional amino acids. While urethan-protected amino acids react free of racemization, the fragment condensation needs N-Hydroxysuccinimide, as shown by the Young test.