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.

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.

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.

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.

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.

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.

[Adhesives in surgery of the skeleton system, part 2: the adhesive strength from 22 adhesives in bone surgery (author's transl)]. / Klebungen am Skelettsystem, Teil 2: Untersuchung der Klebfestigkeit von 22 Klebstoffen am Knochen.

For the first time all relevant adhesive groups were analyzed for their adhesive strength in bone-surgery. After having glued bone pieces in a special designed system, the tensil- and shearing-strength was determined. All adhesives showed a loss of adhesive strength under in vivo-conditions. Moderate moisture increased the tensile strength in some adhesives. While the fibrin adhesive had only a low adhesive strength under simulated in vivo-conditions, the adhesives containing gelatine, epoxy resin or acrylates reached reasonable results. The relationship between chemical structure and adhesive strength will be further used for the development of a special bone adhesive as a new surgeon's tool.

[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.

[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.

[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.

[A bioassay for the testing of the oxytocic effect of hormone analogs of the neurohypophysis and the pineal body]. / Ein Bioassay zur Austestung der oxytocischen Wirksamkeit von Hormonanaloga der Neurohypophyse und Zirbeldrüse.

An improved bioassay for the testing of oxytocic compounds by the use of myometrium layer preparations has been developed and proved with synthetic oxytocin. The oxytocic activity of synthetic analogues was testes with this bioassay and compared to oxytocin. The myometrial layer preparation was demonstrated to be reliable, the vitality of the tissue is found for more than 8 hours. The sensitivity of the bioassay is found in the region of 10(-11) Mol/l oxytocin, the maximum of the stimulation of muscle strips varied from 5 X 10(-5) IU/ml to 10(-4) IU/ml oxytocin in the organ-bath. The concentration of the myometrium strips showed two different phases. Only the initial phase can be used for this bioassay. The calculation of the results was afforded planimetrically and by measuring of the concentration-maximum during the testing period. The synthetic hormone analogues [1-desamino-4-alpha-aminobutyric acid] lysine-vasotocin (2) and [4 alpha-aminobutyric acid] lysine-vasotocin had oxytocic activity and the myometrium was activated to contractions at 10(-8) Mol/1. The analogue 2 was 5-10 more active than 3. The contraction maximum for both analogues was found between 5 X 10(-6) and 5 X 10(-5) Mol/l. The induction of the concentration for both analogues varied with the factor 10(5).

[5-Isothiocyanato-1,8-naphthalenedicarbox-4-methylphenylimide, a new fluorescence reagent for compounds containing amino groups (author's transl)]. / 5-Isothiocyanato-1,8-naphthalindicarbonsäure-4-methylphenylimid, ein neues Fluoreszenzreagenz für Aminogruppen-haltige Stoffe

5-Isothiocyanato-1,3-dioxo-2-p-tolyl-2,3-dihydro-1H-benz[de]isoquinoline (= 5-isothio-cyanato-1,8-naphthalenedicarbox-4-methylphenylimide, compd.5) was synthesized from 1H,3H-naphtho(1,8-cd)pyran-1,3-dione (= 1,8-naphtha-lenedicarboxylic anhydride, compd.1) through nitration, condensation with p-toluidine, reduction with tin(II)-chloride yielding 5-amino-1,3-dioxo-2-p-tolyl-2,3-dihydro-1H-benz(de)isoquino-line (4) as intermediate, and condensation with thiophosgene. Compd. 5 can be used for qualitative and quantitative analyses of compounds containing amino groups.

[Sodium and potassium concentrations in whole blood and the serum of pregnant females and newborn infants]. / Natrium- und Kaliumkonzentrationen im Vollblut und Serum von Schwangeren und Neugeborenen.

The behaviour of sodium-potassium level in whole blood and serum of 464 mothers and their newborns was studied intra partum and immediately after delivery. There is a control group of 100 non pregnant women without hormonal contraception. During pregnancy there is a marked decrease of serum sodium concentration levels. In pathological pregnancies a small increase of serum-sodium-levels is observed in comparison to normal pregnancy. Multiparas and female neonates show increased concentrations of sodium in comparison with the control group. The serum-potassium levels of pregnant women are significantly increased compared with non pregnant women. The sodium-potassium concentration of newborns with Apgar 5-7 is significantly higher than that of children with an Apgar 8-10. The potassium level of all the newborns is increased compared with the potassium concentrations of their mothers. The sodium-level in whole blood of pregnant and non pregnant women is increased, whereas the potassium level is reduced in contrast to a low serum-sodium level and a high serum-potassium level.

[Pathobiochemistry of mineral and trace elements. III. Magnesium concentration in whole blood and serum of pregnant women and newborn (author's transl)]. / Pathobiochemie von Mineral- und Spurenelementen. III Magnesiumkonzentration im Vollblut und Serum von Schwangeren und Neugeborenen.

Magnesium is a constitutional or additive component in over 200 enzymes: its significance in physiological or pathophysiological metabolism is therefore considerable. For this reason this element was also included on our program of studies on cation kinetics in pregnant women and in neonates. The results of our analyses of 158 gravid women with normal and toxic pregnancies can be summarized as follows: 1. Compared with the corresponding values for non-pregnant women, the mother's magnesium during a normal pregnancy is reduced up to 9.2% in the serum and, likewise significantly reduced in whole blood up to 7% of normal values. The same conditions apply to the newborn as well. During a pathological pregnancy the magnesium in serum is reduced by 6.3% and in whole blood 6.6% as opposed to non-pregnant women. 2. Serum magnesium decline is equally great for primi- as well as multipara. 3. The blood group of the pregnant women is without influence on the magnesium concentration. 4. The rhesus factor has no influence on the course of the magnesium concentration during pregnancy. 5. The reduction of the magnesium values in whole blood is significantly greater for newborn males than for newborn females. 6. A concrete statement about the extent to which magnesium affects the Apgar values cannot be made on the basis of the data analysed in these studies due to the lack of symmetry between the groups to be compared: groups 5-7 to 8-10. Further comparative studies are required for this. 7. Magnesium changes in mother and child correlate with pregnancy toxicoses and other parameters.

[Pathobiochemistry of mineral and trace elements. IV Iron concentration in whole blood and serum of pregnant women and newborn (author's transl)]. / Pathobiochemie von Mineral- und Spurenelementen., IV. Eisenkonzentration im Vollblut und Serum von Schwangeren und Neugeborenen.

We determined the iron levels during the 10-40 week of pregnancy in 158 pregnant women to whom no iron supplements had been administered and who had normal pregnancies as well as toxic ones. We also determined the iron levels in 463 mothers during birth and those of their newborn. We obtained the following results. 1. In the course of pregnancy the iron level in the whole blood remains practically constant at the reduced level attained in the first few weeks until the 40th week, while a continuous depression of iron levels occurs in the serum. 2. Iron levels in whole blood are significantly elevated during a normal pregnancy in contrast to a pathological one, but the serum values stay practically identical in both groups. 3. The actual number of pregnancies (primi- or multipara) had no significant influence on the iron levels in either whole blood or serum in both normal or pathological pregnancies. 4. Blood group status had no influence on iron metabolism. 5. With a rhesus positive factor the whole blood level remains unchanged in the blood, but is significantly higher in the serum in comparison to a rhesus negative factor. 6. Comparison of apgar groups 5-7 with 8-10 showed significantly higher iron levels in mature neonates 8-10 only in the case of pathological pregnancy. 7. Sex of the newborn had no effect on the iron concentration in blood drawn from the umbilical cord. 8. Correlation between the iron values in whole blood and those in the serum are ascertainable in the different indications for toxic pregnancy. 9. Patients who had received an iron supplement therapy were excluded from the study.