Genetic counselling for hypertrophic cardiomyopathy: are we ready for it?

Hypertrophic cardiomyopathy (HCM) is a dominant genetic disorder of the myocardium associated with dysfunctional contractile proteins. The major risk of HCM is sudden cardiac death, which may occur even in asymptomatic carriers. Causes are highly heterogeneous. Over 140 different mutations in nine sarcomeric genes have been described to date. The majority of cases (80% or more) may eventually be traced to one of these genes. Although genetic counselling is suggested even if mutations are not known, molecular diagnosis implies new options such as carrier identification or - theoretically - preclinical risk stratification. A scheme according to which cardiologists and clinical and molecular geneticists could cooperate in counselling patients and managing HCM clinically is proposed.     

The polymerase chain reaction: an improved method for the analysis of nucleic acids

Summary The polymerase chain reaction (PCR) is a method for the selective amplification of DNA or RNA segments of up to 2 kilobasepairs (kb) or more in length. Synthetic oligonucleotides flanking sequences of interest are used in repeated cycles of enzymatic primer extension in opposite and overlapping directions. The essential steps in each cycle are thermal denaturation of double-stranded target molecules, primer annealing to both strands and enzymatic synthesis of DNA. The use of the heat-stable DNA polymerase from the archebacterium Thermus aquaticus (Taq polymerase) makes the reaction amenable to automation. Since both strands of a given DNA segment are used as templates, the number of target sequences increases exponentially. The reaction is simple, fast and extremely sensitive. The DNA or RNA content of a single cell is sufficient to detect a specific sequence. This method greatly facilitates the diagnosis of mutations or sequence polymorhisms of various types in human genetics, and the detection of pathogenic components and conditions in the context of clinical research and diagnostics; it is also useful in simplifying complex analytical or synthetic protocols in basic molecular biology. This article describes the principles of the reaction and discusses the applications in different areas of biomedical research.     

Carbonic anhydrase subunits of the mitochondrial NADH dehydrogenase complex (complex I) in plants

The mitochondrial nicotinamide adenine dinucleotide, reduced (NADH) dehydrogenase complex (complex I) of plants has a molecular mass of about 1000 kDa and is composed of more than 40 distinct protein subunits. About three quarter of these subunits are homologous to complex I subunits of heterotrophic eukaryotes, whereas the remaining subunits are unique to plants. Among them are three to five structurally related proteins that resemble an archaebacterial γ-type carbonic anhydrase (γCA). The γCA subunits are attached to the membrane arm of complex I on the matrix-exposed side and form an extra spherical domain. At the same time, they span the inner mitochondrial membrane and are essential for assembly of the protein complex. Expression of the genes encoding γCA subunits is reduced if plants are cultivated in the presence of elevated CO₂ concentration. The functional role of these subunits within plant mitochondria is currently unknown but might be related to photorespiration. We propose that the complex I–integrated γCAs are involved in mitochondrial HCO₃⁻ formation to allow efficient recycling of inorganic carbon for CO₂ fixation in chloroplasts under high light conditions.     

Effects of secondary interactions on the kinetics of peptide and peptide ester hydrolysis by tissue kallikrein and trypsin

Kinetic constants for the hydrolysis by porcine tissue beta-kallikrein B and by bovine trypsin of a number of peptides related to the sequence of kininogen (also one containing a P2 glycine residue instead of phenylalanine) and of a series of corresponding arginyl peptide esters with various apolar P2 residues have been determined under strictly comparative conditions. kcat and kcat/Km values for the hydrolysis of the Arg-Ser bonds of the peptides by trypsin are conspicuously high. kcat for the best of the peptide substrates, Ac-Phe-Arg-Ser-Val-NH2, even reaches kcat for the corresponding methyl ester, indicating rate-limiting deacylation also in the hydrolysis of a peptide bond by this enzyme. kcat/Km for the hydrolysis of the peptide esters with different nonpolar L-amino acids in P2 is remarkably constant (range 1.7), as it is for the pair of the above pentapeptides with P2 glycine or phenylalanine. kcat for the ester substrates varies fivefold, however, being greatest for the P2 glycine compounds. Obviously, an increased potential of a P2 residue for interactions with the enzyme lowers the rate of deacylation. In contrast to results obtained with chymotrypsin and pancreatic elastase, trypsin is well able to tolerate a P3 proline residue. In the hydrolysis of peptide esters, tissue kallikrein is definitely superior to trypsin. Conversely, peptide bonds are hydrolyzed less efficiently by tissue kallikrein and the acylation reaction is rate-limiting. The influence of the length of peptide substrates is similar in both enzymes and indicates an extension of the substrate recognition site from subsite S3 to at least S'3 of tissue kallikrein and the importance of a hydrogen bond between the P3 carbonyl group and Gly-216 of the enzymes. Tissue kallikrein also tolerates a P3 proline residue well. In sharp contrast to the behaviour of trypsin is the very strong influence of the P2 residue in tissue-kallikrein-catalyzed reactions. kcat/Km varies 75-fold in the series of the dipeptide esters with nonpolar L-amino acid residues in P2, a P2 glycine residue furnishing the worst and phenylalanine the best substrate, whereas this exchange in the pentapeptides changes kcat/Km as much as 730-fold. This behaviour, together with the high value of kcat/Km for Ac-Phe-Arg-OMe of 3.75 X 10(7) M-1 s-1, suggests rate-limiting binding (k1) in the hydrolysis of the best ester substrates.(ABSTRACT TRUNCATED AT 400 WORDS)     

A new facultatively nitrite oxidizing bacterium,Nitrobacter vulgaris sp. nov.

A total of 17 facultatively lithoautotrophic strains of Nitrobacter were investigated. They all were found to be related on the species level by DNA hybridizations. The G+C content of DNA ranged between 58.9 and 59.9 mol %. The isolates originated from divers environments. The cells were 0.5–0.8×1.2–2.0 m in size and motile by one polar to subpolar flagellum. Cell-division normally occurred by budding. Polar caps of intracytoplasmic membranes as well as carboxysomes were present. The cells tended to excrete extracellular polymers forming aggregates or biofilms. Heterotrophic growth was slower than mixotrophic but often faster than litoautotrophic growth. In the presence of nitrite and organic substances the organisms often showed diphasic growth. First nitrite and then the organic material was oxidized. In the absence of oxygen growth was possible by dissimilatory nitrate reduction. Nitrite, nitric and nitrous oxide as well as ammonia were formed. Depending on growth conditions the generation times varied from 12 to 140 h. The new Nitrobacter spec. may be one of the most abundant nitrite-oxidizing bacteria in soils, fresh waters and natural as well as artificial stones. For this organism the name Nitrobacter vulgaris is proposed.The type strain is filed with the culture collection of the Institut für Allgemeine Botanik, Universität Hamburg, FRG.     

Glycosaminoglycan accumulation by normal and malignant human mammary epithelial cells in primary culture

The effects of glycosaminoglycans (GAGs) on cell growth and differentiation appear to vary with cell type and stage of development. This study describes the types and distribution of GAGs accumulated by normal and malignant human mammary epithelial cells in primary culture during their exponential and stationary phases of growth. Cultures incubated with [3H]glucosamine or [35S]sulfate were separated into medium, extracellular matrix (ECM), and cell fractions. Labelled GAGs were identified by chemical and enzymatic degradations and cellulose acetate electrophoresis. Cultures of normal cells in the exponential phase of growth released the most labelled GAGs into the medium fraction, the majority of which was hyaluronic acid (HA). The increase in labelled GAG accumulation, the increase in sulfated GAGs localized in the ECM fraction correlated with the reduced proliferative activity and increased cell density of cells in stationary cultures. In contrast, cultures of mammary tumour cells had the same labelled GAG profile, regardless of their growth status. Although there was variation among tumours, in general, the majority of the labelled GAGs were secreted into the medium fraction and the predominant GAG was HA. The results are comparable with those obtained from studies on mammary tissue in vivo. Primary cultures of human mammary epithelial cells should be useful for determining how modulations of GAGs affect growth and differentiation of these cells.     

Modulation of differentiation markers in human choriocarcinoma cells by extracellular matrix: on the role of a three-dimensional matrix structure

Abstract. During spontaneous or chemically induced differentiation human choriocarcinoma cells express typical characteristics of the normal differentiating trophoblast: 1) increased production of peptide and steroid hormones (chorionic gonadotropin, placental lactogen, estrogens, progesterone); 2) increased activity of cellular alkaline phosphatase; 3) morphological transition from cytotrophoblast to syncytiotrophoblast-like cells; and 4) arrested cell proliferation. Since the extracellular matrix is known to control gene expression we have examined the effects of different substrates composed of matrix macromolecules on the differentiation of BeWo choriocarcinoma cells. Matrices tested were; fibronectin, laminin, collagens type I and type IV, the basement membrane-like complex matrix Matrigel, and a complex matrix extracted from human term placenta. Irrespective of the type of molecule(s), it was consistently found that, whenever the matrix molecules were presented as threedimensional structures (as opposed to protein coatings on tissue culture plastic) the response of affected differentiation markers monitored was highly pronounced. Morphology was changed from monolayers to rounded colonies, cell proliferation was reduced, and the secretion of chorionic gonadotropin was increased up to tenfold. Heterogeneous effects were observed on progesterone secretion and on the activity of cellular alkaline phosphatase. Cell adhesion to matrix molecules, however, did not depend on the structure of the matrix. This study demonstrates that gene expression in these tumor cells can be modified by extracellular matrix and highlights that not only the presence of effector molecules in the matrix but also the three-dimensional structure of the matrix is important for the induction of differentiation.