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.     

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.     

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.     

Isolation of brush-border membranes from rat and rabbit colonocytes: Is alkaline phosphatase a marker enzyme?

Summary A method for the isolation of brush-border membranes of large intestinal epithelial cells was developed, which is based on the purification of intact brush-border caps by Percoll^® density-gradient centrifugation followed by separation of the vesiculated brush-border membranes on sucrose gradients. The procedure has two major advantages in comparison to known methods: 1) its first step does not depend on the determination of marker enzymes and 2) the method is applicable to rats as well as rabbits without major modifications. Due to the lack of an accepted marker for the colonic brush-border membrane the validity of the isolation procedure was tested by its application to the small intestine. Rat small intestinal brush-border membranes were enriched 21-fold when compared to the homogenate. The method was used to evaluate alkaline phosphatase as a marker enzyme for the colonic brush-border membrane. The results suggest that alkaline phosphatase is not exclusively localized in the brush-border membrane since this enzyme was also associated with membranes having different physical properties.     

Deoxyribonucleic acid homologies among 96 strains of ammonia-oxidizing bacteria

DNA of 96 strains of the genera Nitrosomonas, Nitrosococcus, Nitrosospira, Nitrosolobus, and Nitrosovibrio was isolated and analysed spectrophotometrically. Percentages of guanine plus cytosine (G+C) content, genome sizes, and DNA-DNA homologies were determined. The results indicated the presence of eight Nitrosomonas species, three or four Nitrosococcus species, five Nitrosospira species, and two species of both Nitrosolobus and Nitrosovibrio. DNA homologies between strains of a separate species ranged from 56–100%. Average homologies between strains of different species were 33% in Nitrosococcus, 36% in Nitrosomonas, 37% in Nitrosolobus, 40% in Nitrosospira, and 42% in Nitrosovibrio. Average homologies between species of different genera were 33% and thus not significantly above the background value of 30% detected between DNA of ammonia-oxidizing bacteria and Escherichia coli. Genome sizes ranged from 1.90–2.74×10⁹ dalton in Nitrosomonas, 2.09–2.37×10⁹ dalton in Nitrosococcus, 1.87–2.15×10⁹ dalton in Nitrosospira, 1.92–2.10×10⁹ dalton in Nitrosolobus, and 1.91–2.15×10⁹ dalton in Nitrosovibrio. Differences in genome sizes were in accordance with DNA homologies.     

Contitions for optimal growth of a PSTV-infected potato cell suspension and detection of viroid-complementary longer-than-unit-length RNA in these cells

A suspension culture from potato spindle tuber viroid (PSTV)-infected cells of the wild type potato (Solanum demissum) has been established, which is a suitable model system for studying PSTV replicationin vivo. The conditions for rapid growth of these cells and for permanent extensive viroid biosynthesis within them are described. Biosynthesis of PSTV in the potato cells was demonstrated by³²P-incorporation into nucleic acids and their subsequent electrophoretic analysis on polyacrylamide gels. Under optimum culture conditions the amount of³²P-orthophosphate incorporation into PSTV reached 10% of that incorporated into the 2 M LiCl-soluble cellular RNA. (+)PSTV and its complementary form, i.e. (?)PSTV were identified after their electrophoretic separation on polyacrylamide and agarose gels by molecular hybridization. This analysis revealed the presence of six high molecular weight(?)PSTV species, which are possibly multimers of the unit length(+)PSTV molecule consisting of 359 nucleotides.     

Fixation requirements for the immunohistochemical reactivity of PCNA antibody PC10 on cryostat sections

Summary In contrast to that in paraffin-embedded tissue, the reactivity of monoclonal PCNA antibody PC10 on cryostat sections requires a special fixation procedure as the target epitope is seemingly not accessible to its antibody. A panel of 18 fixation protocols was investigated. Chilled methanol or acetone, or PLP (paraformaldehyde-lysine-periodate) was found to be unsuitable for skin preparations. A two-step fixation protocol was developed for normal skin and basal cell carcinomas. They were fixed first in 3.4% buffered formaldehyde, followed by fixation in 2:1 v/v ethanol-acetic acid. Following this fixation regime, cryostat sections displayed the same PCNA/PC10 labelling pattern as paraffin sections of formalin-fixed tissue.