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Biomedical subjects

W Bloch

Publications and source records attributed to W Bloch.

At least 91 records · Page 5Linked to original sources
PubMed1997

Introduction to PCR/OLA/SCS, a multiplex DNA test, and its application to cystic fibrosis.

The field of medical, molecular diagnostics has grown rapidly over the last few years, becoming increasingly informative to both clinician and patient. As genes associated with specific diseases have been discovered and sequenced, many genotype-phenotype relationships have been defined. For those genetic diseases with associated, defined, gene mutations, sophisticated DNA diagnostic tests are being developed. As an example, the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, is associated with Cystic Fibrosis (CF). We have developed a new molecular diagnostic technology, PCR/OLA/SCS, and applied it first to the diagnosis of CF. Test design in the field of molecular diagnostics must consider such characteristics as specificity, sensitivity, ease and speed of protocol, multiplex capacity, and cost. PCR/OLA/SCS addresses these requirements. Polymerase Chain Reaction (PCR) is widely used in both diagnostics and research. We have combined well established PCR technology with Oligonucleotide Ligation Assay (OLA) and Sequence-Coded Separation (SCS), two relatively new technologies.

Alleles↗
PubMed1996

Differentiation and integrity of cardiac muscle cells are impaired in the absence of beta 1 integrin.

Cellular interactions with substrata of the microenvironment are one of the major mechanisms for differentiation and morphogenesis. Many of these interactions are mediated via the beta 1 integrin subfamily of cell surface receptors, which are believed to transduce signals upon cell adhesion. We have used beta 1 integrin-deficient embryonic stem cells to test their ability to differentiate into cardiac muscle cells. We show here by several approaches that beta 1 integrin is important for normal cardiogenesis. First, the in vitro differentiation of beta 1 integrin-deficient embryonic stem cells into cardiac muscle cells is retarded. This is demonstrated by the delayed expression of cardiac muscle-specific genes and action potentials. Second, the specification of cardiac precursor cells into pacemaker-, atrial- and ventricular-like cells is significantly impaired in beta 1 integrin-deficient cells. The occurrence of atrial- and ventricular-like cells is reduced and transient. Only cells exhibiting peacemaker-like action potentials of high frequency and arrhythmias survive. Third, the sarcomeric architecture is incomplete and disarranged in the absence of beta 1 integrin. Fourth, beta 1-deficient embryonic stem cells can contribute to the developing heart in chimaeric mice but many areas with beta 1-null cells contain cell debris. The number of beta 1-null cells decrease from prenatal to postnatal stages and is lost completely in 6-month-old hearts. Thus, we conclude that interactions with the extracellular matrix via beta 1 integrin is necessary for differentiation and the maintenance of a specialized phenotype of cardiac muscle cells.

Animals↗
PubMed1996

Long-term intraocular toxic effects of topical mitomycin C in rabbits.

PURPOSE: To detect whether toxic intraocular effects of mitomycin C (MMC) after extraocular application are long-standing or are reversible over time. METHODS: We placed sponges soaked with MMC on the intact sclera of rabbit eyes after incision of the conjunctiva. Controls received sponges soaked with balanced salt solution. Concentrations of MMC were 0.1 and 0.2 mg/ml. The eyes were examined histologically after 6 and 12 months. RESULTS: In all MMC-treated eyes, pathologic changes of the ciliary epithelium were present, which included irregular cell surfaces, intracytoplasmatic vacuoles, swelling of mitochondria, and disorganization of the endoplasmic reticulum. Nerves within the ciliary body also exhibited mild-to-moderate loss of axons in nonmyelinated nerves and increase of interstitial collagen deposition. Capillaries within the ciliary body consistently had a thickened basement membrane, which was arranged in two to three layers in multiple areas. The changes seen were similar in eyes examined at 6 and 12 months after application of MMC. CONCLUSIONS: These results suggest that toxic intraocular changes are dose dependent and do not recover over time. The impact of these findings on humans is unclear, but may further emphasize the high toxic potential of MMC.

Administration, Topical↗
PubMed1995

Nitric oxide mediates microvascular permeability in the isolated perfused rat mesentery?

The permeability influencing effect of nitric oxide was studied by means of an isolated perfused mesentery preparation in combination with a intravital/fluorescence microscopic set up. As a fluorescent marker served FITC-dextran mol wt 70,000 daltons. Tissue treatment was performed by application of NO-donors like SNP or GTN. Additionally the effects of bradykinin, L-nitroarginine or the combination of both were tested in regard to a permeability modulation. The results point out that NO-donors did not enhance microvascular permeability, whereas the permeability increasing effect of bradykinin is mediated by an NO-independent pathway.

Animals↗
PubMed1995

Exogenously supplied nitric oxide influences the dilation of the capillary microvasculature in vivo.

An endogenous NO-release which exceeds the basal endogenous NO-release has a regulatory effect on capillary microvasculature in isolatedly perfused rat hearts. The basal NO-release in contrast has no effect on capillaries. The functional findings are corresponding to the endothelial distribution of NOS in coronary vessels, which displays a lack of NOS in capillary endothelium. An increase of coronary flow by exogenously administered NO-donors does not necessarily lead to a dilation of capillary microvasculature. Local differences in the release of unstable NO by SNP and GTN are responsible for variations in effects. We can conclude: NO influences the dilation of the capillary microvasculature independently of flow regulation.

Animals↗
PubMed1995

Endothelial relaxation is disturbed by oxidative stress in the diabetic rat heart: influence of tocopherol as antioxidant.

Increased oxidative stress has been suggested to contribute to disturbances in the regulation of coronary flow and the increased cardiac risk in diabetes mellitus. Using the isolated perfused heart of streptozotocin-diabetic rats our study shows that basal and maximal coronary flow (tested by infusion of sodium nitroprusside) are not altered in diabetes, but that 5-hydroxytryptamine (5-HT) stimulated endothelium-dependent increase in coronary flow becomes progressively impaired. This defect of the endothelium-dependent vasodilatation was prevented by perfusion of the hearts with superoxide dismutase and pretreatment of the diabetic rats with tocopherol-acetate. Morphological studies also revealed that pretreatment with tocopherol-acetate was cardioprotective, and largely prevented severe alterations of myocardial structure typically observed after a diabetes duration of 3 months; deterioration and fragmentation of myofilament bundles were seen less, and the numbers of areas of focal necrosis and of contraction bands were clearly reduced. In contrast to untreated diabetic hearts the autonomic nerve fibers detected by catecholamine fluorescence were running in parallel in hearts of tocopherol-treated diabetic rats, and the amount of catecholamines was not different from that of healthy control rats. Trichrome staining and immunohistochemical staining of collagen I and III showed a dramatic increase in number and the size of deposits of collagen fibers at precapillary locations in the diabetic hearts which were significantly reduced by anti-oxidative treatment. These findings demonstrate that oxidative stress may not only play a major role in the impairment of endothelium-dependent regulation of coronary flow, but also in the development of perivascular fibrosis and severe changes of the autonomic nerves and contractile system in myocardium.

Aging↗
PubMed1994

High-density multiplex detection of nucleic acid sequences: oligonucleotide ligation assay and sequence-coded separation.

We describe a non-isotopic, semi-automated method for large-scale multiplex analysis of nucleic acid sequences, using the cystic fibrosis transmembrane regulator (CFTR) gene as an example. Products of a multiplex oligonucleotide ligation assay (OLA) are resolved electrophoretically from one another and from unligated probes under denaturing conditions with fluorescence detection. One ligation probe for each OLA target carries a fluorescent tag, while the other probe carries an oligomeric non-nucleotide mobility modifier. Each OLA product has a unique electrophoretic mobility determined by the ligated oligonucleotides and the mobility-modifier oligomer arbitrarily assigned (coded) to its target. The mobility range for practical mobility modifiers is much wider than the accessible range from unmodified ligated oligonucleotides of practical length. Each mobility modifier is built from phosphoramidite monomers in a stepwise manner on its associated oligonucleotide using an automated synthesizer. The resulting mobility modifiers lower the probe-target duplex Tm by less than 3 degrees C and retard probe-target annealing by less than 50%, with negligible effect on OLA yield and specificity. This method is especially useful for allelic discrimination in highly polymorphic genes such as CFTR.

Autoanalysis↗
PubMed1994

Nitric oxide modulates sympathetic neurotransmission at the prejunctional level.

In spite of accumulating evidence for a modulation of sympathetic neurotransmission by endogenously produced nitric oxide (NO), it remains unclear in which parts of the vascular system and at what level this interaction takes place. The aim of the present study was to investigate the distribution of endothelial and neuronal NO synthase (NOS) along the vascular tree of the heart at the light and electron microscopic level using NADPH-diaphorase (NADPH-d) staining as a marker for NOS. In addition, the functional effects of exogenous NO on coronary vascular resistance and cardiac adrenergic nerves was studied using the isolated perfused rat heart as a model. The intraaxonal catecholamine content of adrenergic nerve fibers was visualised and morphometrically assessed by applying glyoxylic acid-induced histofluorescence. The expression of endothelial NOS in the heart was found to depend on the diameter of the blood vessel. Arteries > 100 microns always showed intense staining, whereas staining in smaller arteries and veins was considerably weaker. Smooth-muscle free vessels were essentially devoid of NADPH-d activity. In atrial and ventricular myocardium, neuronal NOS localised in autonomic nerve fibers along the entire vascular tree. Ultrastructurally, NADPH-d staining revealed adjacent localisation of NOS-positive and -negative axons, suggesting and interaxonal modulation of adjacent autonomic nerve fibers by NO. In isolated perfused rat hearts, the intracoronary application of 10(-8) M NO produced a marked decrease of coronary perfusion pressure, which was accompanied by a distinct increase in intraaxonal catecholamine levels of intramural adrenergic nerve fibers.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic Fibers↗
PubMed1993

Allelic discrimination by nick-translation PCR with fluorogenic probes.

Nick-translation PCR was performed with fluorogenic probes. Two probes were used: one complementary to a sequence containing the F508 codon of the normal human cystic fibrosis (CF) gene (wt DNA) and one complementary to a sequence containing the delta F508 three base pair deletion (mut DNA). Each probe contained a unique and spectrally resolvable fluorescent indicator dye at the 5' end and a common quencher dye attached to the seventh nucleotide from the 5' end. The F508/delta F508 site was located between the indicator and quencher. The probes were added at the start of a PCR containing mut DNA, wt DNA or heterozygous DNA and were degraded during thermal cycling. Although both probes were degraded, each probe generated fluorescence from its indicator dye only when the sequence between the indicator and quencher dyes was perfectly complementary to target. The identify of the target DNA could be determined from the post-PCR fluorescence emission spectrum.

Alleles↗
PubMed1993

Importance of different variables for enhancing in situ detection of PCR-amplified DNA.

This study determined the effects of several variables on the in situ signal after PCR amplification in fixed cells. A signal was evident in all human peripheral blood monocytes fixed in buffered formalin using primers for the human proto-oncogene bcl-2 with in situ PCR only after prolonged fixation and protease digestion. A much lower detection rate was noted after ethanol or acetone fixation due to loss of amplified product out of the nucleus into amplifying solution. This observation demonstrates the importance of cross-linking fixatives for retention of amplified DNA at the site of origin. The increased amount of target-specific DNA synthesis evident with the manual hot start modification to in situ PCR was also seen with chemical hot start mediated by the Escherichia coli single-stranded DNA-binding protein. The manual hot start method strongly suppressed in situ unwanted DNA synthesis dictated by nonsense primers; residual nonspecific synthesis was influenced by annealing temperatures and post-fixation protease digestion conditions.

Base Sequence↗
PubMed1992

Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications.

A Hot Start Polymerase Chain Reaction (PCR) entails the withholding of at least one reagent from the reaction mixture until the reaction tube temperature has reached 60-80 degrees C. Hot Start amplification with an AmpliWax vapor barrier uses a layer of solid wax to separate the retained reagent(s) and the test sample from the bulk of the reagents until the first heating step of automated thermal cycling melts the wax and convectively mixes the two aqueous layers. Wax-mediated Hot Start PCR greatly increases the specificity, yield, and precision of amplifying low copy numbers of three HIV targets. In the presence of 1 microgram of human placental DNA (1.6 x 10(5) diploid genomes) the specificity improvement entails considerable to complete reduction in the amplification of mis-primed sequences and putative primer oligomers. When mis-priming is negligible, the procedural improvement still suppresses putative primer oligomerization. Hot Start PCR with an AmpliWax vapor barrier permits routine amplification of a single target molecule with detection by ethidium stained gel electrophoresis; nonisotopically visualized probing suffices for confirmation. The improved amplification performance is evident for target copy numbers below approximately 10(3).

Base Sequence↗
PubMed1991

An improved technique for the in situ detection of DNA after polymerase chain reaction amplification.

In situ detection of polymerase chain reaction (PCR)-amplified DNA in cell and tissue preparations previously required 5 to 7 primer pairs designed to generate a long (greater than 1,000 base pair) product. The authors describe a nonisotopic PCR in situ technique, employing a single primer pair and target sequences as short as 115 base pairs, that can detect one target molecule per cell. The essential procedural change is to withhold the DNA polymerase or primers until the reaction temperature approaches 80 degrees C. The Hot-Start method greatly increased amplification specificity which, more than product size, appears to determine success of in situ PCR. The marked improvement in specificity may permit target detection by direct incorporation of labeled nucleotides.

DNA, Viral↗
PubMed1988

Hepatitis B virus particles contain a polypeptide encoded by the largest open reading frame: a putative reverse transcriptase.

A segment of the largest open reading frame of hepatitis B virus (HBV) was inserted into an open reading frame vector directing the expression in Escherichia coli of a fusion molecule containing 143 HBV-encoded amino acids. The fusion protein was used to generate antiserum which served in immunoblots to identify a polypeptide with a molecular mass of 65 kilodaltons in HBV particles. Because of the small number of molecules in virus particles, unambiguous detection required the development of a highly sensitive immunoblot procedure.

3,3'-Diaminobenzidine↗
PubMed1987

Nonisotopic M13 probes for detecting the beta-globin gene: application to diagnosis of sickle cell anemia.

M13 DNA probes labeled with biotinylated psoralen and a streptavidin-horseradish peroxidase conjugate provide nonradioactive detection of the sickle cell and normal alleles of the beta-globin locus. The two biotinylated probes contain single-stranded sequences complementary to two different Sau3AI restriction fragments from the 5' region of the beta-globin gene and double-stranded M13 vector sequences. These probes are labeled with biotinylated psoralen photochemically linked to DNA. After hybridization, the presence of biotinylated probe bound to target DNA is detected in 3 h by using a streptavidin-horseradish peroxidase conjugate and the substrate, 3,3',5,5'-tetramethylbenzidine. Digestion of the normal (beta A) allele of the beta-globin gene with MstII (or isoschizomers) yields a 1.14-kb restriction fragment, while digestion of the mutant beta S allele yields a 1.34-kb fragment. These fragments can be resolved by gel electrophoresis and detected by Southern blot hybridization. The nonradioisotopic probe system can detect the beta-globin restriction fragment in as little as 0.5 microgram of human DNA and can distinguish heterozygotes (beta A beta S) from homozygotes (beta A beta A or beta S beta S) in 2.0 micrograms of human DNA.

Alleles↗
PubMed1980

Catalytic mechanism of Escherichia coli alkaline phosphatase: resolution of three variants of the acyl-enzyme mechanism.

Three variants of the classical acyl-enzyme mechanism were compared theoretically with respect to the predicted transient kinetics of substrate hydrolysis by Escherichia coli alkaline phosphatase. In all three, acyl-enzyme hydrolysis was assumed to exist initially primarily as a noncovalent complex with the acid product, inorganic phosphate. In one mechanism, the pre-steady-state rate-controlling step was assumed to be the dissociation of acid product from its initial complex with enzyme. In the other two, pre-steady-state rate control was assigned to an enzyme isomerization occurring before or after substrate binding to free enzyme. Under concentration conditions of excess substrate and acid product, integrated rate laws were used to reject the possibility of pre-steady-state rate control by enzyme isomerization between phosphate dissociation and substrate binding. Whereas this mechanism predicts a pre-steady-state noncompetitive relationship between substrate and acid product, the stopped-flow kinetics of 4-methylumbelliferyl phosphate hydrolysis demonstrates a competitive relationship, consistent with either of the other two mechanisms. Under concentration conditions of stoichiometrically limiting substrate, computer simulations eliminated the possibility of rate control by enzyme isomerization after substrate binding. This mechanism predicts a substrate concentration dependence for the apparent first-order rate constant of substrate hydrolysis which disagrees with previously published data [Halford, S. E. (1971) Biochem. J. 125, 319--327]; the other two mechanisms are consistent with experiment. Comparison of transient kinetic theory and experiment under these two contrasting concentration conditions suggests strongly that the rate-controlling step in phosphate ester hydrolysis by E. coli alkaline phosphate is the dissociation of "sticky" acid product from its noncovalent complex with enzyme. This mechanism explains an anomaly in the stopped-flow kinetic trace, a substoichiometric pre-steady-state burst of alcohol product release.

Alkaline Phosphatase↗
PubMed1978

Phosphate binding to Escherichia coli alkaline phosphatase. Evidence for site homogeneity.

The reversible, noncovalent binding of inorganic phosphate to Escherichia coli alkaline phosphatase at pH 8 has been examined by equilibrium dialysis at two temperatures and two ionic strengths. Binding occurs with a stoichiometry of two phosphate ions per dimeric enzyme molecule and a single dissociation constant that is not very sensitive to temperature or ionic strength. These results contradict published evidence for anti-cooperative binding of inorganic phosphate to alkaline phosphatase. Reasons are presented for believing that the apparent anti-cooperativity reported by other workers is artifactual.

Alkaline Phosphatase↗