Electron microscopy of satellite tobacco mosaic virus crystals: metal-coated, negatively stained and stereo pairs.

Highly purified virions of satellite tobacco mosaic virus (STMV) were found to crystallize at relatively low concentrations (300-500 microg ml(-1)) in pure water. Small crystals of these preparations were examined in the transmission electron microscope after either being rotary shadowcast with metal or negatively stained with 4% uranyl acetate. Stereo views were also obtained of both types of preparations. Stereo pairs of metal-coated crystals provided good three-dimensional images. When stereo pairs of negatively stained crystals were printed from second negatives, they provided striking images although the three-dimensional aspect was not so pronounced. Images of both types of preparations were compared with a computer-generated model of the virus. This model was based on data obtained in earlier X-ray diffraction crystallographic studies. Measurements of crystal axes on the EM images were somewhat lower than those of the computer model. It is assumed the reason for this is the dehydration of crystals during preparation for electron microscopy. The EM images did verify the type of crystal lattice determined in the X-ray diffraction studies. Conversely, knowing the exact unit cell parameters and the distribution of virions in the crystal from X-ray diffraction data aids in the further interpretation of electron micrographs of virus crystals.

Human skeletal myosin heavy chain genes are tightly linked in the order embryonic-IIa-IId/x-ILb-perinatal-extraocular.

Myosin heavy chain (MyHC) is the major contractile protein of muscle. We report the first complete cosmid cloning and definitive physical map of the tandemly linked human skeletal MyHC genes at 17p13.1. The map provides new information on the order, size, and relative spacing of the genes. and it resolves uncertainties about the two fastest twitch isoforms. The physical order of the genes is demonstrated to contrast with the temporal order of their developmental expression. Furthermore, nucleotide sequence comparisons allow an approximation of the relative timing of five ancestral duplications that created distinct genes for the six isoforms. A firm foundation is provided for molecular analysis in patients with suspected primary skeletal myosinopathies and for detailed modelling of the hypervariable surface loops which dictate myosin's kinetic properties.

Stable restoration of the sarcoglycan complex in dystrophic muscle perfused with histamine and a recombinant adeno-associated viral vector.

Limb-girdle muscular dystrophies 2C-F represent a family of autosomal recessive diseases caused by defects in sarcoglycan genes. The cardiomyopathic hamster is a naturally occurring model for limb-girdle muscular dystrophy caused by a primary deficiency in delta-sarcoglycan. We show here that acute sarcolemmal disruption occurs in this animal model during forceful muscle contraction. A recombinant adeno-associated virus vector encoding human delta-sarcoglycan conferred efficient and stable genetic reconstitution in the adult cardiomyopathic hamster when injected directly into muscle. A quantitative assay demonstrated that vector-transduced muscle fibers are stably protected from sarcolemmal disruption; there was no associated inflammation or immunologic response to the vector-encoded protein. Efficient gene transduction with rescue of the sarcoglycan complex in muscle fibers of the distal hindlimb was also obtained after infusion of recombinant adeno-associated virus into the femoral artery in conjunction with histamine-induced endothelial permeabilization. This study provides a strong rationale for the development of gene therapy for limb-girdle muscular dystrophy.

Integration of physical, breakpoint and genetic maps of chromosome 22. Localization of 587 yeast artificial chromosomes with 238 mapped markers.

Detailed physical maps of the human genome are important resources for the identification and isolation of disease genes and for studying the structure and function of the genome. We used data from STS content mapping of YACs and natural and induced chromosomal breakpoints to anchor contigs of overlapping yeast artificial chromosome (YAC) clones spanning extensive regions of human chromosome 22. The STSs were assigned to specific regions (bins) on the chromosome using cell lines from a somatic hybrid mapping panel defining a maximum of 25 intervals. YAC libraries were screened by PCR amplification of hierarchical pools of yeast DNA with 238 markers, and a total of 587 YAC clones were identified. These YACs were assembled into contigs based upon their shared STS content using a simulated annealing algorithm. Fifteen contigs, containing between 2 and 74 STSs were assembled, and ordered along the chromosome based upon the cytogenetic breakpoint, meiotic and PFG maps. Additional singleton YACs were assigned to unique chromosomal bins. These ordered YAC contigs will be useful for identifying disease genes and chromosomal breakpoints by positional cloning and will provide the foundation for higher resolution physical maps for large scale sequencing of the chromosome.

A rapid method for measurement of ethylene glycol.

It has been reported that ethylene glycol produces a positive interference in the triglyceride assay on the DuPont aca discrete analyzer. The sensitivity of this method for ethylene glycol was exploited to develop a rapid and convenient method for detecting and quantitating ethylene glycol in serum by the use of a "triglyceride gap." This method is based on the difference between two different enzymatic triglyceride measurements; the DuPont aca triglyceride measurement, which uses lipase and glycerol dehydrogenase, and a Boehringer Mannheim method, which uses lipase, glycerol kinase, glycerolphosphate oxidase, and peroxidase. Serum pools were spiked with increasing concentrations of ethylene glycol to construct a standard curve (linear to 25 mmol/L). Patients specimens and control sera were analyzed using a one point calibration. Within-run and between-run coefficients of variation (CV) of 1.5, 2.8, 5.8, and 3.2%; 3.4%; and 12.6% were obtained at ethylene glycol concentrations of 20.13, 8.37, and 2.18 mmol/L, respectively. The sensitivity of this method is 1.0 mmol/L. Methanol, ethanol, n-propanol, isopropanol, and acetone at 100 mmol/L; 20 mmol/L 1,3-propanediol; and 10 mmol/L glycolic acid, oxalic acid, glyoxylic acid, and lactic acid did not interfere in the quantitation of ethylene glycol. High levels (10 mmol/L) of beta-hydroxybutyrate and glycerol showed a slight positive interference on the quantitation of ethylene glycol, whereas 10 mmol/L glycoaldehyde caused a substantial overestimation of serum ethylene glycol. The presence of propylene glycol, at levels ranging from 5 to 50 mmol/L, resulted in an underestimation of serum ethylene glycol.(ABSTRACT TRUNCATED AT 250 WORDS)

Association of logistic and Poisson models of infection with some physical characteristics of a single component plant virus.

A logistic model was recently formulated to describe the relationship between concentration of a single component plant virus and infections produced by inoculation to a local lesion host. In this paper the logistic is combined with a Poisson model. The logistic makes accurate fitting possible for a variety of infection-dilution series; and the Poisson acts as a base line, indicating whether lesion numbers are compatible with the hypothesis that random infection of similar infection sites has occurred. A logarithmic form of the logistic equation gives a straight line with negative slope (logit slope) which is useful in characterizing dilution series to which the logistic is fitted. A modified Poisson equation can also be fitted to a range of dilution series; it provides an independent estimate of slope for curves not widely divergent from the standard Poisson. Models have also been developed to define the limits of concentration within which single virions are likely to be randomly dispersed in inoculum without immediate contact with other virions, and are therefore more likely to enter inoculated tissue independently and cause random infections. Models are formulated for aggregation of tobacco mosaic virus in monolayers, crystals, and lenticular aggregates. Published and unpublished data are fitted and analyzed using some of these models.

A tobacco mosaic virus-hybrid expresses and loses an added gene.

An additional open reading frame from the chloramphenicol acetyltransferase (CAT) gene was fused behind a tobacco mosaic virus (TMV) subgenomic RNA promoter and inserted into different positions in the complete TMV genome to examine how much this viral genome can be altered with continued replication. One hybrid virus, CAT-CP, with the insertion between the 30K and coat protein genes, replicated efficiently, produced an additional subgenomic RNA and CAT activity, and assembled into 350-nm virions, compared to 300-nm virions of wild-type TMV. However, during systemic infection of plants, the inserted sequences were deleted. This deletion was exact, resulting in progeny wild-type TMV. Another hybrid virus examined was CP-CAT, which had the insertion between the coat protein gene and the nontranslated 3' region. This virus replicated poorly, produced only minimal levels of CAT activity, and did not systemically invade infected plants. These data show that some extensive modifications of the TMV genome still allow efficient virus replication.

Protein dependence of digoxin determination on the Abbott-TDX.

The protein dependence of digoxin quantitation on the Abbott TDX was evaluated. An inverse relationship was found to exist between protein concentration and digoxin levels in samples that were deproteinized with 50 g/L trichloroacetic acid prior to assay. This was especially evident in the protein range from 30-60 g/L where a 30% decrease in digoxin concentration was observed. Recovery studies using 125I-digoxin showed that this decrease could be totally accounted for by a decreased recovery of digoxin in the trichloroacetic acid supernatant. The serum-based calibrators used to establish the standard curve on the instrument showed similar recoveries to serum samples. The reduced recovery does not pose accuracy problems as long as the protein concentrations of the patient samples and calibrators are similar. The clinical and operational implications of this observation are discussed.

Clinical effectiveness of the Du Pont aca measurement of creatine kinase MB in serum from patients in a coronary-care unit.

We evaluated the clinical effectiveness of measuring creatine kinase (CK; EC 2.7.3.2) isoenzyme MB and lactate dehydrogenase (LD; EC 1.1.1.27) isoenzymes in diagnosis of acute myocardial infarction. We used an agarose electrophoresis method to measure CK and LD isoenzymes and the Du Pont aca column method to measure CK-MB. Serial blood specimens were drawn from 100 patients consecutively admitted to our Coronary Care Unit. Because of the low diagnostic specificity for CK-MB measurements by both agarose electrophoresis and the discrete-analysis method, as compared with reported values, we re-evaluated our isoenzyme data by using Receiver Operating Characteristic curves. Such analysis of the data established optimal decision levels of greater than or equal to 25 U/L and greater than or equal to 18 U/L plus greater than or equal to 6% of total CK for serum CK-MB measured by the agarose electrophoresis and the aca methods, respectively, and an optimal decision level of greater than or equal to 0.92 for the ratio of LD 1/2 measured after agarose electrophoresis. At these decision levels we obtained a sensitivity of 100%, 100%, and 95% and a specificity of 94%, 92%, and 90% for CK-MB (agarose electrophoresis), CK-MB (aca), and the LD 1/2 ratio, respectively.

Quantitation of creatine kinase MB using an agarose electrophoresis method; importance of enzyme reagent concentration.

Quantification of CK-MB activity in serum using agarose electrophoresis requires that CK enzyme reagent be layered over the gel in order to develop enzyme activity. We have shown that when commercial enzyme reagents were used, and reconstituted in a normal fashion, that the CK-MM isoenzyme activity is underestimated which results in an overestimation of CK-MB activity. Underestimation of CK-MM occurs because the enzyme reagent are diluted approximately five-fold by the agarose, which results in the non-linear development of enzyme activity under sub-optimal conditions. Reconstitution of these commercial enzyme reagents in a concentrated manner (5 times more concentrated) compensates for the dilution of the reagent by the agarose and allows for the linear development with time of CK-MM and CK-MB enzyme activity. However, use of five-fold concentrated enzyme reagent does not totally prevent underestimation of CK-MM enzyme activity, and care must be taken in limiting the amount of enzyme activity placed on the agarose gel.

Subcellular localization of human heart atypical creatine kinase.

Human heart subcellular fractions were obtained using method A described by Scholte et al [7,8]. It was found that 3.5 +/- 2.5%, 3.4 +/- 1.9%, 8.5 +/- 2.9%, 2.5 +/- 1.3%, and 88 +/- 21% of the total creatine kinase activity was associated with the myofibrillar, nuclear, mitochondrial, microsomal and particle-free soluble fractions respectively. Using DEAE-sephacel chromatography it was determined that the atypical creatine kinase (CK-Z) accounted for approximately 90% of the creatine kinase activity associated with the mitochondrial fraction.