Polymorphisms and deduced amino acid substitutions in the coding sequence of the ryanodine receptor (RYR1) gene in individuals with malignant hyperthermia.

Twenty-one polymorphic sequence variants of the RYR1 gene, including 13 restriction fragment length polymorphisms (RFLPs), were identified by sequence analysis of human ryanodine receptor (RYR1) cDNAs from three individuals predisposed to malignant hyperthermia (MH). All RFLPs were detectable in PCR-amplified products, and their segregation was consistent with our initial finding of linkage to MH in the nine families previously informative for one or more intragenic markers (MacLennan et al., 1990, Nature 343:559-561). Four amino acid substitutions were identified in the study: Arg for Gly248, Cys for Arg470, Leu for Pro1785, and Cys for Gly2059. Of 45 families tested, a single family presented the Arg for Gly248 substitution where it segregated with malignant hyperthermia, making it a candidate mutation for predisposition to MH in man. The other three polymorphic substitutions failed to segregate with malignant hyperthermia in those families in which they occurred, implying that they represent polymorphisms with little or no effect on the function of the RYR1 gene.     

Distribution of linear antigenic sites on glycoprotein gp55 of human cytomegalovirus.

Human convalescent serum and bacterial fusion proteins constructed from overlapping open reading frames of the nucleotide sequence encoding the human cytomegalovirus gp55 component of the major envelope glycoprotein complex, gp55-116 (gB), were used to localize antigenic regions recognized by human antibodies. All donor serum analyzed contained antibody reactivity for an antigenic site(s) located between amino acids (AA) 589 and 645, a region containing a previously defined linear site recognized by neutralizing monoclonal antibodies (U. Utz, B. Britt, L. Vugler, and M. Mach, J. Virol. 63:1995-2001, 1989). Furthermore, in-frame insertion of two different synthetic oligonucleotides encoding four amino acids into the sequence at nucleotide 1847 (AA 616) eliminated antibody recognition of the fusion protein. A second antibody binding site was located within the carboxyl terminus of the protein (AA 703 through 906). A competitive binding inhibition assay in which monoclonal antibodies were used to inhibit human antibody reactivity with recombinant gp55-116 (gB) suggested that the majority of human anti-gp55-116 (gB) antibodies were directed against a single antigenic region located between AA 589 and 645. Furthermore, inoculation of mice with fusion proteins containing this antigenic site led to a boostable antibody response. These results indicated that the antigenic site(s) located between AA 589 and 645 was an immunodominant antibody recognition site on gp55 and likely the whole gp55-116 (gB) molecule. The enhanced immunogenicity of this region in vivo may account for its immunodominance.     

Prostaglandin F-2 alpha causes regression of an hCG-induced corpus luteum before day 5 of its lifespan in cattle

The experimental objective was to evaluate how a spontaneously formed corpus luteum (CL) differed in its response to prostaglandin (PG) F-2 alpha, given during the first 5 days after ovulation, from a CL induced during dioestrus with hCG. Sixteen Holstein heifers were used during each of 2 consecutive oestrous cycles. During the first cycle (sham cycle), heifers were given no PGF-2 alpha (control) or PGF-2 alpha (25 mg, i.m.) on Day 2, 4 or 6 (oestrus = Day 0). During the second cycle (hCG-treated cycle), heifers were given hCG (5000 i.u., i.m.) on Day 10, followed by no PGF-2 alpha (control) or PGF-2 alpha on Day 12, 14 or 16, corresponding to 2, 4 or 6 days after the ovulatory dose of hCG. A new ovulation was induced in 13 of 16 heifers given hCG on Day 10. Luteolysis did not occur immediately in heifers given PGF-2 alpha on Day 2 or 4 during the sham cycle, but concentration of progesterone in serum during the remainder of the cycle was lower in heifers given PGF-2 alpha on Day 4 than in sham controls or heifers given PGF-2 alpha on Day 2 (P less than 0.05). Luteolysis occurred immediately in heifers given PGF-2 alpha on Day 6 of the sham cycle or on Day 12, 14 or 16 of the hCG-treated cycle, with concentration of progesterone in serum decreasing to less than 1 ng/ml within 2 days.(ABSTRACT TRUNCATED AT 250 WORDS)     

Processing of the gp55-116 envelope glycoprotein complex (gB) of human cytomegalovirus.

The processing pathway of the major envelope glycoprotein complex, gp55-116 (gB), of human cytomegalovirus was studied using inhibitors of glycosylation and endoglycosidases. The results of these studies indicated that the mature gp55-116 is synthesized by the addition of both simple and complex N-linked sugars to a nonglycosylated precursor of estimated Mr 105,000. In a rapid processing step, the Mr 105,000 precursor is glycosylated to a protein of Mr 150,000 (gp150) which contains only endoglycosidase H-sensitive sugar linkages. The gp150 is then processed relatively slowly to a Mr 165,000 to 170,000 species (gp165-170), which is then cleaved to yield the mature gp55-116. Monensin prevented the final processing steps of the gp150, including cleavage, suggesting that transport through the Golgi apparatus is required for complete processing. Digestion of the intracellular forms of this complex as well as the virion forms confirmed the above findings and indicated that the mature virion form of gp55 contains 8,000 daltons of N-linked sugars. The virion gp116 contains some 52,000 to 57,000 daltons of N-linked carbohydrates and approximately 5,000 daltons of O-linked sugars.     

Human cytomegalovirus neutralizing antibody-resistant phenotype is associated with reduced expression of glycoprotein H.

We have characterized a neutralizing antibody-resistant mutant human cytomegalovirus (HCMV) obtained from a patient treated with a human monoclonal antiglycoprotein H (gH; unique long region 75) antibody. This virus exhibited resistance to several different neutralizing anti-gH murine monoclonal antibodies (MAbs), as well as to a polyvalent anti-gH serum. The resistant phenotype was unstable and could be maintained only by passage of plaque-purified virus under neutralizing MAb selection. In the absence of a MAb, the resistant phenotype reverted to a neutralizing antibody-sensitive phenotype within one passage. The predicted amino acid sequences of gH from the MAb-resistant and -susceptible parent viruses were identical. Biochemical analysis of the MAb-resistant and -susceptible parent viruses revealed a marked decrease of gH expression in the envelope of the MAb-resistant virus. Furthermore, propagation of the virus in various MAb concentrations resulted in the production of extracellular virions with various levels of resistance to the neutralizing activity of the MAb. These results suggest a mechanism for the generation of neutralizing antibody-resistant viruses which could evade host-derived antiviral antibody responses. In addition, our findings indicate that the stoichiometry of gH in the envelope of infectious HCMV virions is not rigidly fixed and therefore offer a simple explanation for production of phenotypic variants of HCMV through an assembly process in which the content of gH in the envelope of progeny virions varies randomly.     

Ulfrastructure of the histological zones in growing vegetative buds of Salix spp.

Ultrastructural differentiation in the shoot apex of growing vegetative buds of Salix was studied, and some micrographs analysed morphometrically. The distribution of inorganic phospahte (P;) was analysed cytochemically. A distinct histological zona–tion was observed in the apex. The relative volumes of nuclei and plastids were significantly higher in the central tunica zone than in the peripheral one. The corpus differed from the central tunica zone by significantly lower volume density of nuclei and higher of vacuoles and mitochondria. During differentiation of the rib meristem vacuole volume increased significantly, while the relative volumes of nuclei, mitochondria, nucleoli, and heterochromatin decreased. It was not possible to decide whether the vacuoles originate from ER or GERL. Morphogenesis of chloroplasts with large starch grains and grana from proplastids was evident in the rib meristem; dedifferentiation to S–plastids was found in the protophloem. Prolamellar bodies were observed in the procambium plastids. The protophloem was characterized by P–protein and spiny vesicles. P_i was found in the nucleoli of most epidermis cells, several procambium cells, and a few chlorencyma cells, but never in the tunica of the growing apical and developing lateral buds. P_i also occurred in some plasmalemma–somes and occasionally in the walls in connection with plasmodesmata.     

Pronounced inhibition of noradrenaline uptake by 10-hydroxymetabolites of nortriptyline

The potency of the tricyclic antidepressant nortriptyline (NT) relative to that of its major metabolite (E-10-hydroxynortriptyline; E-10-OH-NT) to inhibit the uptake of noradrenaline in rat brain slices incubated in human plasma was 1.75 ± S.D. 0.30. The E- and Z-isomers of 10-OH-NT were equipotent. Hydroxy-metabolites of NT or amitriptyline did not inhibited the neuronal uptake of serotonin.During treatment of 87 patients with NT or amitriptyline the mean ratio between the plasma levels of unconjugated 10-OH-NT and NT was 1.40 ± S.D. 0.86 with a range of 0.32 – 5.0. This ratio increased with age. There was a significant correlation between the plasma levels of the two compounds (r = 0.63; p < 0.001). In three patients treated with NT, the CSF levels of the parent drug and unconjugated 10-OH-NT were similar and about 5 % of the plasma levels.These results indicate that during treatment with NT (or amitriptyline) 10-OH-NT contributes to the effect of these drugs on central noradrenergic neurons. This seems to be of special significance in the elderly.     

Automatic Segmentation of Eight Tissue Classes in Neonatal Brain MRI

Purpose

Volumetric measurements of neonatal brain tissues may be used as a biomarker for later neurodevelopmental outcome. We propose an automatic method for probabilistic brain segmentation in neonatal MRIs.

Materials and Methods

In an IRB-approved study axial T1- and T2-weighted MR images were acquired at term-equivalent age for a preterm cohort of 108 neonates. A method for automatic probabilistic segmentation of the images into eight cerebral tissue classes was developed: cortical and central grey matter, unmyelinated and myelinated white matter, cerebrospinal fluid in the ventricles and in the extra cerebral space, brainstem and cerebellum. Segmentation is based on supervised pixel classification using intensity values and spatial positions of the image voxels. The method was trained and evaluated using leave-one-out experiments on seven images, for which an expert had set a reference standard manually. Subsequently, the method was applied to the remaining 101 scans, and the resulting segmentations were evaluated visually by three experts. Finally, volumes of the eight segmented tissue classes were determined for each patient.

Results

The Dice similarity coefficients of the segmented tissue classes, except myelinated white matter, ranged from 0.75 to 0.92. Myelinated white matter was difficult to segment and the achieved Dice coefficient was 0.47. Visual analysis of the results demonstrated accurate segmentations of the eight tissue classes. The probabilistic segmentation method produced volumes that compared favorably with the reference standard.

Conclusion

The proposed method provides accurate segmentation of neonatal brain MR images into all given tissue classes, except myelinated white matter. This is the one of the first methods that distinguishes cerebrospinal fluid in the ventricles from cerebrospinal fluid in the extracerebral space. This method might be helpful in predicting neurodevelopmental outcome and useful for evaluating neuroprotective clinical trials in neonates.