Genetic heterogeneity in neuronal ceroid lipofuscinosis (NCL): Evidence that the late-infantile subtype (Jansky-Bielschowsky disease; CLN2) is not an allelic form of the juvenile or infantile subtypes

The neuronal ceroid lipofuscinoses (NCLs) are a group of inherited neurodegenerative disorders characterized by the accumulation of autofluorescent lipopigment in neurons and other cell types. Inheritance is autosomal recessive. Three main childhood subtypes are recognized: infantile (Haltia-Santavuori disease; MIM 256743), late infantile (Jansky-Bielschowsky disease; MIM 204500), and juvenile (Spielmeyer-Sjögren-Vogt, or Batten, disease; MIM 204200). The gene loci for the juvenile (CLN3) and infantile (CLN1) types have been mapped to human chromosomes 16p and 1p, respectively, by linkage analysis. Linkage analysis of 25 families segregating for late-infantile NCL has excluded these regions as the site of this disease locus (CLN2). The three childhood subtypes of NCL therefore arise from mutations at distinct loci.     

Inhibition of Ca2+-transport ATPase from synaptosomal vesicles by flavonoids

The inhibitory action of the flavonoid quercetin has been examined on the calcium-transport ATPase of synaptosomal vesicles and compared to that of two other flavonoids, morin and rutin. We have found that while quercetin caused a 50% inhibition of calcium transport at a concentration of 15 μM, morin and rutin had similar effects at concentrations of about 200 μM. A similar order of potency was observed also for ATP hydrolysis, though at higher concentrations. Quercetin also strongly inhibited phosphorylation of membrane proteins by ATP in synaptosomal vesicles. Rutin and morin had an almost negligible effect on membrane protein phosphorylation. The order of inhibitory potency of the flavonoids on the Ca²⁺-transport ATPase from synaptosomal vesicles: quercetin > morin > rutin, could be linked to their possible solubility in the membrane lipid phase since: (1) it paralleled their partitioning between a mixture of oil and water; (2) it paralleled their uptake from the reaction mixture by synaptosomal vesicles and phosphatidylcholine liposomes; (3) they had almost equal potency as inhibitors of the water soluble system of histone phosphorylation by protein kinase.     

Altered Maize Endosperm Adp-Glucose Pyrophosphorylases from Revertants of a SHRUNKEN-2-DISSOCIATION ALLELE

Four phenotypically wild-type seeds were obtained from separate Activator-induced events in the Dissociation-inhibited allele sh2-ml (shrunken-2, mutable-1). Endosperm adenosine diphosphoglucose pyrophosphorylase, the enzyme controlled by sh2, was extracted and partially purified from the four revertants and was compared to enzyme produced by the progenitor Sh2 allele and the sh2-m allele.

The revertants contained 50 to 140% of the activity conditioned by the progenitor allele. Each of the revertants appears to be unique as judged by differences in Km(glucose-1-PO₄), 3-phosphoglycerate(3-PGA) activation, and phosphate-inhibition. In one case the reversion event apparently increased the sensitivity of ADP-glucose pyrophosphorylate to 3-PGA activation.

     

Multiple forms of maize endosperm adp-glucose pyrophosphorylase and their control by shrunken-2 and brittle-2

Heat-labile and heat stable forms of ADP-glucose pyrophosphorylase were identified in the maize endosperm. The heat-labile form is destroyed by normal electrophoretic conditions. The heat-stable form corresponds to pyrophosphorylase B. In wild type, 96% of the total activity is heat labile. Both forms are reduced in 11 brittle-2 (bt2) and 12 shrunken-2 (sh2) mutants. The heat-labile form is reduced to a greater extent than is the heat-stable form in each of the 23 mutants. Deletion of sh2 abolishes both forms. The original ratio of the two forms is restored after sh2 function is expressed via transposition of Dissociation from sh2. The possible roles of these genes in the control of ADP-glucose pyrophosphorylase are discussed.     

Fiber types in rat laryngeal muscles and their transformations after denervation and reinnervation.

The intrinsic laryngeal muscles cricothyroid (CT) and thyroarythenoid (TA) differ in myosin expression. CT expresses limb myosin heavy chains (MyHCs) and TA expresses an MyHC found in extraocular (EO) muscles, in addition to limb isoforms. We used immunohistochemical (IHC) analyses with highly specific monoclonal antibodies (MAbs) against various MyHCs to study muscle fiber types in rat CT and TA and to investigate whether nerves to laryngeal muscles control MyHC expression. CT was found to have the full complement of limb fiber types. TA had three major fiber types: 2b/eo, co-expressing 2B and EO MyHCs, 2x/2b, co-expressing 2X and 2B MyHCs, and 2x, expressing 2X MyHC. Type 2a and slow fibers were absent. TA consisted of two divisions: the external division (TA-X), which is homogeneously 2b/eo, and the vocalis division (TA-V), composed principally of 2x and 2b/eo fibers with a minority of 2x/2b fibers. TA-V had two compartments that differ in fiber type composition. At 4 weeks after cutting and re-uniting the recurrent laryngeal nerve (RLN), many 2b/eo fibers in the TA-X began to express 2X MyHC, while EO and 2B MyHC expression in these fibers progressively declined. By 12 weeks, up to 16.5% of fibers in the TA-X were of type 2x. These findings suggest that nerve fibers originally innervating 2x fibers in TA-V and other muscles have randomly cross-innervated 2b/eo fibers in the TA-X and converted them into 2x fibers. We conclude that CT and TA are distinct muscle allotypes and that laryngeal muscle fibers are subject to neural regulation.     

Interference is controlled by segment 2 and possibly by segment 8 of the nondefective interfering influenza virus variant A/FM/1/47-MA.

On mouse adaption of A/FM/1/47, a variant, A/FM/1/47-MA (FM-MA), that had acquired the properties of increased virulence and interference was produced. Coinfection of cells with FM-MA and prototype strains of influenza virus yielded > 100-fold more FM-MA virus than prototype virus, whereas coinfection with the same prototype strains and the parental A/FM/1/47 virus produced equivalent yields, indicating that FM-MA had acquired mutations that confer the property of interference during mouse adaption. FM-MA is a nondefective interfering virus that grows to a high titer in vivo and in vitro. It has previously been shown that segments 4, 7, and 8 and possibly segment 5 account for the increased virulence. In this study we show by genetic analysis of FM-MA x A/HK/1/68 reassortants that segment 2, coding for the polymerase-associated protein PB1, and possibly segment 8, encoding the NS1 and NS2 proteins, control the ability of FM-MA to interfere. Interference could not be overcome by increasing the titer of the coinfecting strain, but delaying FM-MA infection by 4 to 6 h did avoid interference. During interference of A/HK/1/68, protein synthesis was inhibited by less than 65% throughout coinfection. Given the kinetics of interference and the small perturbation in protein synthesis, interference appeared to occur at the level of late genome replication or virus assembly. Virulence and interference in FM-MA were not linked. An interfering avirulent FM-MA x A/HK/1/68 reassortant, E07, was capable of protecting mice against lethal pneumonia due to a virulent noninterfering reassortant, H04.     

Cloning and sequencing of the structural gene for the porin protein of Bordetella pertussis

Bordetella pertussis produces a porin protein which is a prominent outer membrane component found in both virulent and avirulent strains. N-terminal amino acid analysis of purified B. pertussis porin was performed and this amino acid sequence was used to design an oligonucleotide that was then utilized to screen a lambda gt11 library containing randomly sheared fragments of DNA from B. pertussis strain 347. One clone, lambda BpPor, was identified and subcloned into pUC18. A portion of the DNA insert in this subclone, pBpPor1, was sequenced and shown to contain the N-terminal region of the structural porin gene. This truncated gene sequence was used to design an additional oligonucleotide that was used to identify a clone, pBpPor2, which overlapped with pBpPor1 and contained a termination codon. The structural gene deduced from this sequence would encode a 365-amino-acid polypeptide with a predicted mass of 39,103 daltons. The predicted product also contains a signal sequence of 20 residues that is similar to that found in other porin genes. The predicted B. pertussis porin protein sequence contains regions that are homologous to regions found in porins expressed by Neisseria species and Escherichia coli, including the presence of phenylalanine as the carboxy-terminal amino acid. DNA hybridization studies indicated that both virulent and avirulent strains of B. pertussis contain only one copy of this gene and that Bordetella bronchiseptica and Bordetella parapertussis contain a similar gene.