Characterization of ADP-glucose pyrophosphorylase from shrunken-2 and brittle-2 mutants of maize

Electrophoretic characterization of adenosine diphosphate glucose pyrophosphorylase from the developing endosperms of nine shrunken-2 and four brittle-2 mutants revealed that (1) all mutants had low but detectable levels of activity, (2) mutation at either locus decreased activity of pyrophosphorylases A and B, and (3) differences in mobility were not found. However, pyrophosphorylase B extracted from several shrunken-2 and brittle-2 mutants differed from normal in extent of urea denaturation, K _m (glucose-1-phosphate) or type of glucose-1-phosphate saturation kinetics. Pyrophosphorylase B from sh2-m (association of Dissociation with the sh2 locus) appears to differ from normal in K _m (glucose-1-phosphate).This research was supported by the College of Agricultural and Life Sciences and by National Institutes of Health Grant No. 15422.The investigations reported were included in the thesis submitted by L. C. Hannah to the Graduate School, University of Wisconsin, Madison, Wisconsin, in partial fulfillment of requirement for the Ph.D. degree. Laboratory of Genetics Paper No. 1922.     

Adventive plants from ovules and nucelli in Citrus

Summary 1- to 8-week-old ovules and nucelli from three Citrus cultivars—Shamouti and Valencia (Citrus sinensis) oranges and Marsh Seedless (C. paradisi) grapefruit—were cultured in vitro. No embryo differentiation was observed in the explants prior to culture. The Shamouti ovules had degenerated and were apparently unfertilized. Embryoids formed on Murashige and Tucker nutrient medium supplemented with 500 mg/l malt extract. Whole plants developed on the same basal medium supplemented with kinetin and indole-3-acetic acid (IAA), coconut milk or gibberellic acid (GA₃). A higher kinetin/IAA ratio or the addition of coconut milk favoured stem elongation more than root formation while a lower kinetin/IAA ratio favoured root formation and inhibited stem elongation. The addition of GA₃ to the basal medium stimulated rooting and stem elongation. These results can be of aid in mutation research, allowing irradiation at stages prior to embryonic development.     

The cell substratum modulates skeletal muscle differentiation

During chick embryogenesis, massive alterations occur in the migrating cell's substratum, or extracellular matrix. The possibility that some of the components of this milieu play a regulatory role in cell differentiation was explored in a cell-culture system derived from embryonic chick skeletal muscle tissue. In particular, the effects of collagen and the glycosaminoglycans were studied. Collagen is required for muscle cell attachment and spreading onto plastic and glass tissue-culture dishes. A major constituent of the early embryonic extracellular space, hyaluronate (HA), while having no significant effect on collagen-stimulated cell attachment and spreading, was found to inhibit myogenesis. The muscle-specific M subunit of creatine kinase was preferentially inhibited. Control experiments indicated that the inhibition was specifically caused by HA and not by other glycosaminoglycans. A general metabolic inhibition of the cultures was not observed. Muscle cells could bind to HA-coated beads at all stages of differentiation but were inhibited only when HA was added within the first 24 h of culture. Endogenous GAG in the culture is normally degraded during the first 24 h after plating as well; this may parallel the massive degradation of HA that occurs in the early embryo in vivo. These findings suggest a regulatory role for HA in modulating skeletal muscle differentiation, with degradation of an inhibitory component of the cell substratum a requirement for myogenesis.     

Molecular phylogenetic evidence for the monophyly of Fritillaria and Lilium (Liliaceae; Liliales) and the infrageneric classification of Fritillaria

We present phylogenetic analyses of 37 taxa of Fritillaria (Liliaceae), 15 species of Lilium, and several outgroup taxa from Liliaceae s.s. to investigate the generic delimitation of Fritillaria in relation to Lilium as well as infrageneric relationships within Fritillaria. We used DNA sequences from the maturase-coding plastid matK gene and the trnK intron, the intron of the ribosomal protein-coding rpl16 plastid gene, and the nuclear ribosomal internal transcribed spacers (ITS). Phylogenetic analysis using maximum parsimony defined Fritillaria and Lilium (the latter including Nomocharis) as sister taxa. Fritillaria sections Fritillaria and Liliorhiza are supported in part, and some of the most enigmatic species usually included in Fritillaria (sections Petilium and Theresia and the monotypic genus Korolkowia) are closely related. The results support the new classification of Fritillaria proposed by Rix. We postulate independent origins of the underground bulbils found in Fritillaria davidii and the remainder of subgenus Liliorhiza.     

Evaluation of antibiotic resistance analysis and ribotyping for identification of faecal pollution sources in an urban watershed

AIMS: The accuracy of ribotyping and antibiotic resistance analysis (ARA) for prediction of sources of faecal bacterial pollution in an urban southern California watershed was determined using blinded proficiency samples. METHODS AND RESULTS: Antibiotic resistance patterns and HindIII ribotypes of Escherichia coli (n = 997), and antibiotic resistance patterns of Enterococcus spp. (n = 3657) were used to construct libraries from sewage samples and from faeces of seagulls, dogs, cats, horses and humans within the watershed. The three libraries were analysed to determine the accuracy of host source prediction. The internal accuracy of the libraries (average rate of correct classification, ARCC) with six source categories was 44% for E. coli ARA, 69% for E. coli ribotyping and 48% for Enterococcus ARA. Each library's predictive ability towards isolates that were not part of the library was determined using a blinded proficiency panel of 97 E. coli and 99 Enterococcus isolates. Twenty-eight per cent (by ARA) and 27% (by ribotyping) of the E. coli proficiency isolates were assigned to the correct source category. Sixteen per cent were assigned to the same source category by both methods, and 6% were assigned to the correct category. Addition of 2480 E. coli isolates to the ARA library did not improve the ARCC or proficiency accuracy. In contrast, 45% of Enterococcus proficiency isolates were correctly identified by ARA. CONCLUSIONS: None of the methods performed well enough on the proficiency panel to be judged ready for application to environmental samples. SIGNIFICANCE AND IMPACT OF THE STUDY: Most microbial source tracking (MST) studies published have demonstrated library accuracy solely by the internal ARCC measurement. Low rates of correct classification for E. coli proficiency isolates compared with the ARCCs of the libraries indicate that testing of bacteria from samples that are not represented in the library, such as blinded proficiency samples, is necessary to accurately measure predictive ability. The library-based MST methods used in this study may not be suited for determination of the source(s) of faecal pollution in large, urban watersheds.     

Connexin36 mediates spike synchrony in olfactory bulb glomeruli

Neuronal synchrony is important to network behavior in many brain regions. In the olfactory bulb, principal neurons (mitral cells) project apical dendrites to a common glomerulus where they receive a common input. Synchronized activity within a glomerulus depends on chemical transmission but mitral cells are also electrically coupled. We examined the role of connexin-mediated gap junctions in mitral cell coordinated activity. Electrical coupling as well as correlated spiking between mitral cells projecting to the same glomerulus was entirely absent in connexin36 (Cx36) knockout mice. Ultrastructural analysis of glomeruli confirmed that mitral-mitral cell gap junctions on distal apical dendrites contain Cx36. Coupled AMPA responses between mitral cell pairs were absent in the knockout, demonstrating that electrical coupling, not transmitter spillover, is responsible for synchronization. Our results indicate that Cx36-mediated gap junctions between mitral cells orchestrate rapid coordinated signaling via a novel form of electrochemical transmission.     

Natural resistance to liver cold ischemia-reperfusion injury associated with the hibernation phenotype

The success of liver grafts is currently limited by the length of time organs are cold preserved before transplant. Novel insights to improve viability of cold-stored organs may emerge from studies with animals that naturally experience low body temperatures (T(b)) for extended periods. In this study, we tested whether livers from hibernating ground squirrels tolerate cold ischemia-warm reperfusion (cold I/R) for longer times and with better quality than livers from rats or summer squirrels. Hibernators were used when torpid (T(b) < 10 degrees C) or aroused (T(b) = 37 degrees C). Livers were stored at 4 degrees C in University of Wisconsin solution for 0-72 h and then reperfused with 37 degrees C buffer in vitro. Lactate dehydrogenase (LDH) release after 60 min was increased 37-fold in rat livers after 72 h cold I/R but only 10-fold in summer livers and approximately three- to sixfold in torpid and aroused hibernator livers, despite twofold higher total LDH content in livers from hibernators compared with rats or summer squirrels. Reperfusion for up to 240 min had the least effect on LDH release in livers from hibernators and the greatest effect in rats. Compared with rats or summer squirrels, livers from hibernators after 72 h cold I/R showed better maintenance of mitochondrial respiration, bile production, and sinusoidal lining cell viability, as well as lower vascular resistance and Kupffer cell phagocytosis. These results demonstrate that the hibernation phenotype in ground squirrels confers superior resistance to liver cold I/R injury compared with rats and summer squirrels. Because hibernation-induced protection is not dependent on animals being in the torpid state, the mechanisms responsible for this effect may provide new strategies for liver preservation in humans.     

No overall hyposialylation in hereditary inclusion body myopathy myoblasts carrying the homozygous M712T GNE mutation

Hereditary inclusion body myopathy (HIBM) is a unique group of neuromuscular disorders characterized by adult-onset, slowly progressive distal and proximal muscle weakness, which is caused by mutations in UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE), the key enzyme in the biosynthetic pathway of sialic acid. In order to investigate the consequences of the mutated GNE enzyme in muscle cells, we have established cell cultures from muscle biopsies carrying either kinase or epimerase mutations. While all myoblasts carrying a mutated GNE gene show a reduction in their epimerase activity, only the cells derived from the patient carrying a homozygous epimerase mutation present also a significant reduction in the overall membrane bound sialic acid. These results indicate that although mutations in each of the two GNE domains result in an impaired enzymatic activity and the same HIBM phenotype, they do not equally affect the overall sialylation of muscle cells. This lack of correlation suggests that the pathological mechanism of the disease may not be linked solely to the well-characterized sialic acid pathway.