Lactate dehydrogenase (LDH) gene duplication during chordate evolution: the cDNA sequence of the LDH of the tunicate Styela plicata

L-Lactate dehydrogenase (L-LDH, E.C. 1.1.1.27) is encoded by two or three loci in all vertebrates examined, with the exception of lampreys, which have a single LDH locus. Biochemical characterizations of LDH proteins have suggested that a gene duplication early in vertebrate evolution gave rise to Ldh-A and Ldh-B and that an additional locus, Ldh-C arose in a number of lineages more recently. Although some phylogenetic studies of LDH protein sequences have supported this pattern of gene duplication, others have contradicted it. In particular, a number of studies have suggested that Ldh-C represents the earliest divergence among vertebrate LDHs and that it may have diverged from the other loci well before the origin of vertebrates. Such hypotheses make explicit statements about the relationship of vertebrate and invertebrate LDHs, but to date, no closely related invertebrate LDH sequences have been available for comparison. We have attempted to provide further data on the timing of gene duplications leading to multiple vertebrate LDHs by determining the cDNA sequence of the LDH of the tunicate Styela plicata. Phylogenetic analyses of this and other LDH sequences provide strong support for the duplications giving rise to multiple vertebrate LDHs having occurred after vertebrates diverged from tunicates. The timing of these LDH duplications is consistent with data from a number of other gene families suggesting widespread gene duplication near the origin of vertebrates. With respect to the relationships among vertebrate LDHs, our data are not consistent with previous claims that Ldh-C represented the earliest divergence. However, the precise relationships among some of the main lineages of vertebrate LDHs were not resolved in our analyses.      

Activation of a Latent Measles Virus Infection in Hamster Cells

The characteristics of infectious measles virus released from latently infected hamster embryo fibroblast cells are described. Low levels of virus were released spontaneously when the cultures were incubated at 37 C; this phenomenon was observed 19 passages after the cells had been exposed to the virus and has continued through cell passage 45. The virus yield could be significantly increased by cocultivation of the hamster cells with BSC-1 cells or incubation of the latently infected cells at 33.5 C rather than at 37 C. Measles virus released after cocultivation demonstrated increased cytopathology in cell culture and reduced temperature sensitivity when compared to the virus released at 33.5 C. After cell passage 45, there was an increase in spontaneous release of virus. However, the viruses recovered by cocultivation or temperature release after cell passage 45 were nearly identical. These observations suggest a possible mechanism for measles virus activation in cells latently infected with this virus.     

Lethal and sub‐lethal impacts of respiratory cryptosporidiosis on Red Grouse,a wild gamebird of economic importance

Respiratory cryptosporidiosis was first diagnosed in Red Grouse Lagopus lagopus scotica, the UK subspecies of Willow Ptarmigan, in 2010. In the next 3 years, respiratory infection by Cryptosporidium baileyi had manifested itself in Grouse on half the moors in northern England and 80% of moors in the North Pennine Hills. In this first account of the impact of respiratory cryptosporidiosis on the population dynamics of a wild bird we fitted 111 diseased and 67 healthy Grouse with radio‐transmitters at two North Pennine moors where disease prevalence averaged 8.1% and monitored their survival and fecundity between autumn 2013 and autumn 2015. Six‐month natural survival rates (excluding shooting) were 0.70 in healthy Grouse, but only 0.44 in diseased females, and 0.22 in diseased males. Some 39% of diseased birds died from their infection, whereas 28% of healthy birds were shot. A similar proportion of each group were killed by predators, either by Stoat Mustela erminea or raptors. Diseased females bred 8 days later than healthy females, but clutch size, egg volume and nesting success did not differ in relation to disease status. Productivity was 43% lower among pairs with a diseased member than in healthy pairs, but appeared impaired only if the female was diseased, not the male. Differences in productivity were related to chick survival rather than the proportion of pairs that reared broods, with chick survival being lower in the 10 days after hatching and again when chicks were 20–50 days old. This latter period was when respiratory infection among chicks was first noticed and the onset of infection may have been a contributing factor to higher mortality during this period. Described levels of respiratory infection reduced the number of birds available to shoot in August by 6%, which represented a mean annual loss of £0.9 million in revenue across managed grouse moors. Likely reductions in shoot economics could escalate should prevalence increase. This disease is a welfare concern and potentially a conservation concern, too, should infection cross to other bird species occupying the same moors.     

Expressed sequence tags (ESTs) from desiccated Tortula ruralis identify a large number of novel plant genes.

The desiccation-tolerant moss Tortula ruralis [Hedw.] Gaerten., Meyer & Scherb. has both a constitutive protection system and an active rehydration induced recovery mechanism apparently unique to bryophytes. Immediately following rehydration, desiccated T.ruralis gametophytes produce a set of polypeptides whose synthesis is unique to the rehydrated state. We report the construction of a cDNA expression library from the polysomal mRNA of desiccated gametophytes and the single-pass sequencing of randomly selected clones. 152 expressed sequence tags (ESTs) were generated representing more than 60,000 bp of non-redundant DNA sequence. 44 ESTs (29%) demonstrated significant homology to previously identified nucleotide and/or polypeptide sequences, such as ribosomal proteins, desiccation-related peptides, early light-inducible proteins and a V-type ATPase. Analysis of a subset of these homologous ESTs reveals that codon preference in T.ruralis is similar to that of vascular plants, particularly the Magnoliopsida. 108 ESTs (71%) demonstrated no significant homology to deposited sequences and represent a large number of novel plant genes. Analysis of these ESTs will define the range of genes involved in cellular repair and recovery and may provide greater insight to the complex phenotype of vegetative desiccation-tolerance.     

Composting of pulp and paper mill fly ash with wastewater treatment sludge

Wastewater treatment sludge and power boiler fly ash were combined and composted in mixed and static windrows 50 m long, 4 m high and 6 m wide. Moisture content was maintained above 50%. The final compost had a pH of 8.5, contained high concentrations of specific nutrients, and an average C:N ratio of 43:1. All metal, PCB, chlorophenol and PAH concentrations were below levels stipulated by local regulations. Over the first 8 weeks of the composting period dioxin concentration decreased by 45% to 41 pg/g TEQ. Leachate tests indicated minimal (<0.1 mg/l Cu and Pb; <50 mg/l Na, P, and SO₄⁻²) leaching of contaminants from the composted material. Application of compost (8 cubic yards/acre) at a sod farm improved soil characteristics as measured by a number of parameters. The dioxin concentration in the final soil/compost mixture was 3 pg/g TEQ, allowing the soil/compost mixture to be classified as agricultural soil. It was concluded that composting produced an acceptable soil conditioner attractive for large volume users of inexpensive soil material (sod farms, golf courses, land reclamation sites).     

Role of biotin-containing membranes and nuclear distribution in differentiating human endometrial cells

Human Ishikawa endometrial cells form domes when confluent monolayers are stimulated with fresh fetal bovine serum. Extensive structural and biochemical changes have been detected during the approximately 30 h differentiation period. The earliest detectable change involves the formation of multinucleated structures and the appearance of “granules” that stain for biotin within those structures. Nuclei become associated with each other and are ultimately enclosed within a biotin-containing membrane. Aggregated membrane-sheathed nuclei and the cells containing them begin to elevate from the dish as biotin staining becomes apparent in apical membranes. The elevated structures are called predomes and consist of one or more very large cells containing the sheathed nuclei. Apical membranes of these unusual cells extend far out into the medium in structures that resemble endometrial pinopods. A lumen under the elevated cells fills with transcytosed fluid. As differentiation proceeds, highly concentrated chromatin material that was flattened against apical and lateral membranes of the predome cells begins to disperse. Small mononuclear cells evolve from larger predome cells. Apical membranes of predome and dome cells continue to stain for biotin. Gel electrophoresis of SDS-solubilized biotin-containing membranes, followed by Western blot analysis using avidin-linked peroxidase, resulted in three stained bands with molecular weights similar to those of the mitochondrial carboxylases: propionyl carboxylase, methylmalonyl carboxylase, and pyruvate carboxylase. J. Cell. Biochem. 71:400–415, 1998. © 1998 Wiley-Liss, Inc.     

Endonuclease-like activity of heme proteins

Heme proteins, metmyoglobin, methemoglobin, and metcytochrome c showed unusual affinity for double-stranded DNA. Calorimetric studies show that binding of methemoglobin to calf thymus DNA (CTDNA) is weakly endothermic, and the binding constant is 4.9+/-0.7x10(5) M(-1). The Soret absorption bands of the heme proteins remained unchanged, in the presence of excess CTDNA, but a new circular dichroic band appeared at 210 nm. Helix melting studies indicated that the protein-DNA mixture denatures at a lower temperature than the individual components. Thermograms obtained by differential scanning calorimetry of the mixture indicated two distinct transitions, which are comparable to the thermograms obtained for individual components, but there was a reduction in the excess heat capacity. Activation of heme proteins by hydrogen peroxide resulted in the formation of high valent Fe(IV) oxo intermediates, and CTDNA reacted rapidly under these conditions. The rate was first-order in DNA concentration, and this reactivity resulted in DNA strand cleavage. Upon activation with hydrogen peroxide, for example, the heme proteins converted the supercoiled pUC18 DNA into nicked circular and linear DNA. No reaction occurred in the absence of the heme protein, or hydrogen peroxide. These data clearly indicate a novel property of several heme proteins, and this is first report of the endonuclease-like activity of the heme proteins.