Immobilization of β-galactosidase and other enzymes onto p-amino-carbanilated cellulose derivatives

β-Galactosidase and other enzymes were immobilized on p-amino-carbanilated derivatives of cellulose and methylol cellulose using the diazo method and through glutaraldehyde. The optimum conditions for coupling cellulose tri-(p-amino-carbanilate) (CTAC) to β-galactosidase were established. The diazo coupling method with CTAC gave greater activity than with glutaraldehyde when coupled to β-galactosidase (Escherichia coli). The stability of the CTAC–β-galactosidase system was examined. The disubstituted p-amino-carbanilate derivative (CDAC) gave a lower activity, whereas the methylol analog (MCTAC) gave slightly greater activity. The CTAC was also used to immobilize glucose oxidase, trypsin, pepsin, and papain.     

Treatment of pregnant gilts with a prostaglandin analogue, Cloprostenol, to control oestrus and fertility

Oestrus was induced 4-7 days after treatment with Cloprostenol (ICI 80,996) in gilts between 12 and 40 days pregnant. Fertility at this synchronized oestrus was good (85%).     

The isolation and identification of the female sex pheromones of the red bollworm moth, Diparopsis castanea

The female sex pheromones of the red bollworm moth, Diparopsis castanea, were isolated by solvent extraction of the terminal abdominal segments of the female moth followed by purification of the solvent extract by liquid-solid and gas chromatography. The pheromones were identified by gas chromatographic analysis, micro-ozonolysis, infra-red and mass spectroscopy, and comparison with synthetic material. The major pheromone was 9,11-dodecadien-1-yl acetate (80 : 20 trans : cis). Minor components identified were trans 9-dodecen-1-yl acetate, 11-dodecen-1-yl acetate, and dodecan-1-yl acetate.     

Diffusible signals and fasciculated growth in reticulospinal axon pathfinding in the hindbrain

We have addressed the control of longitudinal axon pathfinding in the developing hindbrain, including the caudal projections of reticular and raphe neurons. To test potential sources of guidance signals, we assessed axon outgrowth from embryonic rat hindbrain explants cultured in collagen gels at a distance from explants of midbrain-hindbrain boundary (isthmus), caudal hindbrain, or cervical spinal cord. Our results showed that the isthmus inhibited caudally directed axon outgrowth by 80% relative to controls, whereas rostrally directed axon outgrowth was unaffected. Moreover, caudal hindbrain or cervical spinal cord explants did not inhibit caudal axons. Immunohistochemistry for reticular and raphe neuronal markers indicated that the caudal, but not the rostral projections of these neuronal subpopulations were inhibited by isthmic explants. Companion studies in chick embryos showed that, when the hindbrain was surgically separated from the isthmus, caudal reticulospinal axon projections failed to form and that descending pioneer axons of the medial longitudinal fasciculus (MLF) play an important role in the caudal reticulospinal projection. Taken together, these results suggest that diffusible chemorepellent or nonpermissive signals from the isthmus and substrate-anchored signals on the pioneer MLF axons are involved in the caudal direction of reticulospinal projections and might influence other longitudinal axon projections in the brainstem.     

Hepatitis Delta Virus RNA Editing Is Highly Specific for the Amber/W Site and Is Suppressed by Hepatitis Delta Antigen

RNA editing at adenosine 1012 (amber/W site) in the antigenomic RNA of hepatitis delta virus (HDV) allows two essential forms of the viral protein, hepatitis delta antigen (HDAg), to be synthesized from a single open reading frame. Editing at the amber/W site is thought to be catalyzed by one of the cellular enzymes known as adenosine deaminases that act on RNA (ADARs). In vitro, the enzymes ADAR1 and ADAR2 deaminate adenosines within many different sequences of base-paired RNA. Since promiscuous deamination could compromise the viability of HDV, we wondered if additional deamination events occurred within the highly base paired HDV RNA. By sequencing cDNAs derived from HDV RNA from transfected Huh-7 cells, we determined that the RNA was not extensively modified at other adenosines. Approximately 0.16 to 0.32 adenosines were modified per antigenome during 6 to 13 days posttransfection. Interestingly, all observed non-amber/W adenosine modifications, which occurred mostly at positions that are highly conserved among naturally occurring HDV isolates, were found in RNAs that were also modified at the amber/W site. Such coordinate modification likely limits potential deleterious effects of promiscuous editing. Neither viral replication nor HDAg was required for the highly specific editing observed in cells. However, HDAg was found to suppress editing at the amber/W site when expressed at levels similar to those found during HDV replication. These data suggest HDAg may regulate amber/W site editing during virus replication.     

Validation of ecological status concepts in UK rivers using historic diatom samples

We assessed the feasibility of using herbarium specimens to validate reference conditions in the UK by comparing diatom community composition of river sites with both recent and historic diatom samples. The question of substrate specificity was addressed by comparing epilithon (stone-derived) and epiphyton (plant-derived) samples from a number of rivers. No significant differences were found between the Trophic Diatom Index (TDI), species richness, species diversity, and percentage of motile valves between paired diatom samples (epilithic and epiphytic) from contemporary samples. Significant differences were recorded between a number of indices derived from analysis of the historic diatom samples on plant material sampled pre-1930 compared with diatoms from stones collected post-1990 from the same river location. The TDI, mean species richness, and species diversity and percentage of motile valves and nutrient tolerant valves were all significantly greater in the contemporary samples (p ≤ 0.05). The percentage of nutrient sensitive valves was significantly lower in the contemporary samples (p ≤ 0.05).The relative abundance of Achnanthidium minutissimum and Cocconeis placentula var. lineata was significantly greater on the herbarium material compared to matched contemporary samples. Calculated values for the TDI (43 ± 3) expected at reference conditions were similar to the observed TDI values derived from herbarium material (44 ± 12) showing no significant deviation in ecological status.     

Glutamine cycling in isolated working rat heart

To what extent does glutamine turnover keep pace with oxidative metabolism in the rat heart? To address this question, the following groups of substrates were presented to the isolated, working rat heart: 1) glucose (5 mM), insulin (40 microU/ml), and [2-13C]acetate (5 mM; high workload, n = 5); 2) pyruvate (2.5 mM) and [2-13C]acetate (5 mM; normal workload, n = 5); or 3) propionate (1 mM) and [2-13C]acetate (2.5 mM; normal workload, n = 3). In a subset of these experiments, the exchange of glutamate and glutamine was quantified by separation with ion exchange chromatography and analysis by GC-MS. There was an apparent equilibration of mass isotopomers of glutamate and glutamine after 50 min of perfusion, although the extent of equilibration was not determined. The fractional enrichment in glutamine was 31% of the enrichment of glutamate with the three different perfusates. From high-resolution nuclear magnetic resonance spectra, we found a ratio of glutamine to glutamate content of 94.1, 53.4, and 96.9%, respectively, for each experimental group. In experiments for which l-[1-13C]glutamine (5 mM) was included in the perfusate of group 2, [1-13C]glutamine was detected in the heart, but transfer of 13C from glutamine to glutamate was not detected (n = 4). We conclude that, in the perfused working heart, production of glutamine by amidation of glutamate takes place and can be detected, whereas the reverse process, generation of glutamate from glutamine, remains undetected.