Pattern of hyperphagia in immature three-spined sticklebacks after short-term food deprivation

Juvenile three-spined sticklebacks Gasterosteus aculeatus were fed live enchytraeid worms, and mean daily ad libitum consumption in the absence of periods of deprivation was 11·7% of initial body weight. Then, six groups of five replicate fish were subjected to 1, 3 and 6 days without food. Each period of deprivation was followed by 1 day of ad libitum feeding. The sequential order of deprivations differed between groups. Over the sequence of deprivations and re-feedings, mean cumulative consumption did not differ between groups. Mean daily consumption was 19·8% of initial body weight after a 1-day deprivation, 22·3% after 3 days and 19·5% after 6 days. However, consumption in the 24 h after a given length of deprivation also depended on the prior history of deprivation. Specific growth rate over the experiment did not differ between groups. The results provide evidence for an effective regulation of appetite in growing sticklebacks.     

Degradation of monomeric and fibrillar type III collagens by human skin collagenase. Kinetic constants using different animal substrates

Human skin collagenase activity was examined against type III collagens, in both soluble and fibrillar form, from different animal species. In either form, human, dog, and cat type III were degraded 10- to 30-fold faster than was that from guinea pig and nearly 100-fold more readily than chick type III. These differences in susceptibility were mirrored by essentially identical differences in the rate of trypsin cleavage of the same substrates. Human, dog, and cat type III were cleaved most rapidly by trypsin, guinea pig III more slowly, and chick III was completely resistant to the serine protease. Arrhenius plots, relating enzyme activity to temperature, revealed differences in the various type III substrates consistent with their collagenase and trypsin susceptibilities. Human, dog, and cat type III collagens yielded nonlinear plots, with accompanying activation energies which decreased at temperatures above 26 degrees C; guinea pig type III displayed a plot which deviated only slightly from linearity while the plot for chick type III was completely linear. These data strongly suggest that type III collagens display substantial variability in the stability of the helix at or near the collagenase cleavage site. The susceptibility of these type III substrates as reconstituted fibrils was also examined. The relative rates of degradation of these substrates by collagenase, and by trypsin, were the same as those observed in solution. The absolute rates of degradation of collagen in fibrillar form, however, were massively lower than predicted by extrapolation from solution values. This reduction in rate is even greater for type III than for type I collagens. Thus, whereas in solution type III substrates are cleaved much faster than type I collagens, in fibrillar form these differences are less than 2-fold. These data, together with values for activation energies and deuterium isotope effects on type III fibrillar substrates, reinforce the concept that helical integrity near the collagenase cleavage site is a major specifier of the rate of collagenase activity. Furthermore, the data suggest that the exclusion of water accompanying the tight packing of monomers into fibrils presents a major energy barrier to collagenase activity, which is particularly large for type III collagen.     

The Klebsiella aerogenes glutamate dehydrogenase (gdhA) gene: cloning, high-level expression and hybrid enzyme formation in Escherichia coli

Summary The NADP-dependent glutamate dehydrogenase gene of Klebsiella aerogenes was cloned in E. coli in the expression plasmid pRK9. The cloned gene shows a high level of expression in E. coli in the hybrid plasmid pKG3 and such expression is independent of the vector promoter, as shown by experiments in which the promoter was deleted. Active hybrid GDH hexamers were shown in cell-free extracts of an E. coli strain carrying cloned gdhA genes of both E. coli and K. aerogenes. The nucleotide sequence of the N-terminal coding region of the K. aerogenes gdhA gene was determined and found to be strongly homologous with that of E. coli.Abbreviations GDH glutamate dehydrogenase - PMS phenazine methosulphate - MTT 3-(4,5-Dimethylthiazolyl-2)-2,5-diphenyltetrazolium-bromide - PMSF phenylmethylsulphonylfluoride - SSC standard saline citrate - DTT dithiothreitol - bp base pairs - kbp kilo base pairs - dNTP deoxynucleoside triphosphate     

Characterization of the umu-complementing operon from R391.

In addition to conferring resistances to antibiotics and heavy metals, certain R factors carry genes involved in mutagenic DNA repair. These plasmid-encoded genes are structurally and functionally related to the chromosomally encoded umuDC genes of Escherichia coli and Salmonella typhimurium. Three such plasmid operons, mucAB, impCAB, and samAB, have been characterized at the molecular level. Recently, we have identified three additional umu-complementing operons from IncJ plasmid R391 and IncL/M plasmids R446b and R471a. We report here the molecular characterization of the R391 umu-complementing operon. The nucleotide sequence of the minimal R plasmid umu-complementing (rum) region revealed an operon of two genes, rumA(R391) and rumB(R391), with an upstream regulatory signal strongly resembling LexA-binding sites. Phylogenetic analysis revealed that the RumAB(R391) proteins are approximately equally diverged in sequence from the chromosomal UmuDC proteins and the other plasmid-encoded Umu-like proteins and represent a new subfamily. Genetic characterization of the rumAB(R391) operon revealed that in recA+ and recA1730 backgrounds, the rumAB(R391) operon was phenotypically indistinguishable from mucAB. In contrast, however, the rumAB(R391) operon gave levels of mutagenesis that were intermediate between those given by mucAB and umuDC in a recA430 strain. The latter phenotype was shown to correlate with the reduced posttranslational processing of the RumA(R391) protein to its mutagenically active form, RumA'(R391). Thus, the rumAB(R391) operon appears to possess characteristics that are reminiscent of both chromosome and plasmid-encoded umu-like operons.     

Identification of sex in Cetaceans by multiplexing with three ZFX and ZFY specific primers

We sequenced 540 nucleotides of the last exon in the ZFY/ZFX gene in two males and two females for eight cetacean species; four odontocetes (toothed whales) and four mysticetes (baleen whales). Based upon the obtained nucleotide sequences, we designed two sets of oligonucleotide primers for specific amplification of the ZFX and the ZFY sequence in odontocetes and mysticetes, respectively. Each primer set consisted of three oligonucleotides; one forward-orientated primer, which anneals to the ZFY as well as the ZFX sequence, and two reverse-orientated primers that anneal to either the ZFX or the ZFY sequence. The resulting two amplification products (specific for the ZFY and ZFX sequences) can be distinguished by gel-electrophoresis through 2% NuSieve™. The accuracy of the technique was tested by determination of gender in 214 individuals of known sex. Finally we applied the technique to determine the sex of 3570 cetacean specimens; 2284 humpback whales, 315 fin whales, 37 blue whales, 7 minke whales, as well as 592 belugas, 335 narwhals and 25 harbour porpoises.     

Efficacy and deficiencies of rapid biomonitoring in biodiversity conservation: a case study in South Africa

Rapid biomonitoring protocols, using biotic indices based on macroinvertebrate diversity to assess river ecosystem health, are widely used globally. Such quick assessment techniques are lauded for the rapid results obtained and the relatively easy protocol used to achieve an answer. However, do such quick assessments of water quality give enough information about ecosystems? Are important details being overlooked? When should a full faunal survey be used in preference? Important research programmes, including environmental impact studies, often misuse biomonitoring techniques, making influential management decisions using superficial, low-level data obtained using biomonitoring tools, inappropriate to address those management objectives. The value of using biomonitoring as a quick tool, versus a more detailed faunal assessment, is considered here. The assessment of teloganodid mayfly fauna occurring in South African rivers provides an example of the value of detailed studies versus superficial family level investigations, showing that a rapid biomonitoring approach should not be used as a shortcut when a more detailed survey is needed. Each situation should be assessed for its own merit in a given set of project circumstances. A checklist of criteria is presented, giving guidance on when rapid biomonitoring alone is valuable and when more detailed assessments would give a more relevant result.     

Reversal of fentanyl/fluanisone neuroleptanalgesia in the rabbit using mixed agonist/antagonist opioids

The reversal of the neuroleptanalgesic combination of fentanyl/fluanisone using mixed agonist/antagonist opioids has been investigated in the rabbit. All of the compounds studied (naloxone, nalbuphine, meptazinol, butorphanol, buprenorphine, pentazocine, doxapram) reversed the respiratory depression and sedation produced by fentanyl/fluanisone. Fentanyl/fluanisone produced profound analgesia for 180 min, which was rapidly and completely antagonized by naloxone. The mixed agonist/antagonist opioids produced a reduction in the degree of analgesia but, in contrast to naloxone, analgesic activity persisted from 120 min (meptazinol) to 420 min (buprenorphine). Administration of buprenorphine to rabbits anaesthetized with fentanyl/fluanisone and midazolam confirmed that the reversal of respiratory depression was accompanied by the return of arterial pH, PCO2 and PCO2 to preanaesthetic values. The use of neuroleptanalgesic anaesthetic regimens, which have been shown to provide effective surgical anaesthesia, combined with reversal using a mixed agonist/antagonist opioid to provide postoperative analgesia, appears to be a valuable refinement of current laboratory animal anaesthetic practice.