The effects of brain-stem section on the breathing and behavioural response to morphine in the fetal sheep

In the unanesthetized fetal sheep the administration of morphine causes initial apnoea followed by hyperpnoea. We thought that a section of the brain at midcollicular level might separate these two effects. Therefore we sectioned the brain stem of five fetuses at 132 +/- 1 (SEM) days of gestation and compared their responses to morphine (17 experiments) with that observed in seven intact fetuses at similar gestational ages (15 experiments). Brain stem sections were confirmed morphologically and histologically. Morphine, 1 mg/kg was injected in the fetal jugular vein during low-voltage electrocortical activity (ECoG). We measured ECoG, eye movements, diaphragmatic activity, blood pressure and amniotic pressure. Sectioned fetuses before the administration of morphine had a complete dissociation between ECoG and breathing activity. With the administration of morphine we found: (i) the length of the apnoea was 139.8 +/- 15.5 min in sectioned fetuses and 17.0 +/- 5.8 min in intact fetuses (P less than 0.01); and (ii) there was no hyperpneic response in the sectioned fetus whereas the length of hyperpnoea in the intact group was 99.1 +/- 11.8 min (P less than 0.001). The results support the idea of two central distinct areas of action of morphine in the fetal brain. The absence of hyperpnoea in the sectioned fetuses suggests that neurons inhibiting the 'respiratory neurons' are located rostrally to the mid-collicular line.     

Site-directed mutagenesis of beta-lactamase I. Single and double mutants of Glu-166 and Lys-73.

Two single mutants and the corresponding double mutant of beta-lactamase I from Bacillus cereus 569/H were constructed and their kinetics investigated. The mutants have Lys-73 replaced by arginine (K73R), or Glu-166 replaced by aspartic acid (E166D), or both (K73R + E166D). All four rate constants in the acyl-enzyme mechanism were determined for the E166D mutant by the methods described by Christensen, Martin & Waley [(1990) Biochem. J. 266, 853-861]. Both the rate constants for acylation and deacylation for the hydrolysis of benzylpenicillin were decreased about 2000-fold in this mutant. In the K73R mutant, and in the double mutant, the rate constants for acylation were decreased about 100-fold and 10,000-fold respectively. All three mutants also had lowered values for the rate constants for the formation and dissociation of the non-covalent enzyme-substrate complex. The specificities of the mutants did not differ greatly from those of wild-type beta-lactamase, but the hydrolysis of cephalosporin C by the K73R mutant gave 'burst' kinetics.     

Regional Reductions of Transketolase in Thiamine-Deficient Rat Brain

Abstract: Thiamine deficiency impairs oxidative metabolism and causes metabolic encephalopathy. An early reduction in transketolase (TK) activity may be an important pathogenic event. To assess the role of TK, we have delineated the regional/cellular distribution of TK protein and mRNA in adult rat brain in pyrithiamine-induced thiamine deficiency. TK activity declined in both vulnerable and spared regions. Immunoblots showed a parallel reduction of TK protein. With a few exceptions, immunocytochemistry indicated an overall decline of TK immunoreactivity and the decrease was not specific to vulnerable areas. In contrast to the pronounced, general decline of TK protein, in situ hybridization revealed a regional decrease of 0–25% of TK mRNA in thiamine deficiency. Northern blots indicated a similar level of TK mRNA in whole brain in thiamine deficiency. These results show that the decline of TK activity results from a proportional decrease of TK protein, and the deficiency may be due to an instability of TK protein or an inhibition of TK mRNA translation. The lack of correlation of the distribution, and the absence of specific alteration, of TK in affected regions suggest that the reduced TK may not be linked directly to selective vulnerability in thiamine deficiency.     

Molecular structure of rare but geographically widespread sn-glycerol-3-phosphate dehydrogenase ‘ultra-fast’ electrophoretic alleles in Drosophila melanogaster

Six naturally occurring but rare alleles of sn-glycerol-3-phosphate dehydrogenase (Gpdh) in Drosophila melanogaster have been investigated in this study. They all belong to a class of Gpdh ^UF (ultra-fast) alleles, because their electrophoretic mobilities are faster than that of the Gpdh ^F (fast) allele. The Gpdh ^UF variants are widespread, and have been reported from five continents. DNA sequence analysis has shown that the change in electrophoretic mobility was in each allele caused by a single amino acid residue substitution in the encoded protein. In the Xiamen ^UF allele it is a substitution of lysine (AAA) to asparagine (AAT) in exon 1 (residue 3). An asparagine (AAT) to aspartate (GAT) change was found in exon 6 (residue 336) in the Iowa ^UF and Netherlands ^UF alleles. The mobility of the Raleigh ^UF allele was altered by a valine (GTG) to glutamate (GAG) substitution in exon 3 (residue 76). Two mutations were detected in the Brazzaville ^UF allele: a lysine (AAG) to methionine (ATG) substitution in exon 2 (residue 68) is responsible for the ultra-fast phenotype of this variant, while a tyrosine (TAT) to phenylalanine (TTT) substitution in exon 4 (residue 244) is not expected to alter the electrophoretic mobility of the encoded protein. These results indicate that the Gpdh ^UF alleles originate from different mutational events, and only two of them — Iowa ^UF and Netherlands ^UF — might share a common ancestry. The GPDH activity of the Iowa ^UF allele is intermediate between those of the Gpdh ^S and Gpdh ^F control stocks. The other Gpdh ^UF variants have lower activities than the controls: Xiamen ^UF -83%, Raleigh ^UF -80% and Brazzaville ^UF -73% of the Gpdh ^F control.     

An experimental demonstration of trophic interactions affecting water quality of Rice Lake, Ontario (Canada)

Eight cylindrical enclosures (3 m diameter, 2.7 m long, V = 20m³) were installed in eutrophic Rice Lake (Ontario, Canada) in late spring of 1987. Fish (yearling yellow perch (Perca flavescens) and macrophytes (Potamogeton crispus) presence and absence were set at the beginning of the experiment to yield four combinations of duplicate treatments. The purpose of the experiment was to determine if the phytoplankton, zooplankton, macrophytes and fish species resident in the lake interact to influence water quality (major ions, phosphorus, algal densities and water clarity).The presence of fish was associated with: (1) decreased biomass of total zooplankton, (2) decreased number of species in the zooplankton, (3) decreased average size of several zooplankton taxa, (4) higher total phosphorus concentrations, (5) higher phytoplankton and chlorophyll a concentrations, (6) lower water clarity, (7) lower potassium levels during macrophyte die-back, (8) lower pH and higher conductivity in the presence of macrophytes. Biomass of large Daphnia species (but not total zooplankton) was highly correlated with the algal response (r ² = 0.995) and was associated with reduced biomass of several algal taxa including some large forms (Mougeotia, Oedogonium) and several colonial blue-green algae. However, no significant control of late summer growth of the bloom-forming blue-green alga Anabaena planctonica Brun. was achieved by the Daphnia presence-fish absence treatment. Release of phosphorus to the water column during the die-back of P. crispus was not an important phenomenon.     

Characterization of exochelins of Mycobacterium avium: evidence for saturated and unsaturated and for acid and ester forms.

Mycobacterium avium secretes iron-binding siderophores called exochelins. The exochelins from M. avium have previously been reported to have unsaturated side chains that terminate in carboxylic acid. In contrast, our data show the side chains to be both saturated and unsaturated and to terminate with either a carboxylate or methyl ester.     

Distribution of sewage indicated by Clostridium perfringens at a deep-water disposal site after cessation of sewage disposal.

Clostridium perfringens, a marker of domestic sewage contamination, was enumerated in sediment samples obtained from the vicinity of the 106-Mile Site 1 month and 1 year after cessation of sewage disposal at this site. C. perfringens counts in sediments collected at the disposal site and from stations 26 nautical miles (ca. 48 km) and 50 nautical miles (ca. 92 km) to the southwest of the site were, in general, more than 10-fold higher than counts from an uncontaminated reference site. C. perfringens counts at the disposal site were not significantly different between 1992 and 1993, suggesting that sewage sludge had remained in the benthic environment at this site. At stations where C. perfringens counts were elevated (i.e., stations other than the reference station), counts were generally higher in the top 1 cm and decreased down to 5 cm. In some cases, C. perfringens counts in the bottom 4 or 5 cm showed a trend of higher counts in 1993 than in 1992, suggesting bioturbation. We conclude that widespread sludge contamination of the benthic environment has persisted for at least 1 year after cessation of ocean sewage disposal at the 106-Mile Site.     

Rapid purification of the oxygenase component of toluene dioxygenase from a polyol-responsive monoclonal antibody.

A monoclonal antibody designated 302 beta that is specific for the beta subunit of the oxygenase component (ISPTOL) of toluene dioxygenase from Pseudomonas putida F1 was used to prepare an immunoaffinity column. ISPTOL in cell extracts of Escherichia coli JM109(pDTG611) bound to the column, and an enzyme-linked immunosorbent elution-screening assay with different combinations of polyols and kosmotropic anions was used to determine the conditions necessary for recovery of active enzyme. Elution from an 8-ml antibody column with 50 mM 2-(N-morpholino)ethanesulfonate buffer (pH 6.8) containing 50% ethylene glycol, 1.0 M ammonium sulfate, 1.0 mM dithiothreitol, and 0.2 mM ferrous ammonium sulfate gave approximately 2 mg of ISPTOL with a specific activity that was more than 300 times the specific activity previously obtained.