The use of (GACA)4 PCR to sex Old World vultures (Aves: Accipitridae)

A PCR method was developed employing a single primer (GACA)₄ to sex Black vultures ( Aegypius monachus ), Lappet-faced vultures ( Torgos tracheliotus ), and Griffon vultures ( Gyps fulvus ). Using the (GACA)₄ primer several PCR products were generated. One or more PCR products displayed a sex-specific pattern, i.e. they were only present in females (probably corresponding to repetitive DNA on the W chromosome) but absent in males. The sex ratio of 85 Griffon vultures from Spain was almost 1.     

Molecular determinants for the action of general anesthetics at recombinant α2β3γ2γ-aminobutyric acidA receptors

General anesthetics modulate the activity of ligand-gated ion channels including the GABA(A) receptor. Mutational studies mainly on the benzodiazepine-insensitive alpha(2)beta(1(M286W)) and alpha(6)beta(3(N289M))gamma(2) GABA(A) receptors revealed that a serine in transmembrane domain 2 and a methionine in transmembrane domain 3 are essential for the action of most general anesthetics. We investigated whether these residues would similarly be relevant for their action at the benzodiazepine-sensitive GABA receptor subtype, alpha(2)beta(3)gamma(2). We found that not only the N265M but also the M286W mutation nearly abolished the modulatory effect of etomidate. However, the anti-convulsant loreclezole, a structural homologue of etomidate, was inactive on the N265M mutant, but displayed normal modulatory activity on the M286W mutant. Both mutations did not affect the modulatory action of the neurosteroid alphaxalone. The direct action of alphaxalone, however, was dramatically increased in the M286W mutant to about twice the maximal GABA current but not significantly affected in the N265M mutant. These data demonstrate that the structural requirements for modulatory and direct actions of various general anesthetics are distinct. The molecular switches induced by these mutations can be exploited to identify the molecular determinants for the action of general anesthetics.     

Cloning and genetic organization of the gene cluster encoding F71 fimbriae of a uropathogenic Escherichia coli and comparison with the F72 gene cluster

Abstract The gene cluster coding for expression of F7₁ fimbriae of the uropathogenic Escherichia coli strain AD110 has been cloned by a cosmid-cloning procedure. A positive clone was further subcloned to a plasmid of 17.5 kilobases (kb), pPIL110-75. Analysis of pPIL110-75 showed that at least six genes are present encoding proteins with apparent M _rs of 75 000, 36 000, 23 000, 20 000, 17 000 and 14 000. The 20-kDa protein, encoding the F7₁ fimbrial subunit is dispensable for expression of the MRHA phenotype. Complementation experiments of mutants in the F7₂ gene cluster by gene products of the F7₁ gene cluster show that the two gene clusters are related.     

Response of plankton communities to whole-lake Ca(OH)2 and CaCO3 additions in eutrophic hardwater lakes

1. The impact of whole-lake lime (slaked lime, Ca(OH)₂, and/or calcite, CaCO₃) addition on plankton communities was evaluated in eutrophic hardwater lakes on the North American Boreal Plain.
2. Two lakes received a single treatment of lime (Ca(OH)₂ at 74 or 107 mg L^–1), two lakes received multiple treatments with Ca(OH)₂ and/or CaCO₃ (5–78 mg L^–1), and four lakes were untreated and served as reference systems.
3. Over the long-term (> 1 year), phytoplankton biomass was reduced in multiple-dose lakes, but not in single-dose lakes. Cyanobacteria typically dominated the algal community in the years before, during and after lime treatment in both single- and multiple-dose lakes.
4. In the single-dose lakes, randomized intervention analysis showed no significant change in the biomass of zooplankton after lime addition.     

Non-association between Rfp-Y major histocompatibility complex-like genes and susceptibility to Marek's disease virus-induced tumours in 63× 72 F2 intercross chickens

Marek's disease (MD) is a lymphoproliferative disease caused by a member of the herpesvirus family, and the best understood genetic resistance to MD involves the chicken major histocompatibility complex (MHC) B -complex. Preliminary observations have suggested that MHC-like Rfp-Y genes might also influence the incidence of MD. This study describes the differentiation and definition of unique Rfp-Y genes in inbred lines 6₃ and 7₂, lines that possess identical B -complex genes, but that are resistant or susceptible to MD, respectively. To assess if Rfp-Y genes affect susceptibility to MD, 265 6₃× 7₂ F₂ chickens were challenged with the JM strain of MD virus at 1 week of age and were evaluated for MD lesions at up to 10 weeks of age. Genotyping of the F₂ chickens for Rfp-Y haplotypes was performed by restriction fragment length polymorphism analysis of genomic DNA using Taq I and a B-FIV probe. Analysis of variance and interval mapping procedures were used to determine association between the Rfp-Y haplotypes and the phenotypic MD values of the F₂ chickens. The cosegregation analysis of 265 F₂ chickens indicated that there was no association between Rfp-Y haplotypes and MD susceptibility. Furthermore, the fact that the Rfp-Y haplotypes fit the 1:2:1 segregation ratio and the Rfp-Y allele frequencies did not differ significantly from 0·5 in the full population or in selected subpopulations (of either 40 MD-resistant or 39 MD-susceptible chickens) also indicated that Rfp-Y haplotypes do not significantly influence MD susceptibility. We conclude that Rfp-Y haplotypes do not play a major role in determining the genetic susceptibility to MD in 6₃× 7₂ F₂ White Leghorn chickens.     

Reduction of Phytophthora Stem Rot Disease on Soybeans by the Application of CaCl2 and Ca(NO3)2

This study investigated the effect of CaCl₂ and Ca(NO₃)₂ on fungal growth of Phytophthora sojae isolates, disease reduction on two cultivars of Glycine max (L.) Merr. cv. Chusei‐Hikarikuro (black soybean) and cv. Sachiyutaka (white soybean) and zoospore release. A concentration of 20–30 mm CaCl₂ or 30 mm Ca(NO₃)₂ led to a slight decrease of the growth rate of two isolates on PDA; however, 0.4 and 4 mm of CaCl₂ and Ca(NO₃)₂ increased growth. The application of 4 mm CaCl₂ or more than 4 mm Ca(NO₃)₂ before inoculation greatly inhibited infection in the two soybean cultivars. Disease suppression recorded in laboratory experiments using pathogen mycelium was because of the response of plant tissues rather than a direct inhibition of pathogen hyphal growth by the application of calcium. Furthermore, Ca(NO₃)₂ was more effective than CaCl₂. The calcium contents in plants increased at the time of inoculation. The extent of disease reduction was related to an increased calcium uptake by plants of the two cultivars, except for some cases involving cv. Chusei‐Hikarikuro. Results showed that the effective element in reducing Phytophthora stem rot was calcium and that differences existed between the two cultivars in terms of the mechanisms of calcium uptake and the effect on disease suppression. The presence of 4–30 mm CaCl₂ and Ca(NO₃)₂ decreased the release of zoospores from isolates on lima bean agar, although 0.4 mm CaCl₂ and Ca(NO₃)₂ significantly induced zoospore release. These results suggest the possibility of applying a solution containing more than 4 mm of calcium to decrease the incidence of disease in agricultural fields by the inhibition of zoospore release.     

Biogenesis of F71 and F72 fimbriae of uropathogenic Escherichia coli: influence of the FsoF and FstFG proteins and localization of the Fso/FstE protein

The F7(1) and F7(2) P-fimbriae of Escherichia coli are encoded by the fso (F seven one) and fst (F seven two) gene clusters, respectively (Van Die et al., 1984; 1985). With the immunocytochemical gold-labelling technique it was demonstrated that both the FsoE and FstE proteins are non-adhesive minor fimbrial subunits located at the tip of the fimbrial structure. The FsoF and FstFG proteins play an important role in the initiation of polymerization of the minor and major subunits into the fimbrial structure.     

Influence of NaCl, Cd(NO3)2 and air humidity on transpiration of Tamarix aphylla

Transpiration rates of young Tamarix aphylla (L.) Karst, plants grown in hydroponics were measured under NaCl- and Cd(NO₃)₂-stress. Transpiration rates were negatively correlated with the relative humidity of the ambient air at all NaCl concentrations investigated. Low and intermediate concentrations of Cd²⁺ (45 and 90 μ M , respectively) in the medium caused an increase in transpiration rates. This was particularly pronounced at low levels of relative humidity. At 180 μ M Cd²⁺, transpiration rates dropped, probably as a result of root damage due to Cd²⁺ toxicity. Since the transpiration rates differed by a factor of ca 3 between day and night, it is concluded that the stomata did not lose their ability to regulate transpiration under the influence of NaCl or of Cd(NO₃)₂. The transpiration behaviour of T. aphylla indicates that the effect of water vapour pressure (presented as relative humidity) on the degree of stomatal opening is small. Under conditions of ample water supply transpiration follows the evaporative demand of the ambient air and is influenced by the water uptake capacity of the root system as well as by other environmental factors, e.g. light.     

Antinociceptive Actions of α2-Adrenoceptor Agonists in the Rat Spinal Cord: Evidence for Antinociceptive α2-Adrenoceptor Subtypes and Dissociation of Antinociceptive α2-Adrenoceptors from Cyclic AMP

The antinociceptive actions of intrathecal injections of two alpha 2-adrenergic agonists, UK-14,304 and guanfacine, were investigated in rats after pretreatment of the animals with the noradrenaline neurotoxin N-2-chloroethyl-N-ethyl-2-bromobenzylamine (DSP4) 14 days in advance. The chronic noradrenaline depletion induced by DSP4 caused a marked increase in sensitivity of the antinociceptive action of UK-14,304 in the tail-flick test. By contrast, the antinociceptive effect of guanfacine was not appreciably affected by the DSP4 treatment. The antinociceptive effects of both UK-14,304 and guanfacine were blocked by intraperitoneal injections of yohimbine, a result indicating that both drugs induced their actions by activating alpha 2-adrenoceptors. Both UK-14,304 and guanfacine were found to reduce the production of cyclic AMP (cAMP) in the spinal cord, as determined using an in vitro radioisotopic method. The cAMP inhibitory effects of both agonists were effectively blocked by yohimbine, but not by prazosin, a finding indicating the alpha 2-adrenergic nature of the response. However, the cAMP inhibitory effect of UK-14,304 was not potentiated by pretreatment with DSP4, a finding in marked contrast with the strong potentiation of the antinociceptive action of UK-14,304 induced by the chronic depletion of endogenous noradrenaline. Moreover, intrathecal injections of forskolin, which increased the endogenous levels of spinal cord cAMP fivefold, did not modify the antinociceptive effects of UK-14,304 or guanfacine in neither normal nor DSP4-treated animals. It is suggested that there exist pharmacologically differing alpha 2-adrenergic receptor pathways capable of mediating antinociceptive effects at the level of the spinal cord. The cAMP inhibitory actions of spinal cord alpha 2-adrenoceptors appear not to be directly linked with the antinociceptive actions of these receptors.     

Functional analysis of the fsoC gene product of the F71 (fso) fimbrial gene cluster

Contrary to what would be expected from data in the literature, mutations in the fsoC gene of the F7(1) (fso) P-fimbrial gene cluster do not completely block fimbrial biogenesis. fsoC mutants still express small amounts of fimbriae of normal length, which carry the non-adhesive minor subunit protein, FsoE, but lack the adhesin, FsoG. The FsoC protein operates at the same stage in fimbrial biogenesis as the FsoF and FsoG proteins. The data suggest that FsoC, FsoF and FsoG interact to form an initiation complex for fimbrial biogenesis.     

Coupling of Astroglial α2-Adrenoreceptors to Second Messenger Pathways

Abstract: We have investigated which α₂-receptor subtypes are expressed in cultured cortical astroglia, and their coupling to second messengers. Binding assays using [³H]rauwolscine showed a very low number of α₂ receptors in the astrocytic cultures. Treatment of cultures with dibutyryl cyclic AMP (dBcAMP) increased significantly the number of receptors. The RNase protection assay was used to investigate which receptor subtype the cells express. The α_2B message was expressed at a low level in both treated and untreated cells, the levels of mRNA for the α_2A/D subtype were up-regulated significantly in cells treated with dBcAMP and no expression of mRNA for the α_2C subtype was detected. The α₂ agonist dexmedetomidine inhibited forskolin-induced increases in cyclic AMP both in treated and untreated cultures in a pertussis toxin-dependent manner. This effect was abolished by the α₂-receptor antagonist rauwolscine. Selective α₂-receptor agonists dexmedetomidine, clonidine, and UK14,304 all increased intracellular calcium only in dBcAMP-treated cells. The antagonist rauwolscine abolished this effect. Ca²⁺ responses were also seen in the absence of extracellular Ca²⁺ and they were inhibited by the phospholipase C inhibitor U-73122, suggesting that astroglial α₂ receptors are coupled to the inositol phospholipid pathway. We therefore also tested the effect of dexmedetomidine directly on inositol 1,4,5-trisphosphate accumulation. A significant increase was seen that was blocked by the antagonist rauwolscine and, as expected, by U-73122. In short, the results demonstrate that the α₂ receptors in astroglia are coupled to multiple second messenger pathways. They are up-regulated in cells treated with dBcAMP, which simultaneously assume a process-bearing morphology. If this morphological change reflects some in vivo process such as reactive gliosis, the up-regulation of α₂-receptor expression could mean an adaptive change in astrocytic responses to a common neurotransmitter, noradrenaline.     

Subtype-Selective Immunoprecipitation of the β2-Adrenergic Receptor

Most antibodies known to interact with beta-adrenergic receptors do not exhibit subtype selectivity, nor do they provide quantitative immunoprecipitation. A monoclonal antibody, G27.1 raised against a synthetic peptide corresponding to the C-terminus of the beta 2-adrenergic receptor of hamster, is selective for the beta 2 subtype. G27.1 provides nearly quantitative immunoprecipitation of the beta 2-adrenergic receptor from hamster lung that has been photoaffinity-labeled and solubilized with sodium dodecyl sulfate. Immunoprecipitation is completely blocked by nanomolar concentrations of the immunizing peptide. This antibody interacts with beta 2-adrenergic receptors from three rodent species, but not with those from humans. When C6 glioma cells, which contain both beta 1- and beta 2-adrenergic receptors, are photoaffinity-labeled in the absence or presence of subtype-selective antagonists, subtype-selective photoaffinity-labeling results. G27.1 can immunoprecipitate beta 2-, but not beta 1-, adrenergic receptors from these cells. Similar results were obtained following subtype-selective photoaffinity-labeling of membranes from rat cerebellum and cerebral cortex. The beta-adrenergic receptors from C6 glioma cells and rat cerebral cortex exist as a mixture of two molecular weight species. These species differ in glycosylation, as shown by endoglycosidase F digestion of crude and immunoprecipitated receptors.     

Possible effects of rising C02 on climate

Abstract Two factors will determine the rate at which CO₂ levels in the atmosphere increase in the future: the rate of input to the atmosphere, primarily from fossil fuel burning, and the way in which this CO₂ is partitioned between atmosphere, ocean and biosphere. A brief review is given of the current state of knowledge of these aspects of the CO₂ issue prior to a discussion of the changes in climate that might be expected from increased levels of CO₂, whenever these might occur. The basis of climate modelling upon which our expectations rest is explained, indicating the nature of the uncertainty that currently exists in the model results. While some of the gross features of the likely climatic change seem reasonably well established qualitatively, considerable model development will be needed before reliable information on the likely regional effects is forthcoming. Observations have yet to confirm the occurrence of temperature change attributable to CO₂ increases. Nevertheless, the possibility exists of a change in climate during the coming century that may be substantial relative to past experience. Although direct measures to control CO₂ emissions would certainly be premature, long-term planning of infrastructures, closely tuned to present climatic conditions, should ensure their robustness in the face of the uncertain climatic changes that may lie ahead.     

The leukotoxin of Pasteurella haemolytica binds to β2 integrins on bovine leukocytes

The putative receptor proteins of Pasteurella haemolytica leukotoxin were isolated from bovine polymorphonuclear neutrophil lysate by affinity chromatography on a leukotoxin-specific monoclonal antibody column to which the leukotoxin was pre-bound. SDS-PAGE of the purified proteins showed four bands at 180 kDa, 170 kDa, 150 kDa and 95 kDa, in addition to the expected 102-kDa leukotoxin band and a series of bands with molecular masses lower than 102 kDa representing the disintegrated leukotoxin. N-terminal amino acid sequencing of the 170-kDa band showed homology with human and murine CD11b. The purified proteins reacted specifically with monoclonal antibodies specific for CD11a, CD11b, CD11c (the alpha chains of beta(2) integrins), and CD18 (the beta chain of beta(2) integrins). Pre-incubation of polymorphonuclear neutrophils with a monoclonal antibody specific for CD18 reduced the cytotoxicity of the leukotoxin to the cells. These results indicate that the leukotoxin binds to the beta(2) integrins on bovine leukocytes, very likely via CD18.