Alloantigen system L affects the outcome of rous sarcomas

This study was designed to examine the alloantigen system L effects on Rous sarcomas in three B complex genotypes. The parental stock was 50% Modified Wisconsin Line 3 x White Leghorn Line NIU 4 and 50% inbred Line 6.15-5. Pedigree matings of two B(2)B(5) L(1)L(2) sires to five B(2)B(5) L(1)L(2) dams per sire produced experimental chicks segregating for B and L genotypes. Chicks were inoculated with 20 pock-forming units (pfu) of Rous sarcoma virus (RSV) at 6 weeks of age. Tumors were scored six times over 10 weeks postinoculation after which the tumor scores were used to assign a tumor profile index (TPI) to each chicken. Tumor growth over time and TPI were evaluated by repeated-measures analysis of variance and analysis of variance, respectively. Six trials were conducted with a total of 151 chickens. The major histocompatibility (B) complex affected the responses as the B(2)B(2) and B(2)B(5) genotypes had significantly lower tumor growth over time and TPI than the B(5)B(5) genotype. Separate analyses revealed no significant L system effect in B(2)B(2) or B(2)B(5) backgrounds. However, L genotype significantly affected (P < 0.05) both tumor growth over time and TPI in B(5)B(5) chickens. B(5)B(5) L(1)L(2) birds had TPI significantly lower than B(5)B(5) L(1)L(1) chickens but not B(5)B(5) L(2)L(2). Mortality was lower in the B(5)B(5) L(1)L(2) birds than in B(5)B(5) L(2)L(2) chickens. The L system, or one closely linked, affects the growth and ultimate outcome of Rous sarcomas. The response may depend upon the genetic background as well as MHC type.     

High agonist-independent clearance of rabbit kinin B1 receptors in cultured cells

We hypothesized that the inducible kinin B(1) receptor (B(1)R) is rapidly cleared from cells when its synthesis subsides. The agonist-independent degradation of the rabbit B(1)Rs and related B(2) receptors (B(2)Rs) was investigated. Endocytosis of the B(1)R-yellow fluorescent protein (YFP) conjugate was more intense than that of B(2)R-green fluorescent protein (GFP) based on fluorescence accumulation in HEK 293 cells treated with a lysosomal inhibitor. The cells expressing B(1)R-YFP contained more GFP/YFP-sized degradation product(s) than those expressing B(2)R-GFP (immunoblot, antibodies equally reacting with both fluorescent proteins). The binding site density of B(1)R-YFP decreased in the presence of protein synthesis or maturation inhibitors (anisomycin, brefeldin A), whereas that of B(2)R-GFP remained constant. Wild-type B(1)Rs were also cleared faster than B(2)Rs in rabbit smooth muscle cells treated with metabolic inhibitors. Contractility experiments based on brefeldin A-treated isolated rabbit blood vessels also functionally support that B(1)Rs are more rapidly eliminated than B(2)Rs (decreased maximal effect of agonist over 2 h). The highly regulated B(1)R is rapidly degraded, relative to the constitutive B(2)R.     

Identification of a key region of kinin B(1) receptor for high affinity binding of peptide antagonists

To investigate the molecular basis for the specificity of ligand recognition in human kinin B(1) (B(1)R) and B(2) (B(2)R) receptors, we constructed a series of chimeric receptors by progressively replacing, from the N to the C terminus, the human B(2)R domains by their B(1) counterparts. The chimeric construct possessing the C-terminal tail and the transmembrane domain VII (TM VII) of the B(2)R (construct 6) displayed 7- and 20- fold decreased affinities for the B(1) agonist [(3)H]desArg(10)-kallidin (desArg(10)-KD) and the B(1) antagonist [(3)H]desArg(10)-[Leu(9)]-KD respectively, as compared with the wild-type B(1)R. Moreover, the substitution of the B(1) TM VII by its B(2) homologue TM increased the affinity for the pseudopeptide antagonists, Hoe140 and NPC 567. High affinity for desArg(10)-KD binding was fully regained when the B(2) residue Thr(287) was replaced in construct 6 by the corresponding B(1) Leu(294) residue. When the B(2) residue Tyr(295) was exchanged with the corresponding B(1) Phe(302), high affinity binding for both agonist and antagonist was recovered. Moreover, the L294T and F302Y mutant B(1)R exhibited 69- and 6.5-fold increases, respectively, in their affinities for the B(2) receptor antagonist, Hoe140. Therefore we proposed that Leu(294) and Phe(302) residues, which may not be directly involved in the binding of B(1)R ligands and, hence, their Thr(287) and Tyr(295) B(2) counterparts, are localized in a receptor region, which plays a pivotal role in the binding selectivity of the peptide or pseudopeptide kinin ligands.     

Numerical taxonomy of Bacteroides and other genera of Gram-negative anaerobic rods.

A numerical taxonomy was performed on 157 cultures (141 different strains) of species of Bacteroides, Polyphyromonas, Prevotella [not Prevotella (Labroue, 1976)] and Fusobacterium. Isolates were each tested for 111 phenotypic characters which included possession of constitutive enzymes, fermentation of specific carbohydrates, gas chromatographic analysis of metabolic end-products and of cellular carboxylic acid composition. Computation of similarity coefficients was followed by a single-linkage cluster analysis. At the 94% similarity level, the following groupings at genus level were apparent: (1) Bacteroides ureolyticus; (2) Fusobacterium mortiferum, F. necrogenes, F. necrophorum, F. nucleatum and F. varium; (3) B. caccae, B. distasonis, B. eggerthii, B. fragilis, B. merdae, B. ovatus, B stercoris, B. thetaiotaomicron, B. uniformus and B. vulgatus; (4) B. splanchnicus; (5) Porphyromonas asaccharolytica; (6) B. bivius (Prevotella bivia); (7) B. disiens (P. disiens); (8) B. intermedius (P. intermedia);and (9) B. melaninogenicus (P. melaninogenica). Single isolates of B. ruminicola (P. ruminicola), B. denticola (P. denticola) and B. capillosus did not cluster with other strains.     

The B chromosome polymorphism of the grasshopper Eyprepocnemis plorans in North Africa. IV. Transmission of rare B chromosome variants

In addition to the principal B chromosome (B(1)) in Moroccan populations of the grasshopper Eyprepocnemis plorans, nine B chromosome variants appeared at low frequency. The transmission of five of these rare B chromosome variants through females was analysed in three natural populations. Sixteen controlled crosses provided useful information on the transmission of B(M2), B(M6) and B(M7) in Smir, B(M3) and B(M6) in SO.DE.A. (Société de Développement Agricole lands near Ksar-el-Kebir city), and B(M2) and B(M10) in Mechra, all located in Morocco. Since six female parents carried two different B variants, a total of 22 progeny analyses could be studied. Intraindividual variation in B transmission rate (k(B)) was observed among the successive egg pods in 26.7 % of the females, but this variation did not show a consistent temporal pattern. Only the B(M2) and B(M6) variants in Smir showed net drive, although variation was high among crosses, especially for B(M2). These two variants are thus good candidates for future regenerations (the replacement of a neutralized B, B(1) in this case, by a new driving variant, B(M2) or B(M6)) in Smir, the northern population where the B polymorphism is presumably older. The analysis of all crosses performed in the three populations, including those reported previously for the analysis of B(1) transmission, showed that the largest variance in k(B) among crosses stands at the individual level, and not at population or type of B levels. The implications of these findings for the occurrence of possible regeneration processes in Moroccan populations are discussed.     

Anti-Rous sarcoma response of major histocompatibility (B) complex haplotypes B23, B24 and B30

Responses to Rous sarcoma virus (RSV) induced tumours were studied in UNH 105, a non-inbred line of New Hampshire chickens. Six single male matings encompassing a total of 50 dams produced 345 progeny which segregated for B complex genotypes B23/B23, B23/B24, B23/B30, B24/B24, B24/B30 and B30/B30. Six-week-old chicks were wingweb inoculated with a pseudotype of Bryan high titre Rous sarcoma virus, BH RSV (RAV-1). Tumours were scored for size six times over a 10-week period post-inoculation. Each chick was assigned a tumour profile index (TPI) as an indicator of immunological response. The number of days to death (DTD) was recorded for 148 chicks with terminal tumours. Genotypes B23/B23, B23/B24 and B23/B30, with TPIs of 1.8, 1.7 and 2.0 respectively, did not differ significantly from each other, suggesting dominance of response of B23 over B24 and B30 haplotypes. B24/B30 chicks with the highest TPI (3.4) and shortest DTD (34.6) were significantly different from B30/B30 (2.8; 41.6) but not from B24/B24 (3.1; 34.9) suggesting dominance of response of the B24 haplotype over B30 in the absence of B23.     

Phosphorylation of the B1 (CD20) molecule by normal and malignant human B lymphocytes

The B1 molecule (CD20) is a phosphoprotein found only on B lymphocytes. Multiple isoforms of the B1 molecule are expressed with Mr of 33,000, B1(33) and Mr of 34,500-36,000, B1(35). In this study it was found that nonproliferating B cells did not incorporate 32PO4 into B1 although phosphorylated class I histocompatibility molecules were easily detected. In contrast B1 isolated from proliferating or malignant B cells or B cell lines was heavily phosphorylated. Cross-linking B1 on the cell surface by antibody resulted in enhanced phosphorylation of B1 as did exposure to phorbol esters, and the membrane permeable diacylglycerol analog 1,2,-dioctanoylglyceron. B1(33) and B1(35) produced identical peptide maps following limited proteinase digestion. However, B1(35) contained both phosphoserine and phosphothreonine, while B1(33) only contained phosphoserine. In addition alkaline phosphatase was able to remove the phosphate residue(s) that resulted in generation of the B1(35) form of B1 but was unable to remove the phosphorylation of B1(33). These results suggest that phosphorylation of B1 molecules is associated with proliferation and that the different Mr forms of B1 result from the phosphorylation of B1 at different sites. Also, the finding that antibody binding to B1 generated a transmembrane signal may explain why antibody binding to B1 alters B cell function.     

Hetero-oligomerization between GABAA and GABAB receptors regulates GABAB receptor trafficking

The neurotransmitter gamma-aminobutyric acid (GABA) mediates inhibitory signaling in the brain via stimulation of both GABA(A) receptors (GABA(A)R), which are chloride-permeant ion channels, and GABA(B) receptors (GABA(B)R), which signal through coupling to G proteins. Here we report physical interactions between these two different classes of GABA receptor. Association of the GABA(B) receptor 1 (GABA(B)R1) with the GABA(A) receptor gamma2S subunit robustly promotes cell surface expression of GABA(B)R1 in the absence of GABA(B)R2, a closely related GABA(B) receptor that is usually required for efficient trafficking of GABA(B)R1 to the cell surface. The GABA(B)R1/gamma2S complex is not detectably functional when expressed alone, as assessed in both ERK activation assays and physiological analyses in oocytes. However, the gamma2S subunit associates not only with GABA(B)R1 alone but also with the functional GABA(B)R1/GABA(B)R2 heterodimer to markedly enhance GABA(B) receptor internalization in response to agonist stimulation. These findings reveal that the GABA(B)R1/gamma2S interaction results in the regulation of multiple aspects of GABA(B) receptor trafficking, allowing for cross-talk between these two distinct classes of GABA receptor.     

Acquisition of VanB-type vancomycin resistance by Bacillus subtilis: the impact on gene expression, cell wall composition and morphology

The vancomycin resistance operons from Enterococci, Staphylococci and Actinomycetes encode a VanRS two-component signal transduction system (TCS) and a suite of enzymes to modify the peptidoglycan biosynthetic precursor lipid II and to eliminate the D-Ala-D-Ala from the cell. Commingling of these regulatory and enzymatic activities with host functions has the potential to significantly impact host gene expression and cell wall metabolism. Here we report the effects of individually expressing the VanR(B) S(B) TCS and the VanY(B) WH(B) BX(B) resistance proteins in Bacillus subtilis. VanY(B) WH(B) BX(B) expression confers resistance to 2 μg ml(-1) of vancomycin with concomitant reduced Van-FL staining and leads to a cell division defect. In contrast to E. faecalis and S. aureus, VanS(B) is active in B. subtilis without vancomycin addition. Individual expression of the VanR(B) S(B) TCS and the VanY(B) WH(B) BX(B) resistance proteins repress and increase, respectively, expression of PhoPR regulon genes in the phosphate-limited state. When vancomycin-resistant cells are exposed to elevated vancomycin levels, mutant strains with increased resistance to vancomycin and a growth dependency on vanY(B) WH(B) BX(B) expression frequently arise. Mutation of the endogenous Ddl ligase is the necessary and sufficient cause of both phenotypes. We discuss how these effects may influence establishment of van operons in new host bacteria.     

Occurrence of macrolide-lincosamide-streptogramin B resistant isolates of Staphylococcus aureus in clinical specimens in 2003-2004

The aim of the study was the analysis of frequency of macrolide-lincosamide-streptogramin B (MLS(B)) resistance among MSSA (n=1682) and MRSA (n=272) strains which were isolated in 2002-2004 from various clinical materials from patients hospitalized in the University Hospital at the L. Rydygier Collegium Medicum in Bydgoszcz, University of Nicolaus Copernicus in Toruń. Susceptibility testing and examination of methicillin-resistant strains were performed by the disc diffusion techniques according to recommendation of NCCLS. Resistance to the MLS(B) antimicrobials agents was higher among MRSA compared to MSSA isolates. The MLS, constitutive phenotype was more prevalent than the MLS(B) inducible phenotype among investigated MRSA (65.4%) and MSSA (7.6%) isolates. Inducible resistance had only 2.5% of the MSSA and 2.6% of the MRSA strains. Moreover in 2004 there were found increasing frequency of inducible MLS(B) resistance from 1.1% to 5.7% and decreasing frequency of constitutive MLS(B) resistance from 9.2% to 4.7% among MSSA strains, in comparison to 2003. The investigated MSSA MLS(B)-, MLS(B)- and MRSAMLS(B)+, MLS(B)- strains were the most frequently isolated from pus (adequately 5.2%, 28.8% and 30.5%, 10.7%) and also from nosopharynx swabs (1.7% MSSA MLS(B)+ and 22.9% MSSA MLS(B)-) and biomaterials (15,1% MRSA MLS(B)+ and 9.6% MRSA MLS(B)-). They mainly came from patients of the outpatient clinic (2,4% MSSA MLS(B)+ and 19.9% MSSA MLS(B)-) and patients treated at the neurosurgical ward (20.6% MRSA MLS(B)+ and 12.1% MRSA MLS(B)-).     

The heptahelical domain of GABA(B2) is activated directly by CGP7930, a positive allosteric modulator of the GABA(B) receptor

The gamma-aminobutyric acid, type B (GABA(B)) receptor is well recognized as being composed of two subunits, GABA(B1) and GABA(B2). Both subunits share structural homology with other class-III G-protein-coupled receptors. They are composed of two main domains: a heptahelical domain (HD) typical of all G-protein-coupled receptors and a large extracellular domain (ECD). Although GABA(B1) binds GABA, GABA(B2) is required for GABA(B1) to reach the cell surface. However, it is still not demonstrated whether the association of these two subunits is always required for function in the brain. Indeed, GABA(B2) plays a major role in the coupling of the heteromer to G-proteins, such that it is possible that GABA(B2) can transmit a signal in the absence of GABA(B1). Today only ligands interacting with GABA(B1) ECD have been identified. Thus, the compounds acting exclusively on the GABA(B2) subunit will be helpful in analyzing the specific role of this subunit in the brain. Here, we explored the mechanism of action of CGP7930, a compound described as a positive allosteric regulator of the GABA(B) receptor. We showed that it activates the wild type GABA(B) receptor but with a low efficacy. The GABA(B2) HD is necessary for this effect, although one cannot exclude that CGP7930 could also bind to GABA(B1). Of interest, CGP7930 could activate GABA(B2) expressed alone and is the first described agonist of GABA(B2). Finally, we show that CGP7930 retains its agonist activity on a GABA(B2) subunit deleted of its ECD. This demonstrates that the HD of GABA(B2) behaves similar to a rhodopsin-like receptor, because it can reach the cell surface alone, can couple to G-protein, and be activated by agonists. These data open new strategies for studying the mechanism of activation of GABA(B) receptor and examine any possible role of homomeric GABA(B2) receptors.     

Role of bradykinin B2 and B1 receptors in the local, remote, and systemic inflammatory responses that follow intestinal ischemia and reperfusion injury

The administration of bradykinin may attenuate ischemia and reperfusion (I/R) injury by acting on B(2)Rs. Blockade of B(2)R has also been shown to ameliorate lesions associated with I/R injury. In an attempt to explain these contradictory results, the objective of the present work was to investigate the role of and interaction between B(1) and B(2) receptors in a model of intestinal I/R injury in mice. The bradykinin B(2)R antagonist (HOE 140) inhibited reperfusion-induced inflammatory tissue injury and delayed lethality. After I/R, there was an increase in the expression of B(1)R mRNA that was prevented by HOE 140. In mice that were deficient in B(1)Rs (B(1)R(-/-) mice), inflammatory tissue injury was abrogated, and lethality was delayed and partially prevented. Pretreatment with HOE 140 reversed the protective anti-inflammatory and antilethality effects provided by the B(1)R(-/-) phenotype. Thus, B(2)Rs are a major driving force for B(1)R activation and consequent induction of inflammatory injury and lethality. In contrast, activation of B(2)Rs may prevent exacerbated tissue injury and lethality, an effect unmasked in B(1)R(-/-) mice and likely dependent on the vasodilatory actions of B(2)Rs. Blockade of B(1)Rs could be a more effective strategy than B(2) or B(1)/B(2) receptor blockade for the treatment of the inflammatory injuries that follow I/R.     

The sushi domains of secreted GABA(B1) isoforms selectively impair GABA(B) heteroreceptor function

GABA(B) receptors are the G-protein-coupled receptors for gamma-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the brain. GABA(B) receptors are promising drug targets for a wide spectrum of psychiatric and neurological disorders. Receptor subtypes exhibit no pharmacological differences and are based on the subunit isoforms GABA(B1a) and GABA(B1b). GABA(B1a) differs from GABA(B1b) in its ectodomain by the presence of a pair of conserved protein binding motifs, the sushi domains (SDs). Previous work showed that selectively GABA(B1a) contributes to heteroreceptors at glutamatergic terminals, whereas both GABA(B1a) and GABA(B1b) contribute to autoreceptors at GABAergic terminals or to postsynaptic receptors. Here, we describe GABA(B1j), a secreted GABA(B1) isoform comprising the two SDs. We show that the two SDs, when expressed as a soluble protein, bind to neuronal membranes with low nanomolar affinity. Soluble SD protein, when added at nanomolar concentrations to dissociated hippocampal neurons or to acute hippocampal slices, impairs the inhibitory effect of GABA(B) heteroreceptors on evoked and spontaneous glutamate release. In contrast, soluble SD protein neither impairs the activity of GABA(B) autoreceptors nor impairs the activity of postsynaptic GABA(B) receptors. We propose that soluble SD protein scavenges an extracellular binding partner that retains GABA(B1a)-containing heteroreceptors in proximity of the presynaptic release machinery. Soluble GABA(B1) isoforms like GABA(B1j) may therefore act as dominant-negative inhibitors of heteroreceptors and control the level of GABA(B)-mediated inhibition at glutamatergic terminals. Of importance for drug discovery, our data also demonstrate that it is possible to selectively impair GABA(B) heteroreceptors by targeting their SDs.     

Studies on the Constituents of Aristolochia mollissima Hance

Ten constituents have been isolated from the ethanolie extract of the stems and leaves of Aristolochia mollissima Hanee. Based on speetrai analysis and the determination of physieo-ehemieal constants they have been identified as: (A1) aristoloehie acid A, (A2) vanillie acid, (A3) aristoloehie acid D, (B1) pahnitone, (B2) n-triacontanol, (B3) β-sitosterol, (B1) aristololaetam, (B11)6-methoxy aristololaetam, (B7) β- sitosterol D-glueoside, (B8) stearic acid. A1 and B1 have been found to have an inhibition action of implantation in mice.     

Developing oligodendrocytes express functional GABA(B) receptors that stimulate cell proliferation and migration

GABA(B) receptors (GABA(B)Rs) are involved in early events during neuronal development. The presence of GABA(B)Rs in developing oligodendrocytes has not been established. Using immunofluorescent co-localization, we have identified GABA(B)R proteins in O4 marker-positive oligodendrocyte precursor cells (OPCs) in 4-day-old mouse brain periventricular white matter. In culture, OPCs, differentiated oligodendrocytes (DOs) and type 2 astrocytes (ASTs) express both the GABA(B1abcdf) and GABA(B2) subunits of the GABA(B)R. Using semiquantitative PCR analysis with GABA(B)R isoform-selective primers we found that the expression level of GABA(B1abd) was substantially higher in OPCs or ASTs than in DOs. In contrast, the GABA(B2) isoform showed a similar level of expression in OPCs and DOs, and a significantly higher level in ASTs. This indicates that the expression of GABA(B1) and GABA(B2) subunits are under independent control during oligodendroglial development. Activation of GABA(B)Rs using the selective agonist baclofen demonstrated that these receptors are functionally active and negatively coupled to adenylyl cyclase. Manipulation of GABA(B)R activity had no effect on OPC migration in a conventional agarose drop assay, whereas baclofen significantly increased OPC migration in a more sensitive transwell microchamber-based assay. Exposure of cultured OPCs to baclofen increased their proliferation, providing evidence for a functional role of GABA(B)Rs in oligodendrocyte development. The presence of GABA(B)Rs in developing oligodendrocytes provides a new mechanism for neuronal-glial interactions during development and may offer a novel target for promoting remyelination following white matter injury.     

Induction of B1 receptors in streptozotocin diabetic rats: possible involvement in the control of hyperglycemia-induced glomerular Erk 1 and 2 phosphorylation

We investigated the effects of a 3-week treatment with various combinations of angiotensin-converting enzyme inhibitor (ACEI) and B1 and B2 bradykinin receptor (B1R and B2R) antagonists (B1A and B2A) and AT1 receptor antagonist on ERK 1 and 2 phosphorylation in isolated glomeruli from streptozotocin-treated diabetic rats (STZ rats). Body weight, glycemia, and blood pressure were monitored. The rats were divided into nine groups: (1) control; and groups 2-9 were STZ treated with (3) insulin, (4) ACEI, (5) ACEI + B1A, (6) ACEI + B2A, (7) B2A, (8) B1A, (9) AT1 antagonist. ERK 1 and 2 phosphorylation and expression of B1R and B2R were assessed by Western blot analysis. ERK 1 and 2 phosphorylation was higher in STZ rats; this activation was normalized by insulin and reduced by ACEI but not by AT1 antagonist. The reduction of ERK 1 and 2 phosphorylation by the ACEI was reversed by B1A and B2A. The induction of B1R was confirmed by increased expression of mRNA and B1 receptor protein. Since ERK 1 and 2 phosphorylation is an early event in the induction of matrix secretion and hyperproliferation associated with diabetic nephropathy, activation of B1R and B2R appears to be a useful pharmacological target in the management of this pathology.