Attempt to detect recombination between B-F and B-L genes within the chicken B complex by serological typing,in vitro MLR,and RFLP analyses

In search for recombinants within the chicken major histocompatibility B complex, 1155 animals from crosses between the congenic lines CB (B¹²) and CC (B⁴) were tested with alloantibodies and monoclonal antibodies for the B-F (class I), B-L (class II), and B-G (class IV) antigens and by mixed lymphocyte reaction. The absence of detectable recombination was confirmed by restriction fragment length polymorphism analysis with B-L and B-F probes. Together with previous reports, this indicates that the distance between the B-F and B-L loci is below 0.01 centimorgan.     

Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.      

The behavioral and somatic effects of ovariectomy and replacement therapy in female collared lemmings (Dicrostonyx groenlandicus)

Reproductive behavior and organ weights in intact estrous and diestrous, and in ovariectomized treated and untreated female collared lemmings (Dicrostonyx groenlandicus) were examined. Sexual behavior of intact diestrous and untreated ovariectomized females was similar, and females in both groups behaved differently from intact estrous females in scores for lordosis and mounting the male. Receptivity was observed in some females within 3 days after the first daily dose of as little as 0.1 μg of estradiol benzoate (EB) and in 83% of all females treated with 0.1 μg or more of EB for 5 days. Vaginal perforation and cornification were related to the dose of EB and length of treatment. The doses of EB used were not related to the lordosis quotient. Ovariectomy and subsequent EB treatment influenced uterine but not preputial gland weight. Adrenal weight was not influenced by ovariectomy or EB treatment, but the increased adrenal weights observed may have been related to the daily pairing of females with males.     

Approval of trainers and training practices in the Oxford region: assessment.

This is the second paper of three on criteria for approval of trainers of general practitioners drawn up for the Oxford region. This paper describes assessment of trainers and training practices by a team of general practitioners who visit for one day.     

Rest-activity rhythms characteristics and seasonal changes in seasonal affective disorder

ABSTRACT

Identifying objectively measurable seasonal changes in 24-h activity patterns (rest-activity rhythms or RARs) that occur in seasonal affective disorder (SAD) could help guide research and practice towards new monitoring tools or prevention targets. We quantified RARs from actigraphy data using non-parametric and extended cosine based approaches, then compared RARs between people with SAD and healthy controls in the summer (n = 70) and winter seasons (n = 84). We also characterized the within-person seasonal RAR changes that occurred in the SAD (n = 19) and control (n = 26) participants who contributed repeated measures. Only controls had significant winter increases in RAR fragmentation (intra-daily variability; in controls mean winter-summer changes (log scale) = 0.05, 0.21 standard deviation, p = 0.03). In SAD participants only, estimated evening settling times (down-mesor) were an average of 30 min earlier in the winter compared with the summer (1-h standard deviation, p = 0.045). These RAR characteristics correlated with greater fatigue (Spearman r = 0.36) but not depression symptom severity. Additional research is needed to ascertain why healthy controls, but not people with SAD, appear to have increased RAR fragmentation in the winter. People with SAD lacked this increase in RAR fragmentation, and instead had earlier evening setting in the winter. Prospective and intervention studies with greater temporal resolution are warranted to ascertain how these seasonal behavioral differences relate to fatigue pathophysiology in SAD. Future research is needed to determine whether extending the winter active period, even in relatively fragmented bouts, could help reduce the fatigue symptoms common in SAD.     

Structural and functional features of the transmembrane domain of the Na,K-ATPase beta subunit revealed by tryptophan scanning

In oligomeric P2-ATPases such as Na,K- and H,K-ATPases, beta subunits play a fundamental role in the structural and functional maturation of the catalytic alpha subunit. In the present study we performed a tryptophan scanning analysis on the transmembrane alpha-helix of the Na,K-ATPase beta1 subunit to investigate its role in the stabilization of the alpha subunit, the endoplasmic reticulum exit of alpha-beta complexes, and the acquisition of functional properties of the Na,K-ATPase. Single or multiple tryptophan substitutions in the beta subunits transmembrane domain had no significant effect on the structural maturation of alpha subunits expressed in Xenopus oocytes nor on the level of expression of functional Na,K pumps at the cell surface. Furthermore, tryptophan substitutions in regions of the transmembrane alpha-helix containing two GXXXG transmembrane helix interaction motifs or a cysteine residue, which can be cross-linked to transmembrane helix M8 of the alpha subunit, had no effect on the apparent K(+) affinity of Na,K-ATPase. On the other hand, substitutions by tryptophan, serine, alanine, or cysteine, but not by phenylalanine of two highly conserved tyrosine residues, Tyr(40) and Tyr(44), on another face of the transmembrane helix, perturb the transport kinetics of Na,K pumps in an additive way. These results indicate that at least two faces of the beta subunits transmembrane helix contribute to inter- or intrasubunit interactions and that two tyrosine residues aligned in the beta subunits transmembrane alpha-helix are determinants of intrinsic transport characteristics of Na,K-ATPase.     

Purification and electron microscopic characterization of the membrane subunit (IICB(Glc)) of the Escherichia coli glucose transporter

The glucose transporter of the bacterial phosphotransferase system mediates sugar transport across the cytoplasmic membrane concomitant with sugar phosphorylation. It consists of a cytoplasmic subunit IIA(Glc) and the transmembrane subunit IICB(Glc). IICB(Glc) was purified to homogeneity by urea/alkali washing of membranes and nickel-chelate affinity chromatography. About 1.5 mg highly pure IICB(Glc) representing 77% of the total activity present in the membranes was obtained from 8g (wet weight) of cells. IICB(Glc) was reconstituted into lipid bilayers by temperature-controlled dialysis to yield small 2D crystals and by a rapid detergent-dilution procedure to yield densely packed vesicles. Electron microscopy and digital image processing of the negatively stained 2D crystals revealed a trigonal lattice with a unit cell size of a = b = 14.5 nm. The unit cell morphology exhibited three dimers of IICB(Glc) surrounding the threefold symmetry center. Single particle analysis of IICB(Glc) in proteoliposomes obtained by detergent dialysis also showed predominantly dimeric structures.     

Repetitive versus monomeric antigen presentation: direct visualization of antibody affinity and specificity

The concept of presenting antigens in a repetitive array to obtain high titers of specific antibodies is increasingly applied by using surface-engineered viruses or bacterial envelopes as novel vaccines. A case for this concept was made 25 years ago, when producing high-titer antisera against ordered arrays of gp23, the major capsid protein of bacteriophage T4 (Aebi et al., Proc. Natl. Acad. Sci. USA, 74 (1977) 5514-5518). In view of the current interest in this concept we thought it useful to employ this system to directly visualize the dependence of antibody affinity and specificity on antigen presentation. We compared antibodies raised against T4 polyheads, a tubular variant of the bacteriophage T4 capsid, which have gp23 hexamers arranged in a crystalline lattice (gp23(repetitive)), with those raised against the hexameric gp23 subunits (gp23(monomeric)). The labeling patterns of Fab-fragments prepared from these antibodies when bound to polyheads were determined by electron microscopy and image enhancement. Anti-gp23(repetitive) bound in a monospecific, stoichiometric fashion to the gp23 units constituting the polyhead surface. In contrast, anti-gp23(monomeric) decorated the polyhead surface randomly and with a 40-fold lower occupancy. These results concur with the difference in titers established by ELISA for the antisera against the repetitively displayed form of antigen (anti-gp23(repetitive)) and the randomly presented antigen (gp23(monomeric)), and they constitute a compelling visual documentation of the concept of repetitive antigen presentation to elicite a serotype-like immune response.     

Effect of the two conserved prolines of human growth inhibitory factor (metallothionein-3) on its biological activity and structure fluctuation: comparison with a mutant protein

Human neuronal growth inhibitory factor, a metalloprotein classified as metallothionein-3 (MT-3), impairs the survival and the neurite formation of cultured neurons. In these studies the double P7S/P9A mutant (mutMT-3) and single mutants P7S and P9A of human Zn(7)-MT-3 were generated, and their effects on the biological activity and the structure of the protein were examined. The biological results clearly established the necessity of both proline residues for the inhibitory activity, as even single mutants were found to be inactive. Using electronic absorption, circular dichroism (CD), magnetic CD (MCD), and (113)Cd NMR spectroscopy, the structural features of the metal-thiolate clusters in the double mutant Cd(7)-mutMT-3 were investigated and compared with those of wild-type Cd(7)-MT-3 [Faller, P., Hasler, D. W., Zerbe, O., Klauser, S., Winge, D. R., and Vasák, M. (1999) Biochemistry 38, 10158] and the well characterized Cd(7)-MT-2a from rabbit liver. Similarly to (113)Cd(7)-MT-3 the (113)Cd NMR spectrum of (113)Cd(7)-mutMT-3 at 298 K revealed four major and three minor resonances (approximately 20% of the major ones) between 590 and 680 ppm, originating from a Cd(4)S(11) cluster in the alpha-domain and a Cd(3)S(9) cluster in the beta-domain, respectively. Due to the presence of dynamic processes in the structure of MT-3 and mutMT-3, all resonances showed the absence of resolved homonuclear [(113)Cd-(113)Cd] couplings and large apparent line widths (between 140 and 350 Hz). However, whereas in (113)Cd(7)-mutMT-3 the temperature rise to 323 K resulted in a major recovery of the originally NMR nondetectable population of the Cd(3)S(9) cluster resonances, no such temperature effect was observed in (113)Cd(7)-MT-3. To account for the observed NMR features, a dynamic structural model for the beta-domain is proposed, which involves a folded and a partially unfolded state. It is suggested that in the partially unfolded state a slow cis/trans isomerization of Cys-Pro(7) or Cys-Pro(9) amide bonds in (113)Cd(7)-MT-3 takes place and that this process represents a rate-limiting step in a correct domain refolding. In addition, closely similar apparent stability constants of human MT-3, mutMT-3, and rabbit MT-2a with Cd(II) and Zn(II) ions were found. These results suggest that specific structural features dictated by the repetitive (Cys-Pro)(2) sequence in the beta-domain of MT-3 and not its altered metal binding affinity compared to MT-1/MT-2 isoforms are responsible for the biological activity of this protein.     

Determinants of topogenesis and glycosylation of type II membrane proteins. Analysis of Na,K-ATPase beta 1 AND beta 3 subunits by glycosylation mapping

The structural and molecular determinants that govern the correct membrane insertion and folding of membrane proteins are still ill-defined. By following the addition of sugar chains to engineered glycosylation sites (glycosylation mapping) in Na,K-ATPase beta isoforms expressed in vitro and in Xenopus oocytes, in combination with biochemical techniques, we have defined the C-terminal end of the transmembrane domain of these type II proteins. N-terminal truncation and the removal of a single charged residue at the N-terminal start of the putative transmembrane domain influence the proper positioning of the transmembrane domain in the membrane as reflected by a repositioning of the transmembrane domain, the exposure of a putative cryptic signal peptidase cleavage site, and the production of protein species unable to insert into the membrane. Glycosylation mapping in vivo revealed that the degree of glycosylation at acceptor sites located close to the membrane increases with the time proteins spend in the endoplasmic reticulum. Furthermore, core sugars added to such acceptor sites cannot be processed to fully glycosylated species even when the protein is transported to the cell surface. Thus, the glycosylation mapping strategy applied in intact cells is a useful tool for the study of determinants for the correct membrane insertion of type II and probably other membrane proteins, as well as for the processing of sugar chains in glycoproteins.