Shoshana Tel-Or

Chicken immunoglobulin gamma-heavy chains: limited VH gene repertoire, combinatorial diversification by D gene segments and evolution of the heavy chain locus.

cDNA clones encoding the variable and constant regions of chicken immunoglobulin (Ig) gamma‐chains were obtained from spleen cDNA libraries. Southern blots of kidney DNA show that the variable region sequences of eight cDNA clones reveal the same set of bands corresponding to approximately 30 cross‐hybridizing VH genes of one subgroup. Since the VH clones were randomly selected, it is likely that the bulk of chicken H‐chains are encoded by a single VH subgroup. Nucleotide sequence determinations of two cDNA clones reveal VH, D, JH and the constant region. The VH segments are closely related to each other (83% homology) as expected for VH or the same subgroup. The JHs are 15 residues long and differ by one amino acid. The Ds differ markedly in sequence (20% homology) and size (10 and 20 residues). These findings strongly indicate multiple (at least two) D genes which by a combinatorial joining mechanism diversify the H‐chains, a mechanism which is not operative in the chicken L‐chain locus. The most notable among the chicken Igs is the so‐called 7S IgG because its H‐chain differs in many important aspects from any mammalian IgG. The sequence of the C gamma cDNA reported here resolves this issue. The chicken C gamma is 426 residues long with four CH domains (unlike mammalian C gamma which has three CH domains) and it shows 25% homology to the chicken C mu. The chicken C gamma is most related to the mammalian C epsilon in length, the presence of four CH domains and the distribution of cysteines in the CH1 and CH2 domains. We propose that the unique chicken C gamma is the ancestor of the mammalian C epsilon and C gamma subclasses, and discuss the evolution of the H‐chain locus from that of chicken with presumably three genes (mu, gamma, alpha) to the mammalian loci with 8‐10 H‐chain genes.

Structural basis for the enhanced thermal stability of alcohol dehydrogenase mutants from the mesophilic bacterium Clostridium beijerinckii: contribution of salt bridging

Previous research in our laboratory comparing the three‐dimensional structural elements of two highly homologous alcohol dehydrogenases, one from the mesophile Clostridium beijerinckii (CbADH) and the other from the extreme thermophile Thermoanaerobacter brockii (TbADH), suggested that in the thermophilic enzyme, an extra intrasubunit ion pair (Glu224‐Lys254) and a short ion‐pair network (Lys257‐Asp237‐Arg304‐Glu165) at the intersubunit interface might contribute to the extreme thermal stability of TbADH. In the present study, we used site‐directed mutagenesis to replace these structurally strategic residues in CbADH with the corresponding amino acids from TbADH, and we determined the effect of such replacements on the thermal stability of CbADH. Mutations in the intrasubunit ion pair region increased thermostability in the single mutant S254K‐ and in the double mutant V224E/S254K‐CbADH, but not in the single mutant V224E‐CbADH. Both single amino acid replacements, M304R‐ and Q165E‐CbADH, in the region of the intersubunit ion pair network augmented thermal stability, with an additive effect in the double mutant M304R/Q165E‐CbADH. To investigate the precise mechanism by which such mutations alter the molecular structure of CbADH to achieve enhanced thermostability, we constructed a quadruple mutant V224E/S254K/Q165E/M304R‐CbADH and solved its three‐dimensional structure. The overall results indicate that the amino acid substitutions in CbADH mutants with enhanced thermal stability reinforce the quaternary structure of the enzyme by formation of an extended network of intersubunit ion pairs and salt bridges, mediated by water molecules, and by forming a new intrasubunit salt bridge.

A single proline substitution is critical for the thermostabilization of Clostridium beijerinckii alcohol dehydrogenase

Analysis of the three‐dimensional structures of three closely related mesophilic, thermophilic, and hyperthermophilic alcohol dehydrogenases (ADHs) from the respective microorganisms Clostridium beijerinckii (CbADH), Entamoeba histolytica (EhADH1), and Thermoanaerobacter brockii (TbADH) suggested that a unique, strategically located proline residue (Pro100) might be crucial for maintaining the thermal stability of EhADH1. To determine whether proline substitution at this position in TbADH and CbADH would affect thermal stability, we used site‐directed mutagenesis to replace the complementary residues in both enzymes with proline. The results showed that replacing Gln100 with proline significantly enhanced the thermal stability of the mesophilic ADH: ΔT  1/260 min = + 8°C (temperature of 50% inactivation after incubation for 60 min), ΔT  1/2CD = +11.5°C (temperature at which 50% of the original CD signal at 218 nm is lost upon heating between 30° and 98°C). A His100 → Pro substitution in the thermophilic TbADH had no effect on its thermostability. An analysis of the three‐dimensional structure of the crystallized thermostable mutant Q100P‐CbADH suggested that the proline residue at position 100 stabilized the enzyme by reinforcing hydrophobic interactions and by reducing the flexibility of a loop at this strategic region. Proteins 2007.

Analyses of chicken immunoglobulin light chain cDNA clones indicate a few germline V lambda genes and allotypes of the C lambda locus.

cDNA libraries of chicken spleen and Harder gland (a gland enriched with immunocytes) constructed in pBR322 were screened by differential hybridization and by mRNA hybrid‐selected translation. Eleven L‐chain cDNA clones were identified from which VL probes were prepared and each was annealed with kidney DNA restriction digests. All VL probes revealed the same set of bands, corresponding to about 15 germline VL genes of one subgroup. The nucleotide sequences of six VL clones showed greater than or equal to 85% homology, and the predicted amino acid sequences were identical or nearly identical to the major N‐terminal sequence of L‐chains in chicken serum. These findings, and the fact that the VL clones were randomly selected from normal lymphoid tissues, strongly indicate that the bulk of chicken L‐chains is encoded by a few germline VL genes, probably much less than 15 since many of the VL genes are known to be pseudogenes. Therefore, it is likely that somatic mechanisms operating prior to specific triggering by antigen play a major role in the generation of antibody diversity in chicken. Analysis of the constant region locus (sequencing of CL gene and cDNAs) demonstrate a single CL isotype and suggest the presence of CL allotypes.

Respiratory activity in the marine cyanobacterium Spirulina subsalsa and its role in salt tolerance

Intracellular ion concentration and respiratory activity in the marine cyanobacterium Spirulina subsalsa was analyzed during cell transition from saline to hypersaline medium. During salt upshock, an early phase of Na+ and Cl- influx was observed, followed by an adaptation phase where both Na+ and Cl- were excluded from the cell. Respiration in intact cells was enhanced during salt upshock. S. subsalsa spheroplasts exhibited a high rate of O2 uptake, which was further enhanced in cells grown in hypersaline medium, upon addition of NaCl to the assay mixture. This effect was found to be specific to sodium ions. Plasma membrane fractions from cells grown in hypersaline medium exhibited a high rate of cytochrome oxidase activity, which was further stimulated by NaCl, and was sensitive to DCCD. Immunoblot analysis of Spirulina plasma membrane polypeptides with anti-cytochrome oxidase serum demonstrated high content of 53.4 kDa polypeptide of cytochrome oxidase, which was enriched in membranes obtained from hypersaline Spirulina cells. The enhanced respiration, and more specifically the enrichment of cytochrome oxidase activity in salt-adapted cells in situ, as well as its stimulation by NaCl in vitro and inhibition by DCCD, suggest that cytochrome oxidase is involved in the extrusion of sodium ions from cells of the salt-tolerant Spirulina subsalsa.

Development of a highly sensitive quantitative competitive PCR assay for the detection of murine cytomegalovirus DNA.

Viral persistence and molecular latency are characteristic of infection by the human cytomegalovirus (HCMV). Using the murine cytomegalovirus (MCMV) as a model for human infection, a quantitative-competitive polymerase chain reaction (QC-PCR) assay was developed to detect and quantify MCMV-DNA in the salivary glands of infected mice. The QC-PCR detected high numbers of MCMV DNA copies in the absence of infectious virus. By comparing the DNA content and the results obtained from a standard semiquantitative plaque assay, it is concluded that 1 plaque-forming unit (pfu) is the equivalent of approximately 1500 viral genomes. By day 42-post infection (pi) 4x10(3) copies of DNA/1 mg tissue were sufficient to reactivate infectious virions after cyclophosphamide immunosupression. By day 90 pi, however, when the DNA load was decreased to <1.2x10(2), reactivation was not observed. These results indicate that viral reactivation will occur when the number of infectious DNA copies is equivalent about 2-3 pfu. This quantitative test may therefore help to detect CMV and the risk of reactivation in immunosupressed patients.

Biochemical and Structural Properties of Chimeras Constructed by Exchange of Cofactor-Binding Domains in Alcohol Dehydrogenases from Thermophilic and Mesophilic Microorganisms

The cofactor-binding domains (residues 153-295) of the alcohol dehydrogenases from the thermophile Thermoanaerobacter brockii (TbADH), the mesophilic bacterium Clostridium beijerinckii (CbADH), and the protozoan parasite Entamoeba histolytica (EhADH1) have been exchanged. Three chimeras have been constructed. In the first chimera, the cofactor-binding domain of thermophilic TbADH was replaced with the cofactor-binding domain of its mesophilic counterpart CbADH [chimera Chi21((TCT))]. This domain exchange significantly destabilized the parent thermophilic enzyme (DeltaT(1/2) = -18 degrees C). The reverse exchange in CbADH [chimera Chi22((CTC))], however, had little effect on the thermal stability of the parent mesophilic protein. Furthermore, substituting the cofactor-binding domain of TbADH with the homologous domain of EhADH1 [chimera Chi23((TET))] substantially reduced the thermal stability of the thermophilic ADH (DeltaT(1/2) = -51 degrees C) and impeded the oligomerization of the enzyme. All three chimeric proteins and one of their site-directed mutants were crystallized, and their three-dimensional (3D) structures were determined. Comparison of the 3D structures of the chimeras and the chimeric mutant with the structures of their parent ADHs showed no significant changes to their Calpha chains, suggesting that the difference in the thermal stability of the three parent ADHs and their chimeric mutants could be due to a limited number of substitutions located at strategic positions, mainly at the oligomerization interfaces. Indeed, stabilization of the chimeras was achieved, to a significant extent, either by introduction of a proline residue at a strategic position in the major horse liver ADH-type dimerization interface (DeltaT(1/2) = 35 degrees C) or by introduction of intersubunit electrostatic interactions (DeltaT(1/2) = 6 degrees C).

Expression of the gene for the receptor of gonadotropin-releasing hormone in the rat mammary gland

Recent findings have demonstrated that the GnRH gene is expressed in the mammary gland of pregnant and lactating rats but not of virgin rats. Indeed, significant concentrations of biologically active GnRH have been found in milk of human, cow, sheep and rat. We have, therefore, looked for expression of the GnRH receptor in the rat mammary gland. By reverse transcription (RT)‐PCR amplification, we have demonstrated the presence of GnRH receptor mRNA in mammary gland samples derived from virgin, pregnant and lactating rats. The GnRH receptor transcript cloned from the mammary gland was sequenced and found to have an identical coding region to the one cloned from the pituitary gland. In addition, we have found that the mammary gland, as the pituitary gland, contains at least two transcripts having the same coding region but different 5′ non‐coding regions. Binding studies, however, could demonstrate only low‐affinity binding sites. These results, therefore, suggest that the regulation of the GnRH receptor occurs posttranscriptionally rather than at the level of transcription.

Treatment of murine cytomegalovirus salivary-gland infection by combined therapy with ganciclovir and thymic humoral factor γ2

An optimal therapeutic regimen against primary CMV salivary-gland infection has not yet been developed. We used a murine CMV (MCMV) model system to assess the ability of combined thymic humoral factor THF-gamma 2 immunotherapy and ganciclovir (GCV) antiviral chemotherapy to eliminate detectable viral DNA from salivary glands of infected animals. Mice in different experimental groups were inoculated intraperitoneally with MCMV, treated, and then sacrificed either 2 weeks or 3 months later. To amplify and detect MCMV DNA in infected salivary-gland tissue, we developed a sensitive polymerase chain reaction (PCR) using a glycoprotein B gene primer pair that amplifies a 356 bp segment. During the acute phase of the infection, the detection of high titers of infectious virus in the salivary glands correlated with a strong PCR amplification signal. Although active virions could not be recovered from untreated animals 3 months after viral inoculation, the PCR assay detected a latent MCMV genome. Treatment with either GCV alone or THF-gamma 2 alone had little or no effect on the presence of MCMV DNA. By contrast, combined treatment with THF-gamma 2 and GCV significantly reduced the amount of salivary-gland MCMV DNA to below the limit of PCR detection. The results presented here, and experimental data from previous MCMV research in our laboratories, imply that elimination of the virus from the salivary glands could be due in part to THF-gamma 2 restoration of the various MCMV-suppressed, cell mediated immune-responses. Combining THF-gamma 2 immunotherapy and GCV antiviral chemotherapy may be an important step toward an effective therapeutic regimen that has the potential to prevent the establishment of viral latency ensuing from primary MCMV salivary-gland infection.

Thymic humoral factor, THF-γ2, enhances immunotherapy of murine cytomegalovirus (MCMV) infection by both CD4+ and CD8+ immune T cells

Infection of mice with murine cytomegalovirus (CMV) presents a model for the study of the role of the immune system in the pathogenesis of human CMV. The contribution of the different spleen cell subsets in conferring curative immunocytotherapy to fatally MCMV-infected immunosuppressed mice was assessed using adoptive immunotherapy. It was found that the efficacy of passively transferred immune spleen cells is dose dependent and that the therapeutic effect can be enhanced considerably by treating donor mice with thymic humoral factor (THF-gamma 2). Polymerase chain reaction (PCR) of the donor spleen population was negative, indicating that no MCMV-DNA was transferred with the immune cells. Analysis of the donor mice after THF-gamma 2 treatment showed increased levels of CMV-neutralizing antibodies, while enhancement of natural killer (NK) activity was transient and lasted only during the early phase of the infection. FACS analysis demonstrated that treatment with THF-gamma 2 restored the size of both cell subsets CD4+ and CD8+ that were decreased following MCMV infection. It is shown that both CD4+ and CD8+ T-cell subsets participate in controlling the development of the fatal disease in MCMV-infected immunosuppressed recipients. It is suggested that the enhancement of the immunocompetence of both populations of spleen cells from treated donors is mediated in part by the restoration of Interleukin-2 (IL-2) production by THF-gamma 2.