Induction of mucosal immune responses against a heterologous antigen fused to filamentous hemagglutinin after intranasal immunization with recombinant Bordetella pertussis.

Live vaccine vectors are usually very effective and generally elicit immune responses of higher magnitude and longer duration than nonliving vectors. Consequently, much attention has been turned to the engineering of oral pathogens for the delivery of foreign antigens to the gut-associated lymphoid tissues. However, no bacterial vector has yet been designed to specifically take advantage of the nasal route of mucosal vaccination. Herein we describe a genetic system for the expression of heterologous antigens fused to the filamentous hemagglutinin (FHA) in Bordetella pertussis. The Schistosoma mansoni glutathione S-transferase (Sm28GST) fused to FHA was detected at the cell surface and in the culture supernatants of recombinant B. pertussis. The mouse colonization capacity and autoagglutination of the recombinant microorganism were indistinguishable from those of the wild-type strain. In addition, and in contrast to the wild-type strain, a single intranasal administration of the recombinant strain induced both IgA and IgG antibodies against Sm28GST and against FHA in the bronchoalveolar lavage fluids. No anti-Sm28GST antibodies were detected in the serum, strongly suggesting that the observed immune response was of mucosal origin. This demonstrates, to our knowledge, for the first time that recombinant respiratory pathogens can induce mucosal immune responses against heterologous antigens, and this may constitute a first step toward the development of combined live vaccines administrable via the respiratory route.

Mouse mammary tumor virus/v-Ha-ras transgene-induced mammary tumors exhibit strain-specific allelic loss on mouse chromosome 4

Hybrid mice carrying oncogenic transgenes afford powerful systems for investigating loss of heterozygosity (LOH) in tumors. Here, we apply this approach to a neoplasm of key importance in human medicine: mammary carcinoma. We performed a whole genome search for LOH using the mouse mammary tumor virus/v-Ha-ras mammary carcinoma model in female (FVB/N × Mus musculus castaneus)F1 mice. Mammary tumors developed as expected, as well as a few tumors of a second type (uterine leiomyosarcoma) not previously associated with this transgene. Genotyping of 94 anatomically independent tumors revealed high-frequency LOH (≈38%) for markers on chromosome 4. A marked allelic bias was observed, with M. musculus castaneus alleles almost exclusively being lost. No evidence of genomic imprinting effects was noted. These data point to the presence of a tumor suppressor gene(s) on mouse chromosome 4 involved in mammary carcinogenesis induced by mutant H-ras expression, and for which a significant functional difference may exist between the M. musculus castaneus and FVB/N alleles. Provisional subchromosomal localization of this gene, designated Loh-3, can be made to a distal segment having syntenic correspondence to human chromosome 1p; LOH in this latter region is observed in several human malignancies, including breast cancers. Evidence was also obtained for a possible second locus associated with LOH with less marked allele bias on proximal chromosome 4.

Isolation of quelling-defective (qde) mutants impaired in posttranscriptional transgene-induced gene silencing in Neurospora crassa

We report the isolation of 15 Neurospora crassa mutants defective in “quelling” or transgene-induced gene silencing. These quelling-defective mutants (qde) belonging to three complementation groups have provided insights into the mechanism of posttranscriptional gene silencing in N. crassa. The recessive nature of the qde mutations indicates that the encoded gene products act in trans. We show that when qde genes are mutated in a transgenic-induced silenced strain containing many copies of the transgene, the expression of the endogenous gene is maintained despite the presence of transgene sense RNA, the molecule proposed to trigger quelling. Moreover, the qde mutants failed to show quelling when tested with another gene, suggesting that they may be universally defective in transgene-induced gene silencing. As such, qde genes may be involved in sensing aberrant sense RNA and/or targeting/degrading the native mRNA. The qde mutations may be used to isolate the genes encoding the first components of the quelling mechanism. Moreover, these quelling mutants may be important in applied and basic research for the creation of strains able to overexpress a transgene.

Strain-specific differences in mouse hepatic wound healing are mediated by divergent T helper cytokine responses

Hepatic fibrosis represents the generalized response of the liver to injury and is characterized by excessive deposition of extracellular matrix. The cellular basis of this process is complex and involves interplay of many factors, of which cytokines are prominent. We have identified divergent fibrosing responses to injury among mouse strains and taken advantage of these differences to examine and contrast T helper (Th)-derived cytokines during fibrogenesis. Liver injury was induced with carbon tetrachloride, fibrosis was quantitated, and Th1/Th2 cytokine mRNAs measured. Liver injury in BALB/c mice resulted in severe fibrosis, whereas C57BL/6 mice developed comparatively minimal fibrosis. Fibrogenesis was significantly modified in T and B cell-deficient BALB/c and C57BL/6 severe combined immunodeficient (SCID) mice compared with wild-type counterparts, suggesting a role of Th subsets. Fibrogenic BALB/c mice exhibited a Th2 response during the wounding response, whereas C57BL/6 mice displayed a Th1 response, suggesting that hepatic fibrosis is influenced by different T helper subsets. Moreover, mice lacking interferon γ, which default to the Th2 cytokine pathway, exhibited more pronounced fibrotic lesions than did wild-type animals. Finally, shifting of the Th2 response toward a Th1 response by treatment with neutralizing anti-interleukin 4 or with interferon γ itself ameliorated fibrosis in BALB/c mice. These data support a role for immune modulation of hepatic fibrosis and suggest that Th cytokine subsets can modulate the fibrotic response to injury.

Identification of an ATP-binding cassette transporter involved in bicarbonate uptake in the cyanobacterium Synechococcus sp. strain PCC 7942

Exposure of cells of cyanobacteria (blue–green algae) grown under high-CO2 conditions to inorganic C-limitation induces transcription of particular genes and expression of high-affinity CO2 and HCO3− transport systems. Among the low-CO2-inducible transcription units of Synechococcus sp. strain PCC 7942 is the cmpABCD operon, encoding an ATP-binding cassette transporter similar to the nitrate/nitrite transporter of the same cyanobacterium. A nitrogen-regulated promoter was used to selectively induce expression of the cmpABCD genes by growth of transgenic cells on nitrate under high CO2 conditions. Measurements of the initial rate of HCO3− uptake after onset of light, and of the steady-state rate of HCO3− uptake in the light, showed that the controlled induction of the cmp genes resulted in selective expression of high-affinity HCO3− transport activity. The forced expression of cmpABCD did not significantly increase the CO2 uptake capabilities of the cells. These findings demonstrated that the cmpABCD genes encode a high-affinity HCO3− transporter. A deletion mutant of cmpAB (M42) retained low CO2-inducible activity of HCO3− transport, indicating the occurrence of HCO3− transporter(s) distinct from the one encoded by cmpABCD. HCO3− uptake by low-CO2-induced M42 cells showed lower affinity for external HCO3− than for wild-type cells under the same conditions, showing that the HCO3− transporter encoded by cmpABCD has the highest affinity for HCO3− among the HCO3− transporters present in the cyanobacterium. This appears to be the first unambiguous identification and description of a primary active HCO3− transporter.

Genome scan for teratogen-induced clefting susceptibility loci in the mouse: Evidence of both allelic and locus heterogeneity distinguishing cleft lip and cleft palate

Nonsyndromic clefting of the lip and palate in humans has a highly complex etiology, with both multiple genetic loci and exposure to teratogens influencing susceptibility. Previous studies using mouse models have examined only very small portions of the genome. Here we report the findings of a genome-wide search for susceptibility genes for teratogen-induced clefting in the AXB and BXA set of recombinant inbred mouse strains. We compare results obtained using phenytoin (which induces cleft lip) and 6-aminonicotinamide (which induces cleft palate). We use a new statistical approach based on logistic regression suitable for these categorical data to identify several chromosomal regions as possible locations of clefting susceptibility loci, and we review candidate genes located within each region. Because cleft lip and cleft palate do not frequently co-aggregate in human families and because these structures arise semi-independently during development, these disorders are usually considered to be distinct in etiology. Our data, however, implicate several of the same chromosomal regions for both forms of clefting when teratogen-induced. Furthermore, different parental strain alleles are usually associated with clefting of the lip versus that of the palate (i.e., allelic heterogeneity). Because several other chromosomal regions are associated with only one form of clefting, locus heterogeneity also appears to be involved. Our findings in this mouse model suggest several priority areas for evaluation in human epidemiological studies.

The PDE1-encoded Low-Affinity Phosphodiesterase in the Yeast Saccharomyces cerevisiae Has a Specific Function in Controlling Agonist-induced cAMP Signaling

The yeast Saccharomyces cerevisiae contains two genes, PDE1 and PDE2, which respectively encode a low-affinity and a high-affinity cAMP phosphodiesterase. The physiological function of the low-affinity enzyme Pde1 is unclear. We show that deletion of PDE1, but not PDE2, results in a much higher cAMP accumulation upon addition of glucose or upon intracellular acidification. Overexpression of PDE1, but not PDE2, abolished the agonist-induced cAMP increases. These results indicate a specific role for Pde1 in controlling glucose and intracellular acidification-induced cAMP signaling. Elimination of a putative protein kinase A (PKA) phosphorylation site by mutagenesis of serine252 into alanine resulted in a Pde1ala252 allele that apparently had reduced activity in vivo. Its presence in a wild-type strain partially enhanced the agonist-induced cAMP increases compared with pde1Δ. The difference between the Pde1ala252 allele and wild-type Pde1 was strongly dependent on PKA activity. In a RAS2val19 pde2Δ background, the Pde1ala252 allele caused nearly the same hyperaccumulation of cAMP as pde1Δ, while its expression in a PKA-attenuated strain caused the same reduction in cAMP hyperaccumulation as wild-type Pde1. These results suggest that serine252 might be the first target site for feedback inhibition of cAMP accumulation by PKA. We show that Pde1 is rapidly phosphorylated in vivo upon addition of glucose to glycerol-grown cells, and this activation is absent in the Pde1ala252 mutant. Pde1 belongs to a separate class of phosphodiesterases and is the first member shown to be phosphorylated. However, in vitro the Pde1ala252 enzyme had the same catalytic activity as wild-type Pde1, both in crude extracts and after extensive purification. This indicates that the effects of the S252A mutation are not caused by simple inactivation of the enzyme. In vitro phosphorylation of Pde1 resulted in a modest and variable increase in activity, but only in crude extracts. This was absent in Pde1ala252, and phosphate incorporation was strongly reduced. Apparently, phosphorylation of Pde1 does not change its intrinsic activity or affinity for cAMP but appears to be important in vivo for protein-protein interaction or for targeting Pde1 to a specific subcellular location. The PKA recognition site is conserved in the corresponding region of the Schizosaccharomyces pombe and Candida albicans Pde1 homologues, possibly indicating a similar control by phosphorylation.

Localization of non-Mhc collagen-induced arthritis susceptibility loci in DBA/1j mice

One approach to understanding common human diseases is to determine the genetic defects responsible for similar diseases in animal models and place those defective genes in their corresponding biochemical pathways. Our laboratory is working with an animal model for human rheumatoid arthritis called collagen-induced arthritis (CIA). We are particularly interested in determining the location of disease-predisposing loci. To that end, we performed experiments to localize susceptibility loci for CIA in an F2 cross between the highly susceptible mouse strain DBA/1j and the highly resistant mouse strain SWR/j. Specifically, a quantitative trait locus analysis was performed to localize regions of the mouse genome responsible for susceptibility/severity to CIA. One susceptibility locus, Cia1 in the major histocompatibility locus, had been identified previously. Two additional loci were detected in our analysis that contribute to CIA severity (Cia2, Cia3) on chromosomes 2 and 6. A third locus was detected that contributes to the age of onset of the disease. This locus (Cia4) was located on chromosome 2 and was linked to the same region as Cia2. Determining the identity of these loci may provide insights into the etiology of human rheumatoid arthritis.

Mapping the parameters of prion-induced neuropathology

We present a theoretical framework that enables us to dissect out the parametric dependencies of the pathogenesis of prion diseases. We are able to determine the influence of both host-dependent factors (connectivity, cell density, protein synthesis rate, and cell death) and strain-dependent factors (cell tropism, virulence, and replication rate). We use a model based on a linked system of differential equations on a lattice to explore how the regional distribution of central nervous system pathology in Creutzfeldt-Jakob disease, Gerstmann-Sträussler-Scheinker syndrome, and fatal familial insomnia relates to each of these factors. The model then is used to make qualitative predictions about the pathology for two possible hypothetical triggers of neuronal loss in prion diseases. Pathological progression in overexpressing mouse models has been shown to depend on the site of initial infection. The model allows us to compare the pathologies resulting from different inoculation routes.

An Arabidopsis GSK3/shaggy-Like Gene That Complements Yeast Salt Stress-Sensitive Mutants Is Induced by NaCl and Abscisic Acid

GSK3/shaggy-like genes encode kinases that are involved in a variety of biological processes. By functional complementation of the yeast calcineurin mutant strain DHT22-1a with a NaCl stress-sensitive phenotype, we isolated the Arabidopsis cDNA AtGSK1, which encodes a GSK3/shaggy-like protein kinase. AtGSK1 rescued the yeast calcineurin mutant cells from the effects of high NaCl. Also, the AtGSK1 gene turned on the transcription of the NaCl stress-inducible PMR2A gene in the calcineurin mutant cells under NaCl stress. To further define the role of AtGSK1 in the yeast cells we introduced a deletion mutation at the MCK1 gene, a yeast homolog of GSK3, and examined the phenotype of the mutant. The mck1 mutant exhibited a NaCl stress-sensitive phenotype that was rescued by AtGSK1. Also, constitutive expression of MCK1 complemented the NaCl-sensitive phenotype of the calcineurin mutants. Therefore, these results suggest that Mck1p is involved in the NaCl stress signaling in yeast and that AtGSK1 may functionally replace Mck1p in the NaCl stress response in the calcineurin mutant. To investigate the biological function of AtGSK1 in Arabidopsis we examined the expression of AtGSK1. Northern-blot analysis revealed that the expression is differentially regulated in various tissues with a high level expression in flower tissues. In addition, the AtGSK1 expression was induced by NaCl and exogenously applied ABA but not by KCl. Taken together, these results suggest that AtGSK1 is involved in the osmotic stress response in Arabidopsis.

A Cyanobacterium Lacking Iron Superoxide Dismutase Is Sensitized to Oxidative Stress Induced with Methyl Viologen but Is Not Sensitized to Oxidative Stress Induced with Norflurazon1

A strain of Synechococcus sp. strain PCC 7942 with no functional Fe superoxide dismutase (SOD), designated sodB−, was characterized by its growth rate, photosynthetic pigments, and cyclic photosynthetic electron transport activity when treated with methyl viologen or norflurazon (NF). In their unstressed conditions, both the sodB− and wild-type strains had similar chlorophyll and carotenoid contents and catalase activity, but the wild type had a faster growth rate and higher cyclic electron transport activity. The sodB− was very sensitive to methyl viologen, indicating a specific role for the FeSOD in protection against superoxide generated in the cytosol. In contrast, the sodB− mutant was less sensitive than the wild type to oxidative stress imposed with NF. This suggests that the FeSOD does not protect the cell from excited singlet-state oxygen generated within the thylakoid membrane. Another up-regulated antioxidant, possibly the MnSOD, may confer protection against NF in the sodB− strain. These results support the hypothesis that different SODs have specific protective functions within the cell.

Break-induced replication: A review and an example in budding yeast

Break-induced replication (BIR) is a nonreciprocal recombination-dependent replication process that is an effective mechanism to repair a broken chromosome. We review key roles played by BIR in maintaining genome integrity, including restarting DNA replication at broken replication forks and maintaining telomeres in the absence of telomerase. Previous studies suggested that gene targeting does not occur by simple crossings-over between ends of the linearized transforming fragment and the target chromosome, but involves extensive new DNA synthesis resembling BIR. We examined gene targeting in Saccharomyces cerevisiae where only one end of the transformed DNA has homology to chromosomal sequences. Linearized, centromere-containing plasmid DNA with the 5′ end of the LEU2 gene at one end was transformed into a strain in which the 5′ end of LEU2 was replaced by ADE1, preventing simple homologous gene replacement to become Leu2+. Ade1+ Leu2+ transformants were recovered in which the entire LEU2 gene and as much as 7 kb of additional sequences were found on the plasmid, joined by microhomologies characteristic of nonhomologous end-joining (NHEJ). In other experiments, cells were transformed with DNA fragments lacking an ARS and homologous to only 50 bp of ADE2 added to the ends of a URA3 gene. Autonomously replicating circles were recovered, containing URA3 and as much as 8 kb of ADE2-adjacent sequences, including a nearby ARS, copied from chromosomal DNA. Thus, the end of a linearized DNA fragment can initiate new DNA synthesis by BIR in which the newly synthesized DNA is displaced and subsequently forms circles by NHEJ.

Characterization of the gene cluster of high-molecular-mass nitrile hydratase (H-NHase) induced by its reaction product in Rhodococcus rhodochrous J1.

The 4.6-kb region 5'-upstream from the gene encoding a cobalt-containing and amide-induced high molecular mass-nitrile hydratase (H-NHase) from Rhodococcus rhodochrous J1 was found to be required for the expression of the H-NHase gene with a host-vector system in a Rhodococcus strain. Sequence analysis has revealed that there are at least five open reading frames (H-ORF1 approximately 5) in addition to H-NHase alpha- and beta-subunit genes. Deletion of H-ORF1 and H-ORF2 resulted in decrease of NHase activity, suggesting a positive regulatory role of both ORFs in the expression of the H-NHase gene. H-ORF1 showed significant similarity to a regulatory protein, AmiC, which is involved in regulation of amidase expression by binding an inducer amide in Pseudomonas aeruginosa. H-ORF4, which has been found to be uninvolved in regulation of H-NHase expression by enzyme assay for its deletion transformant and Northern blot analysis for R. rhodochrous J1, showed high similarity to transposases from insertion sequences of several bacteria. Determination of H-NHase activity and H-NHase mRNA levels in R. rhodochrous J1 has indicated that the expression of the H-NHase gene is regulated by an amide at the transcriptional level. These findings suggest the participation of H-ORF4 (IS1164) in the organization of the H-NHase gene cluster and the involvement of H-ORF1 in unusual induction mechanism, in which H-NHase is formed by amides (the products in the NHase reaction), but not by nitriles (the substrates).

Anti-idiotypic antibodies mimicking glycoprotein D of herpes simplex virus identify a cellular protein required for virus spread from cell to cell and virus-induced polykaryocytosis.

Glycoprotein D (gD) of herpes simplex virus 1 (HSV-1) is required for stable attachment and penetration of the virus into susceptible cells after initial binding. We derived anti-idiotypic antibodies to the neutralizing monoclonal antibody HD1 to gD of HSV-1. These antibodies have the properties expected of antibodies against a gD receptor. Specifically, they bind to the surface of HEp-2, Vero, and HeLa cells susceptible to HSV infection and specifically react with a Mr 62,000 protein in these and other (143TK- and BHK) cell lines. They neutralize virion infectivity, drastically decrease plaque formation by impairing cell-to-cell spread of virions, and reduce polykaryocytosis induced by strain HFEM, which carries a syncytial (syn-) mutation. They do not affect HSV growth in a single-step cycle and plaque formation by an unrelated virus, indicating that they specifically affect the interaction of HSV gD) with a cell surface receptor. We conclude that the Mr 62,000 cell surface protein interacts with gD to enable spread of HSV-1 from cell to cell and virus-induced polykaryocytosis.

Mice lacking inducible nitric oxide synthase are not resistant to lipopolysaccharide-induced death.

Nitric oxide produced by cytokine-inducible nitric oxide synthase (iNOS) is thought to be important in the pathogenesis of septic shock. To further our understanding of the role of iNOS in normal biology and in a variety of inflammatory disorders, including septic shock, we have used gene targeting to generate a mouse strain that lacks iNOS. Mice lacking iNOS were indistinguishable from wild-type mice in appearance and histology. Upon treatment with lipopolysaccharide and interferon gamma, peritoneal macrophages from the mutant mice did not produce nitric oxide measured as nitrite in the culture medium. In addition, lysates of these cells did not contain iNOS protein by immunoblot analysis or iNOS enzyme activity. In a Northern analysis of total RNA, no iNOS transcript of the correct size was detected. No increases in serum nitrite plus nitrate levels were observed in homozygous mutant mice treated with a lethal dose of lipopolysaccharide, but the mutant mice exhibited no significant survival advantage over wild-type mice. These results show that lack of iNOS activity does not prevent mortality in this murine model for septic shock.