A chemically diverse conducting polymer-based "electronic nose".

We describe a method for generating a variety of chemically diverse broadly responsive low-power vapor sensors. The chemical polymerization of pyrrole in the presence of plasticizers has yielded conducting organic polymer films whose resistivities are sensitive to the identity and concentration of various vapors in air. An array of such sensing elements produced a chemically reversible diagnostic pattern of electrical resistance changes upon exposure to different odorants. Principal component analysis has demonstrated that such sensors can identify and quantify different airborne organic solvents and can yield information on the components of gas mixtures.

Tumor burden and clonality in multiple intestinal neoplasia mouse/normal mouse aggregation chimeras

Aggregation chimeras were formed between C57BL/6 mice heterozygous for the Apcmin (Min) mutation and wild-type SWR mice, that differ in their Pla2g2a status, a modifier of Apcmin, and also in their resistance to intestinal polyp formation. Variation in the dolichos biflorus agglutinin-staining patterns of the intestines of these mouse strains was used to determine the chimeric composition of the intestine in individual mice and to examine the clonal composition of adenomas. Macroscopic adenoma numbers in chimeric mice were compared with the expected adenoma numbers based on the percentage of C57BL/6J-Apcmin/+ epithelium in individual mice. These results unexpectedly show that there was no apparent inhibitory effect of the SWR-derived (Pla2g2a wild-type) tissue on adenoma formation in the C57BL/6J-Apcmin/+ epithelium. This suggests that the main genetic modifiers of the Min phenotype act at a cellular or crypt-restricted level with no discernable systemic effect. All adenomas were seen to contain C57BL/6J-Apcmin/+-derived epithelium, confirming that the germ-line mutation of the mApc gene is necessary to initiate tumorigenesis in this model system, and that the mApc gene acts in a cell autonomous fashion.

Inorganic polyphosphate kinase is required to stimulate protein degradation and for adaptation to amino acid starvation in Escherichia coli

Inorganic polyphosphate (polyP) kinase was studied for its roles in physiological responses to nutritional deprivation in Escherichia coli. A mutant lacking polyP kinase exhibited an extended lag phase of growth, when shifted from a rich to a minimal medium (nutritional downshift). Supplementation of amino acids to the minimal medium abolished the extended growth lag of the mutant. Levels of the stringent response factor, guanosine 5′-diphosphate 3′-diphosphate, increased in response to the nutritional downshift, but, unlike in the wild type, the levels were sustained in the mutant. These results suggested that the mutant was impaired in the induction of amino acid biosynthetic enzymes. The expression of an amino acid biosynthetic gene, hisG, was examined by using a transcriptional lacZ fusion. Although the mutant did not express the fusion in response to the nutritional downshift, Northern blot analysis revealed a significant increase of hisG-lacZ mRNA. Amino acids generated by intracellular protein degradation are very important for the synthesis of enzymes at the onset of starvation. In the wild type, the rate of protein degradation increased in response to the nutritional downshift whereas it did not in the mutant. Supplementation of amino acids at low concentrations to the minimal medium enabled the mutant to express the hisG-lacZ fusion. Thus, the impaired regulation of protein degradation results in the adaptation defect, suggesting that polyP kinase is required to stimulate protein degradation.

The onset and extent of genomic instability in sporadic colorectal tumor progression

Cancer cell genomes contain alterations beyond known etiologic events, but their total number has been unknown at even the order of magnitude level. By sampling colorectal premalignant polyp and carcinoma cell genomes through use of the technique inter-(simple sequence repeat) PCR, we have found genomic alterations to be considerably more abundant than expected, with the mean number of genomic events per carcinoma cell totaling approximately 11,000. Colonic polyps early in the tumor progression pathway showed similar numbers of events. These results indicate that, as with certain hereditary cancer syndromes, genomic destabilization is an early step in sporadic tumor development. Together these results support the model of genomic instability being a cause rather than an effect of malignancy, facilitating vastly accelerated somatic cell evolution, with the observed orderly steps of the colon cancer progression pathway reflecting the consequences of natural selection.

Phosphohistidyl active sites in polyphosphate kinase of Escherichia coli

In the synthesis of inorganic polyphosphate (polyP) from ATP by polyphosphate kinase (PPK; EC 2.7.4.1) of Escherichia coli, an N—P-linked phosphoenzyme was previously identified as the intermediate. The phosphate is presumed to be linked to N3 of the histidine residue because of its chemical stabilities and its resemblance to other enzymes known to contain N3-phosphohistidine. Tryptic digests of [32P]PPK contain a predominant 32P-labeled peptide that includes His-441. Of the 16 histidine residues in PPK of E. coli, 4 are conserved among several bacterial species. Mutagenesis of these 4 histidines shows that two (His-430 and His-598) are unaffected in function when mutated to glutamine, whereas two others (His-441 and His-460) mutated to glutamine or alanine fail to be phosphorylated, show no enzymatic activities, and fail to support polyP accumulation in cells bearing these mutant enzymes.

Expression of galanin and a galanin receptor in several sensory systems and bone anlage of rat embryos

Using in situ hybridization and immunohistochemistry the expression of, respectively, prepro-galanin (prepro-GAL) mRNA and GAL receptor-1 mRNA, as well as GAL-like and GAL message-associated peptide-like immunoreactivities, were studied in rats from embryonic day 14 (E14) to postnatal day 1. GAL expression was observed already at E14 in trigeminal and dorsal root ganglion neurons and at E15 in the sensory epithelia in developing ear, eye, and nose, as well as at E19 during bone formation. Also, GAL receptor-1 mRNA was expressed in the sensory ganglia of embryos but appeared later than the ligand. These findings suggest that GAL and/or GAL message-associated peptide may have a developmental role in several sensory systems and during bone formation.

Expression of a Hox gene, Cnox-2, and the division of labor in a colonial hydroid

We report the isolation and expression of the Hox gene, Cnox-2, in Hydractinia symbiolongicarpus, a hydrozoan displaying division of labor. We found different patterns of aboral-to-oral Cnox-2 expression among polyp polymorphs, and we show that experimental conversion of one polyp type to another is accompanied by concordant alteration in Cnox-2 expression. Our results are consistent with the suggestion that polyp polymorphism, characteristic of hydractiniid hydroids, arose via evolutionary modification of proportioning of head to body column.

Thymus transplantation, a critical factor for correction of autoimmune disease in aging MRL/+mice.

MRL/MP-+/+ (MRL/+) mice develop pancreatitis and sialoadenitis after they reach 7 months of age. Conventional bone marrow transplantation has been found to be ineffective in the treatment of these forms of apparent autoimmune disease. Old MRL/+ mice show a dramatic thymic involution with age. Hematolymphoid reconstitution is incomplete when fetal liver cells (as a source of hemopoietic stem cells) plus fetal bone (FB; which is used to recruit stromal cells) are transplanted from immunologically normal C57BL/6 donor mice to MRL/+ female recipients. Embryonic thymus from allogeneic C57BL/6 donors was therefore engrafted along with either bone marrow or fetal hematopoietic cells (FHCs) plus fragments of adult or fetal bone. More than seventy percent of old MRL/+ mice (> 7 months) that had been given a fetal thymus (FT) transplant plus either bone marrow or FHCs and also bone fragments survived more than 100 days after treatment. The mice that received FHCs, FB, plus FT from allogeneic donors developed normal T cell and B cell functions. Serum amylase levels decreased in these mice whereas they increased in the mice that received FHCs and FB but not FT. The pancreatitis and sialoadenitis already present at the time of transplantations were fully corrected according to histological analysis by transplants of allogeneic FHCs, FB and FT in the MRL/+ mice. These findings are taken as an experimental indication that perhaps stem cell transplants along with FT grafts might represent a useful strategy for treatment of autoimmune diseases in aged humans.

The chromosomal distribution of mouse odorant receptor genes.

Odorant receptors (ORs) on nasal olfactory sensory neurons are encoded by a large multigene family. Each member of the family is expressed in a small percentage of neurons that are confined to one of several spatial zones in the nose but are randomly distributed throughout that zone. This pattern of expression suggests that when the sensory neuron selects which OR gene to express it may be confined to a particular zonal gene set of several hundred OR genes but select from among the members of that set via a stochastic mechanism. Both locus-dependent and locus-independent models of OR gene choice have been proposed. To investigate the feasibility of these models, we determined the chromosomal locations of 21 OR genes expressed in four different spatial zones. We found that OR genes are clustered within multiple loci that are broadly distributed in the genome. These loci lie within paralogous chromosomal regions that appear to have arisen by duplications of large chromosomal domains followed by extensive gene duplication and divergence. Our studies show that OR genes expressed in the same zone map to numerous loci; moreover, a single locus can contain genes expressed in different zones. These findings raise the possibility that OR gene choice may be locus-independent or involve consecutive stochastic choices.

Molecular cloning of a preprohormone from sea anemones containing numerous copies of a metamorphosis-inducing neuropeptide: a likely role for dipeptidyl aminopeptidase in neuropeptide precursor processing.

Neuropeptides are an important group of hormones mediating or modulating neuronal communication. Neuropeptides are especially abundant in evolutionarily "old" nervous systems, such as those of cnidarians, the lowest animal group having a nervous system. Cnidarians often have a life cycle including a polyp, a medusa, and a planula larva stage. Recently, a neuropeptide, < Glu-Gln-Pro-Gly-Leu-Trp-NH2, has been isolated from sea anemones that induces metamorphosis in a hydroid planula larva to become a hydropolyp [Leitz, T., Morand, K. & Mann, M. (1994) Dev. Biol. 163, 440-446]. Here, we have cloned the precursor protein for this metamorphosis-inducing neuropeptide from sea anemones. The precursor protein is 514-amino acid residues long and contains 10 copies of the immature, authentic neuropeptide (Gln-Gln-Pro-Gly-Leu-Trp-Gly). All neuropeptide copies are preceded by Xaa-Pro or Xaa-Ala sequences, suggesting a role for dipeptidyl aminopeptidase in neuropeptide precursor processing. In addition to these neuropeptide copies, there are 14 copies of another, closely related neuropeptide sequence (Gln-Asn-Pro-Gly-Leu-Trp-Gly). These copies are flanked by basic cleavage sites and, therefore, are likely to be released from the precursor protein. Furthermore, there are 13 other, related neuropeptide sequences having only small sequence variations (the most frequent sequence: Gln-Pro-Gly-Leu-Trp-Gly, eight copies). These variants are preceded by Lys-Arg, Xaa-Ala, or Xaa-Pro sequences, and are followed by basic cleavage sites, and therefore, are also likely to be produced from the precursor. Thus, there are at least 37 closely related neuropeptides localized on the precursor protein, making this precursor one of the most productive preprohormones known so far. This report also shows that unusual processing sites are common in cnidarian preprohormones.

Accumulation of plasma cells in atherosclerotic lesions of Watanabe heritable hyperlipidemic rabbits.

By screening a cDNA library constructed from aortic total RNA derived from Watanabe heritable hyperlipidemic (WHHL) rabbits by differential hybridization, we have obtained a cDNA encoding the kappa light chain of immunoglobulin. Northern blot analysis of total RNA prepared from aortas of WHHL and normal rabbits of various ages revealed that this light-chain mRNA accumulates gradually with age in aortas in WHHL rabbits. Northern blotting and in situ hybridization with an antisense oligonucleotide specific to rabbit immunoglobulin gamma heavy-chain mRNA also detected accumulation of this heavy-chain mRNA in advanced lesions of WHHL rabbit aortas. Moreover, immunohistochemical and electron microscopic analyses demonstrated the presence of plasma cells in the atherosclerotic lesions.

Loss of Apc heterozygosity and abnormal tissue building in nascent intestinal polyps in mice carrying a truncated Apc gene.

Mutations in the APC (adenomatous polyposis coli) gene appear to be responsible for not only familial adenomatous polyposis but also many sporadic cases of gastrointestinal cancers. Using homologous recombination in mouse embryonic stem cells, we constructed mice that contained a mutant gene encoding a product truncated at a 716 (Apc delta 716). Mendelian transmission of the gene caused most homozygous mice to die in utero before day 8 of gestation. The heterozygotes developed multiple polyps throughout the intestinal tract, mostly in the small intestine. The earliest polyps arose multifocally during the third week after birth, and new polyps continued to appear thereafter. Surprisingly, every nascent polyp consisted of a microadenoma covered with a layer of the normal villous epithelium. These microadenomas originated from single crypts by forming abnormal outpockets into the inner (lacteal) side of the neighboring villi. We carefully dissected such microadenomas from nascent polyps by peeling off the normal epithelium and determined their genotype by PCR: all microadenomas had already lost the wild-type Apc allele, whereas the mutant allele remained unchanged. These results indicate that loss of heterozygosity followed by formation of intravillous microadenomas is responsible for polyposis in Apc delta 716 intestinal mucosa. It is therefore unlikely that the truncated product interacts directly with the wild-type protein and causes the microadenomas by a dominant negative mechanism.