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Biomedical subjects

B J Baker

Publications and source records attributed to B J Baker.

At least 19 recordsLinked to original sources
PubMed2006

Three fluorescent protein voltage sensors exhibit low plasma membrane expression in mammalian cells.

Three first-generation fluorescent protein voltage sensitive probes (FP-voltage sensors) were characterized in mammalian cells. Flare, a Kv1.4 variant of FlaSh [Siegel MS, Isacoff EY. Neuron 1997;19(October (4)):735-41], SPARC [Ataka K, Pieribone VA. Biophys J 2002;82(January (1 Pt 1)):509-16], and VSFP-1 [Sakai R, Repunte-Canonigo V, Raj CD, Knopfel T. Eur J Neurosci 2001;13(June (12)):2314-18] were expressed, imaged and voltage clamped in HEK 293 cells and in dissociated hippocampal neurons. We were unable to detect a signal in response to changes in membrane potential after averaging16 trials with any of the three constructs. Using the hydrophobic voltage sensitive dye, di8-ANEPPS, as a surface marker, confocal analyses demonstrated poor plasma membrane expression for Flare, SPARC and VSFP-1 in both HEK 293 cells and dissociated hippocampal neurons. Almost all of the expressed FP-voltage sensors reside in internal membranes in both cell types. This internal expression generates a background fluorescence that increases the noise in the optical measurement.

Animals↗
PubMed2006

Activators of viral gene expression in polarized epithelial monolayers identified by rapid-throughput drug screening.

Epithelial polarity and tight junction formation limit the ability of adenovirus, retrovirus and adeno-associated virus (AAV) to deliver and express virally encoded genes. Using an extended half-life luciferase assay and high-throughput luminometry, we screened 23 000 compounds and natural product extracts as potentiators to overcome this barrier. Seven strong activators were discovered (up to several hundred fold above control) and two of these exhibited spectrum of activity in multiple cell types (HeLa (human cervical carcinoma), cystic fibrosis bronchial epithelial (human bronchial), HT29 (human colonic carcinoma), Calu3 (airway serous glandular)). Enhanced transduction by unrelated gene transfer vectors (adenovirus, lentivirus, AAV, liposomal) was also observed. These results establish a strategy for identifying compounds that improve viral gene transfer to resistant cell types, and provide new tools for examining epithelial defense against viral infection. The compounds should have broad usefulness in experimental therapies for cancer and genetic diseases.

Adenoviridae↗
PubMed2001

Chromosome landing at the tomato Bs4 locus.

The tomato (Lycopersicon esculentum) Bs4 gene confers resistance to strains of Xanthomonas campestris pathovar vesicatoria that express the avirulence protein AvrBs4. As part of a map-based cloning strategy for the isolation of Bs4, we converted Bs4-linked amplified fragment length polymorphism (AFLP) and restriction fragment length polymorphism (RFLP) markers into locus-specific sequence-tagged-site (STS) markers. The use of these markers for the analysis of 1972 meiotic events allowed high-resolution genetic mapping within a 1.2-cM interval containing the target gene. Two tomato yeast artificial chromosome (YAC) clones, each harboring inserts of approximately 250 kb, were identified using the marker most closely linked to Bs4. YAC end-specific markers were established and employed to construct a local YAC contig. The ratio of physical to genetic distance at Bs4 was calculated to be 280 kb/cM, revealing that recombination rates in this region are about three times higher than the genome-wide average. Mapping of YAC end-derived markers demonstrated that the Bs4 locus maps within a region of 250 kb, corresponding to a genetic interval of 0.9 cM.

Chromosome Mapping↗
PubMed2001

Microbiological, molecular biological and stable isotopic evidence for nitrogen fixation in the open waters of Lake Michigan.

We have used a combination of microbiological, molecular biological and stable isotope methods to relate specific microbial populations to elemental cycling at an offshore site in Lake Michigan. Several lines of evidence suggest that atmospheric N2 may be a significant source of nitrogen to the lake. Particulate organic nitrogen (PON) at approximately equals 10-15m depth in July and October had a delta15N of 0.5-1.5%o. These values closely reflect the 15N composition of atmospheric N2, suggesting biological nitrogen fixation. Historical data show a developing late-summer N:P minimum at approximately equals 15 m; low abundance of inorganic nitrogen relative to phosphorus favours species able to acquire atmospheric nitrogen. Microscopic examination of October water samples revealed abundant heterocystous cyanobacteria, including Nodularia sp. Potentially nitrogen-fixing Anabaena spp. have been found in Lake Michigan before but, to our knowledge, this is the first report of Nodularia. Finally, we have amplified both cyanobacterial and non-cyanobacterial nifH sequences (encoding the nitrogenase iron protein) from lakewater samples, evidence for the presence of bacteria capable of nitrogen fixation. The surface waters of Lake Michigan are considered to be phosphate limited in the stratified season and, under these conditions, energetically expensive nitrogen fixation is expected to be uncompetitive with assimilation of combined nitrogen. Our results suggest that, from both microbiological and biogeochemical perspectives, this may be an oversimplification.

Animals↗
PubMed2001

Seasonal and spatial variability in Lake Michigan sediment small-subunit rRNA concentrations.

We have used molecular biological methods to study the distribution of microbial small-subunit rRNAs (SSU rRNAs), in relation to chemical profiles, in offshore Lake Michigan sediments. The sampling site is at a depth of 100 m, with temperatures of 2 to 4 degrees C year-round. RNA extracted from sediment was probed with radiolabeled oligonucleotides targeting bacterial, archaeal, and eukaryotic SSU rRNAs, as well as with a universal probe. The coverage of these probes in relation to the present sequence database is discussed. Because ribosome production is growth rate regulated, rRNA concentrations are an indicator of the microbial populations active in situ. Over a 1-year period, changes in sedimentary SSU rRNA concentrations followed seasonal changes in surface water temperature and SSU rRNA concentration. Sedimentary depth profiles of oxygen, reduced manganese and iron, and sulfate changed relatively little from season to season, but the nitrate concentration was approximately fivefold higher in April and June 1997 than at the other times sampling was done. We propose that sediment microbial SSU rRNA concentrations at our sampling site are influenced by seasonal inputs from the water column, particularly the settling of the spring diatom bloom, and that the timing of this input may be modulated by grazers, such that ammonia becomes available to sediment microbes sooner than fresh organic carbon. Nitrate production from ammonia by autotrophic nitrifying bacteria, combined with low activity of heterotrophic denitrifying bacteria in the absence of readily degradable organic carbon, could account for the cooccurrence of high nitrate and low SSU rRNA concentrations.

Animals↗
PubMed2000

Structure-function analysis of the tobacco mosaic virus resistance gene N.

The tobacco N gene is a member of the Toll-interleukin-1 receptor/nucleotide-binding site/leucine-rich repeat (TIR-NBS-LRR) class of plant resistance (R) genes and confers resistance to tobacco mosaic virus (TMV). We investigated the importance of specific domains of N in inducing TMV resistance, by examining various N deletion and point mutations that introduce single amino acid substitution mutants in vivo. Our deletion analysis suggests that the TIR, NBS, and LRR domains play an indispensable role in the induction of resistance responses against TMV. We show that amino acids conserved among the Toll/IL-1R/plant R gene TIR domain and NBS-containing proteins play a critical role in N-mediated TMV resistance. Some loss-of-function N alleles such as the TIR deletion and point mutations in the NBS (G216A/E/V/R, G218R, G219D, K222E/N, and T223A/N) interfere with the wild-type N function and behave like dominant negative mutations. These F(1) plants mount a hypersensitive response (HR) that is indistinguishable from that of the wild-type N plants, yet TMV was able to move systemically, causing a systemic hypersensitive response (SHR). Many amino acid substitutions in the TIR, NBS, and LRR domains of N lead to a partial loss-of-function phenotype. These mutant plants mount delayed HR compared with the wild-type N plants and fail to contain the virus to the infection site. In addition, some partial loss-of-function alleles (W82S/A, W141S/A, G218V/S, and G219V) interfere with the wild-type N function, leading to SHR. The partial loss-of-function and dominant negative mutant alleles described in this report will be useful in furthering our understanding of the TIR-NBS-LRR class of R genes.

Amino Acid Sequence↗
PubMed2000

Alternatively spliced N resistance gene transcripts: their possible role in tobacco mosaic virus resistance.

The N gene, a member of the Toll-IL-1 homology region-nucleotide binding site-leucine-rich repeat region (LRR) class of plant resistance genes, encodes two transcripts, N(S) and N(L), via alternative splicing of the alternative exon present in the intron III. The N(S) transcript, predicted to encode the full-length N protein containing the Toll-IL-1 homology region, nucleotide binding site, and LRR, is more prevalent before and for 3 hr after tobacco mosaic virus (TMV) infection. The N(L) transcript, predicted to encode a truncated N protein (N(tr)) lacking 13 of the 14 repeats of the LRR, is more prevalent 4-8 hr after TMV infection. Plants harboring a cDNA-N(S) transgene, capable of encoding an N protein but not an N(tr) protein, fail to exhibit complete resistance to TMV. Transgenic plants containing a cDNA-N(S)-bearing intron III and containing 3' N-genomic sequences, encoding both N(S) and N(L) transcripts, exhibit complete resistance to TMV. These results suggest that both N transcripts and presumably their encoded protein products are necessary to confer complete resistance to TMV.

Alternative Splicing↗
PubMed1999

Interactions between tobacco mosaic virus and the tobacco N gene.

The interaction between tobacco mosaic virus (TMV) and tobacco harbouring the N gene is a classical system for studying gene-for-gene interactions in disease resistance. The N gene confers resistance to TMV by mediating defence responses that function to limit viral replication and movement. We isolated the N gene and determined that N belongs to the nucleotide-binding-site-leucine-rich-repeat (NBS-LRR) class of plant disease resistance genes, and encodes both full-length and truncated proteins. Sequence homologies and mutagenesis studies indicated a signalling role for the N protein similar to that seen for proteins involved in defence responses in insects and mammals. The N gene confers resistance to TMV in transgenic tomato, demonstrating the use of the NBS-LRR class of disease resistance genes in engineering crop resistance. From the pathogen side of this interaction, the TMV 126 kDa replicase protein has been implicated as the avirulence factor that triggers N-mediated defence responses. We employed Agrobacterium-mediated expression strategies to demonstrate that expression of the putative helicase region of the replicase protein is sufficient to elicit N-mediated defences. The thermosensitivity of the N-mediated response to TMV is retained when induced by expression of this replicase fragment. Thus, both components of this gene-for-gene interaction are now available for studies that address the molecular mechanisms involved in N-mediated TMV resistance.

Gene Transfer Techniques↗
PubMed1998

Purine and nucleoside metabolites from the Antarctic sponge Isodictya erinacea.

The bright yellow sponge Isodictya erinacea is one of several chemically defended sponges found on the benthos of McMurdo Sound, Antarctica. An investigation of the metabolites from this sponge has resulted in the isolation of purine and nucleoside metabolites, including the previously unreported erinacean (1) and p-hydroxybenzaldehyde. The latter metabolite has been demonstrated to cause a feeding deterrence behavior in Perknaster fuscus, the major predator of antarctic sponges.

Animals↗
PubMed1997

Isolation, structure elucidation, and biological activity of the steroid oligoglycosides and polyhydroxysteroids from the Antarctic starfish Acodontaster conspicuus.

A total of 19 steroids, of which 13 steroidal oligoglycosides (nine new and four known) and six polyhydroxylated steroids (four new and two known), has been isolated from the Antarctic starfish Acodontaster conspicuus. The mixture is dominated by glycosides composed of steroidal aglycons having the hydroxyl groups typically disposed on one side of the tetracyclic nucleus, i.e., 3 beta,4 beta,6 alpha,8,15 beta-, with some having a sulfate at C-6, and differing in the side chains and/or in the disaccharide moieties that are usually attached at C-26, with some at C-28 and C-29. Those compounds are accompanied by minute amounts of glycosides with a delta 8(14)-double bond in the steroid, which is a structural feature not previously found among polyhydroxysteroids derived from starfish. Small amounts of six related unglycosidated polyhydroxysteroids and three higher-molecular-weight asterosaponins complete the composition of the mixture. The structures of the new compounds were determined by interpretation of their spectral data and by comparison with spectral data of known compounds. Eighteen of these compounds were evaluated for their ability to inhibit growth in Antarctic marine bacteria isolated from either the water column or the surfaces of benthic marine invertebrates. Of these compounds, 50% were active against at least one Antarctic marine bacterium. This suggests that these compounds may play an important role in deterring microbial fouling.

Animals↗
PubMed1996

Metabolites from an Antarctic sponge-associated bacterium, Pseudomonas aeruginosa.

In an ongoing survey of the bioactive potential of microorganisms associated with marine invertebrates, the culture media of a sponge-associated bacterial strain of Pseudomonas aeruginosa was found to contain metabolites which inhibit the growth of several Gram-positive microorganisms. A series of diketopiperazines (1-6) including a new natural product (6) and two known phenazine alkaloid antibiotics (7 and 8) were isolated from the culture broth of this bacterium.

Alkaloids↗
PubMed1995

Molecular genetics of plant disease resistance.

Plant breeders have used disease resistance genes (R genes) to control plant disease since the turn of the century. Molecular cloning of R genes that enable plants to resist a diverse range of pathogens has revealed that the proteins encoded by these genes have several features in common. These findings suggest that plants may have evolved common signal transduction mechanisms for the expression of resistance to a wide range of unrelated pathogens. Characterization of the molecular signals involved in pathogen recognition and of the molecular events that specify the expression of resistance may lead to novel strategies for plant disease control.

Amino Acid Sequence↗
PubMed1995

Nucleotide sequence encoding the snake venom fibrinolytic enzyme atroxase obtained from a Crotalus atrox venom gland cDNA library.

Atroxase, isolated from the venom of Crotalus atrox (western diamondback rattlesnake), is a nonhemorrhagic protease which has fibrinolytic activity in vivo. The primary structure of atroxase was deduced from the cDNA encoding the atroxase protein. The venom glands of Crotalus atrox were used to prepare a cDNA library. Degenerate oligonucleotides were synthesized based on the partial amino acid sequence of atroxase and were used as primers in the polymerase chain reaction to amplify overlapping cDNA fragments from the C. atrox cDNA library. The resulting cDNA fragments were subcloned, sequenced, and translated. The final nucleotide sequence shows high homology to previously described primary structures of nonhemorrhagic fibrinolytic proteases isolated from snake venom.

Amino Acid Sequence↗
PubMed1995

Complement component C4 gene intron 9 as a phylogenetic marker for primates: long terminal repeats of the endogenous retrovirus ERV-K(C4) are a molecular clock of evolution.

The complement component C4 genes of Old World primates exhibit a long/short dichotomous size variation, except that chimpanzee and gorilla only contain short C4 genes. In human it has been shown that the long C4 gene is attributed to the integration of an endogenous retrovirus, HERV-K(C4), into intron 9. This 6.36 kilobase retroviral element is absent in short C4 genes. Here it is shown that the homologous endogenous retrovirus, ERV-K(C4), is present precisely at the same position in the long C4 gene of orangutan and African green monkey. Determination of the short C4 gene intron 9 sequences from human, three apes, two Old World monkeys, and a New World monkey allowed the establishment of consistent phylogenetic trees for primates, which favors a chimpanzee-gorilla clade. The 5' long terminal repeats (LTR) and 3' LTR of ERV-K(C4) in long C4 genes of human, orangutan, and African green monkey have similar sequence divergence values of 9.1%-10.5%. These values are more than five-fold higher than the sequence divergence of the homologous intron 9 sequences between the long and short C4 genes in higher primates. The latter is probably a result of homogenization or concerted evolution. We suggest that the 5' LTR and 3' LTR of an endogenous retrovirus can serve as a reliable reference point or a molecular clock for studies of gene duplication and gene evolution. This is because the 5'/3' LTR sequences were identical at the time of retroviral integration and evolved independently of each other afterwards. Our data provides strong evidence for the short C4 gene being the ancestral form in primates, trans-species evolution, and the "slow-down" phenomenon of the sequence divergence in great apes.

Amino Acid Sequence↗
PubMed1994

Allografts of CNS tissue possess a blood-brain barrier: III. Neuropathological, methodological, and immunological considerations.

Development of a blood-brain barrier (BBB) within mammalian CNS grafts, placed either intracerebrally or peripherally, has been controversial. Published data from this laboratory have emphasized the presence or the absence of a BBB within solid mammalian tissue or cell suspension grafts is determined intrinsically by the graft and not by the surrounding host parenchyma (e.g., brain, kidney, testis, etc.). Nevertheless, correctly interpreting whether or not a BBB exists within brain grafts is manifested by methodologies employed to answer the question and by ensuing neuropathological and immunological consequences of intracerebral grafting. The present study addresses these issues and suggests misinterpretation for the absence of a BBB in brain grafts can be attributed to: (1) rupture of interendothelial tight junctional complexes in vessels of CNS grafts fixed by perfusion of the host; (2) damage to host vessels and BBB during the intracerebral grafting procedure; (3) graft placement in proximity to inherently permeable vessels (e.g., CNS sites lying outside the BBB) supplying the subarachnoid space/pial surface and circumventricular organs such as the median eminence, area postrema, and choroid plexus; and (4) graft rejection associated with antigen presenting cells and the host immune response. The latter is prevalent in xenogeneic grafts and exists in allogeneic grafts with donor-host mismatch in the major and/or minor histocompatibility complex. CNS grafts between non-immunosuppressed outbred donor and host rats of the same strain (e.g., Sprague Dawley or Wistar rats) can be rejected by the host; these grafts exhibit populations of immunohistochemically identifiable major histocompatibility complex class I+ and class EE+ cells (microglia, macrophages, etc.) and CD4+ T-helper and CD8+ T-cytotoxic lymphocytes. PC12 cell suspension grafts placed within the CNS of non-immunosuppressed Sprague Dawley rats are rejected similarly. Donor cells from solid CNS grafts placed intracerebrally and stained immunohistochemically for donor major histocompatibility complex (MHC) class I expression are identified within the host spleen and lymph nodes; these donor MHC expressing cells may initiate the host immune response subsequent to the cells entering the general circulation through host cerebral vessels damaged during graft placement. Rapid healing of damaged cerebral vessels is stimulated with exogenously applied basic fibroblast growth factor, which may prove helpful in reducing the potential entry of donor cells to the host circulation. These results have implication clinically for the intracerebral grafting of human fetal CNS cell suspensions.

Animals↗
PubMed1994

The dichotomous size variation of human complement C4 genes is mediated by a novel family of endogenous retroviruses, which also establishes species-specific genomic patterns among Old World primates.

The human complement C4 genes in the HLA exhibit an unusual, dichotomous size polymorphism and a four-gene, modular variation involving novel gene RP, complement C4, steroid 21-hydroxylase (CYP21), and tenascin-like Gene X (RCCX). The C4 gene size dichotomy is mediated by an endogenous retrovirus, HERV-K(C4). Nearly identical sequences for this retrotransposon are present precisely at the same location in the long C4 genes from the tandem RCCX Module I and Module II. Specific nucleotide substitutions between the long and short C4 genes have been identified and used for diagnosis. Southern blot analyses revealed that HERV-K(C4) is present at more than 30 locations in the human genome, exhibits variations in the population, and its analogs exist in the genomes of Old World primates with species-specific patterns. Evidence of intrachromosomal recombination between the two long terminal repeats of HERV-K(C4) is found near the huntingtin locus on chromosome 4. It is possible that members of HERV-K(C4) are involved in genetic instabilities including the RCCX modules, and in protecting the host genome from retroviral attack through an antisense strategy.

Amino Acid Sequence↗