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J R Haynes

Publications and source records attributed to J R Haynes.

At least 37 records · Page 2Linked to original sources
PubMed1994

Accell particle-mediated DNA immunization elicits humoral, cytotoxic, and protective immune responses.

Accell particle-mediated gene delivery technology was employed for the intracellular delivery of antigen-encoding expression vectors in epidermal tissues in laboratory animals. Delivery of plasmid DNA-coated gold microparticles using the Accell gene delivery system resulted in de novo antigen expression in epidermal cells that stimulated the induction of antigen-specific humoral and cytotoxic cellular immune responses. Optimal DNA delivery conditions favoring maximal humoral responses required the delivery of 5 x 10(7) micron-sized gold particles containing 300 plasmid copies per particle (80 ng of vector total) into a 4-cm2 area of abdominal skin. Comparison of immune responses between animals that received intramuscular injections of relatively large quantities of vector DNA (100 micrograms) and those that received intracellular deliveries of submicrogram quantities of the same DNA to the epidermis demonstrated that the latter approach was considerably more effective at eliciting strong humoral responses. In addition, cytotoxic cellular immune responses were elicited to HIV-1 gp120 following epidermal delivery of HIV-1 gp160 or gp120 expression constructs. A qualitative shift from predominantly cytotoxic cellular to predominantly humoral immune responses with continued immunization indicated the potential for optimizing delivery conditions to favor specifically one type of response over the other.

AIDS Vaccines↗
PubMed1993

DNA vaccines: protective immunizations by parenteral, mucosal, and gene-gun inoculations.

Plasmid DNAs expressing influenza virus hemagglutinin glycoproteins have been tested for their ability to raise protective immunity against lethal influenza challenges of the same subtype. In trials using two inoculations of from 50 to 300 micrograms of purified DNA in saline, 67-95% of test mice and 25-63% of test chickens have been protected against a lethal influenza challenge. Parenteral routes of inoculation that achieved good protection included intramuscular and intravenous injections. Successful mucosal routes of vaccination included DNA drops administered to the nares or trachea. By far the most efficient DNA immunizations were achieved by using a gene gun to deliver DNA-coated gold beads to the epidermis. In mice, 95% protection was achieved by two immunizations with beads loaded with as little as 0.4 micrograms of DNA. The breadth of routes supporting successful DNA immunizations, coupled with the very small amounts of DNA required for gene-gun immunizations, highlight the potential of this remarkably simple technique for the development of subunit vaccines.

Animals↗
PubMed1993

Examination of parameters affecting the elicitation of humoral immune responses by particle bombardment-mediated genetic immunization.

A human growth hormone expression construct was delivered intracellularly into the abdominal skin of mice by particle bombardment-mediated gene transfer. Using this technology, the in vivo delivery of antigen-encoding expression vectors affixed to gold microprojectiles results in de novo antigen production in target skin and development of specific antibody responses. In this study, we examined the contribution of various delivery parameters to the resultant protein expression and related antibody responses. The highest levels of both protein expression and antibody production were correlated with particle delivery to the epidermis while deliveries extending into the dermis resulted in decreased protein and antibody production. Optimal immune responses were also shown to be dependent upon the delivery of a sufficient number of DNA-coated gold particles, indicating that a dose-response relationship exists between the number of particles delivered and the resultant protein expression and antibody production. Further, maximal protein expression and associated antibody titers were elicited with surprisingly small amounts of DNA. The practicality of targeting skin and the use of three to four orders of magnitude less DNA than is typically required in direct DNA inoculation studies demonstrates the potential utility of this emerging technology for the rapid production of antibodies in laboratory animals, and in the development of a new class of human clinical vaccines based upon direct, intracellular DNA delivery.

Administration, Topical↗
PubMed1992

Cloning and characterization of ribosomal RNA genes from Opisthorchis viverrini.

The ribosomal DNA (rDNA) unit of the liver fluke Opisthorchis viverrini has been cloned and characterized. The results demonstrated that the total length of this unit is approximately 13 kb, containing 4.2 kb of large subunit (LSU) rDNA, 2.0 kb of small subunit (SSU) rDNA, 1.0 kb of transcribed spacer DNA and 5.8 kb of non-transcribed + external transcribed spacer DNA. Examination of the non-transcribed spacer region between different rDNA units showed variation in the restriction sites rather than in the length. Judging from hybridization of the rDNA plasmid to the restriction endonuclease digest of genomic DNA, rDNA units represent 6.1% of the total genomic DNA. At the RNA level, the LSU rRNA of O. viverrini and Fasciola gigantica contained a hidden break. The molecule consisted of two fragments co-migrating with a SSU rRNA, when electrophoresis was carried out under denaturing conditions. Ribosomal RNA sequence comparison has been previously used to determine phylogenetic classification of parasitic organisms. The sequence of 381 nucleotides at the 5' terminus of the LSU rRNA gene was determined and compared with those from species previously reported by other investigators. Phylogenetic classification of O. viverrini, as determined by rRNA gene sequence comparison, is comparable with the conventional taxonomic classification.

Animals↗
PubMed1992

Expression and characterization of genetically engineered human immunodeficiency virus-like particles containing modified envelope glycoproteins: implications for development of a cross-protective AIDS vaccine.

Noninfectious human immunodeficiency virus type 1 (HIV-1) viruslike particles containing chimeric envelope glycoproteins were expressed in mammalian cells by using inducible promoters. We engineered four expression vectors in which a synthetic oligomer encoding gp120 residues 306 to 328 (amino acids YNKRKRIHIGP GRAFYTTKNIIG) from the V3 loop of the MN viral isolate was inserted at various positions within the endogenous HIV-1LAI env gene. Expression studies revealed that insertion of the heterologous V3(MN) loop segment at two different locations within the conserved region 2 (C2) of gp120, either 173 or 242 residues away from the N terminus of the mature subunit, resulted in the secretion of fully assembled HIV-like particles containing chimeric LAI/MN envelope glycoproteins. Both V3 loop epitopes were recognized by loop-specific neutralizing antibodies. However, insertion of the V3(MN) loop segment into other regions of gp120 led to the production of envelope-deficient viruslike particles. Immunization with HIV-like particles containing chimeric envelope proteins induced specific antibody responses against both the autologous and heterologous V3 loop epitopes, including cross-neutralizing antibodies against the HIV-1LAI and HIV-1MN isolates. This study, therefore, demonstrates the feasibility of genetically engineering optimized HIV-like particles capable of eliciting cross-neutralizing antibodies.

AIDS Vaccines↗
PubMed1991

Nucleotide sequence of the small subunit ribosomal RNA-encoding gene from Opisthorchis viverrini.

The complete nucleotide (nt) sequence of the small subunit ribosomal RNA-encoding gene of Opisthorchis viverrini reported in this study is the first nt sequence reported for a trematode. The gene is 1992 nt long and has a G + C content of 50.94%. It is made up of alternated constant and variable regions that are similar to the gene organization of other eukaryotes. It is also of interest to note an unexpectedly high degree of sequence homology between O. viverrini and human genes.

Animals↗
PubMed1991

Strategy for developing a genetically-engineered whole-virus vaccine against HIV.

The production of genetically-engineered, noninfectious virions of human immunodeficiency virus (HIV) represents a novel approach to the development of a safe and effective vaccine for the acquired immune deficiency syndromes (AIDS). Insofar as preparations of inactivated simian immunodeficiency virus (SIV) are now demonstrating protection in immunization-challenge studies in rhesus monkeys, a safe preparation of noninfectious HIV virions produced in a genetically-engineered cell line becomes a logical candidate vaccine for studies in humans. These particles, or pseudovirions, offer distinct advantages over the use of inactivated HIV for human AIDS vaccines. Guarantees of safety without the requirement for inactivation and their potential for structural modification for the modulation of immunogenicity are compelling reasons for the acceptance of HIV pseudovirions as a candidate vaccine in humans.

Acquired Immunodeficiency Syndrome↗
PubMed1991

Production of immunogenic HIV-1 viruslike particles in stably engineered monkey cell lines.

A proviral fragment from human immunodeficiency virus type 1 (HIV-1) (LAV-1BRU) containing only protein-coding information, was expressed in COS cells using constitutive promoters in transient and stable transfection experiments. The presence of viruslike particles in cell supernatants was verified by Western blot analysis, density gradient centrifugation, and electron microscopy. Transfection of Vero cells with a similar construct employing the human metallothionein promoter led to the isolation of stable cell lines exhibiting inducible viruslike particle expression in response to cadmium chloride treatment. Induction ratios for viruslike particle expression were in excess of 1000-fold with production levels of p24 core antigen as high as 0.6 mg/L per 24 h. HIV-1 viruslike particles were immunogenic in mice, leading to strong envelope and core-specific humoral responses after two immunizations. The development of stable cell lines expressing significant quantities of HIV-1 viruslike particles offers an alternative to the use of live virus vectors for the production and evaluation of particle-based AIDS vaccines.

Animals↗
PubMed1988

A recessive cellular mutation in v-fes-transformed mink cells restores contact inhibition and anchorage-dependent growth.

A contact-inhibited revertant of mink cells transformed by the Gardner-Arnstein strain of feline sarcoma virus was isolated by fluorescence-activated sorting of cells stained with the mitochondria-specific dye rhodamine 123. The revertant cell line exhibited a decrease in its proliferative rate and saturation density and a complete loss of its capacity for anchorage-independent growth, but it remained tumorigenic when inoculated into nude mice. The revertant cells retained a rescuable Gardner-Arnstein feline sarcoma provirus, expressed high levels of the v-fes oncogene product and its associated tyrosine kinase activity, manifested elevated levels of phosphotyrosine-containing cellular proteins similar to those observed in v-fes-transformed cells, and were refractory to retransformation by retroviruses containing the v-fes, v-fms, and v-ras oncogenes. Fusion of the revertant and parental cells generated somatic cell hybrids which formed colonies in semisolid medium, indicating that the block in transformation was recessive. These data together with the observation that the revertant phenotype is unstable in continuous culture suggest that the loss of transformation is due to the presence of limiting quantities of a gene product which functions downstream of the v-fes-coded kinase in the mitogenic pathway.

Animals↗
PubMed1987

High-level expression of alpha-human atrial natriuretic peptide from multiple joined genes in Escherichia coli.

A method is described which allows alpha-human atrial natriuretic peptide to be synthesized in stable form and with high yield in Escherichia coli. In the final expression system, eight copies of the synthetic alpha-hANP gene were linked in tandem, separated by codons specifying a 4-amino-acid (aa) linker with lysine residues flanking the authentic N and C termini of the 28-aa hormone. This sequence was in turn joined to the 3' end of a fragment containing the lac promoter and a leader sequence coding for the first seven N-terminal amino acids of beta-galactosidase. The expressed multidomain protein accumulated intracellularly into stable inclusion bodies and was easily purified by urea extraction of the insoluble cell fraction. The purified protein was cleaved into monomers by digestion with endoproteinase Lys-C, trimmed to expose the authentic C terminus by digestion with carboxypeptidase-B and a single disulfide bond was formed by gentle oxidation with potassium ferricyanide. The fully processed recombinant peptide was shown by reverse phase liquid chromatography to be indistinguishable from the chemically synthesized standard alpha-hANP in both the reduced and in the folded form.

Atrial Natriuretic Factor↗
PubMed1986

Construct validity of the Kendrick Battery with institutionalized aged.

In this study, we attempted to explore the construct validity of the Kendrick Battery by using an American sample and psychometric tests as indexes of diffuse organicity, depression, and normality. Institutionalized residents (N = 53) were tested twice (6-week interval). When organicity was defined by disorientation and memory deficits, then both the Object Learning test and the Digit Copying test were accurate in differentiating preestablished criterion groups. When organicity was defined more broadly, including sensorimotor function, the Digit Copying test alone was more accurate when depression was defined in terms of irritability, restlessness, and despair. These data suggest that although the Kendrick scales appeared to be sensitive to organicity and depression in this sample, their validity varied with the criteria for each when such were defined psychometrically.

Aged↗
PubMed1985

Structural relationship of human interferon alpha genes and pseudogenes.

We have isolated and characterized DNA segments containing IFN-alpha-related sequences from human lambda and cosmid clone banks. We describe six linkage groups comprising 18 distinct IFN-alpha-related loci, and report the nucleotide sequences of nine chromosomal IFN-alpha-genes with intact reading frames, as well as of five pseudogenes. Taking into account as yet unsequenced genes as well as clones described by others, there are now seven linkage groups and 23 loci, of which 15 correspond to potentially functional genes and six to non-functional genes; two loci remain unsequenced. Eighteen additional sequences are likely to be allelic to the above. The finding that at least two IFN-alpha genes appear to be natural hybrids of other IFN-alpha genes, and that two distinct IFN-alpha loci have completely identical coding sequences, although their flanking regions are different, is evidence for information exchange between the individual genes.

Amino Acid Sequence↗
PubMed1981

Transcription of the beta-like globin genes and pseudogenes of the goat in a cell-free system.

We have examined the transcription in a cell-free system of all the members of a highly regulated gene family, the beta-like hemoglobin genes of the goat. These five genes, which code for embryonic, fetal, juvenile, and adult beta-globin proteins, are all transcribed to roughly the same extent in the in vitro system. In all cases initiation of transcription is accurate. However, two goat beta-globin pseudogenes, as well as several artificially constructed deletion mutants, are not transcribed in vitro. A common feature of the transcriptionally inactive genes is the lack of an AT-rich consensus sequence just upstream of the presumptive initiation site.

Animals↗
PubMed1981

Structure and evolution of goat gamma-, beta C- and beta A-globin genes: three developmentally regulated genes contain inserted elements.

We have determined the complete nucleotide sequence of the goat fetal (gamma), preadult (beta C) and adult (beta A) globin genes. In contrast to other globin genes expressed in different stages of development, these three genes are highly homologous (approximately 90%) in both their coding and noncoding regions. The only major difference between them results from elements inserted into their large introns: gamma contains a 247 bp insertion, beta A contains a 318 bp insertion, and beta C contains both a 252 bp insertion and a 60 bp deletion. Based on comparisons of these three genes to each other and to two goat globin pseudogenes, psi beta X and psi beta Z, we conclude that gamma, beta A and beta C have diverged relatively recently, and that the two gene pairs psi beta X - beta C and psi beta Z-beta A arose via a block duplication of an ancestral pseudogene-functional gene pair. Moreover, as opposed to the human fetal genes (G gamma and A gamma), the goat gamma gene appears to be a true beta-like gene that has fetal-specific function. The insertions in gamma, beta A and beta C contain sequence features characteristic of transposon-like elements, and appear to be represented in multiple copies throughout the genome.

Animals↗
PubMed1980

Identification by nucleotide sequence analysis of a goat pseudoglobin gene.

We have recently prepared a recombinant library of goat genomic DNA and have isolated clones containing th known goat globin genes. These include the alpha, gamma, beta C and beta A genes. In addition to these, another beta-like sequence has been observed. In this communication we report the complete nucleotide sequence of this gene, excluding a portion of the large intervening sequence. Several features suggest that this is a non-functional or pseudoglobin gene. The alterations include a frameshift mutation, substitution of the heme-binding histidines, a mutated termination codon, a change in the GT/AG excision sequence of the 5' end of the first intervening sequence, an AT rich sequence in the 3' untranslated region, and a mutated Hogness-Goldberg box. We conclude that this gene cannot function in the synthesis of globin.

Animals↗
PubMed1980

The isolation of the beta A-, beta C-, and gamma-globin genes and a presumptive embryonic globin gene from a goat DNA recombinant library.

As an approach to understand how the expression of globin genes are regulated during development, clones containing globin DNA sequences were selected from a recombinant library of goat genomic DNA. The type of globin gene present in each of the recombinants was determined by cross-hybridization to the DNA of mouse alpha- and beta-globin cDNA-containing plasmids. Of 11 clones isolated, eight hybridized specifically to the DNA of the mouse beta-globin plasmid, while one clone hybridized only to the DNA of the alpha globin plasmid. The location of each globin sequence within its DNA insert was determined by a combination of restriction enzyme mapping and Southern transfer-hybridizations. Selected fragments were sequenced; comparisons of the amino acids coded for by these regions with those of the goat globins identified clones carrying beta A-, beta C-, and gamma-globin genes. Another recombinant coded for amino acid sequences resembling, but not identical with, the known goat globins, and was identified tentatively as containing an embryonic or epsilon-gene. Detailed analysis of the clone containing the beta C gene and an overlapping clone revealed that three other beta-like sequences are located 6, 12, and 21 kilobases on the 5'-side of the beta C gene. The globin sequence of the locus nearest to the beta C gene has an altered translation termination codon and, if transcribed and translated, would give a globin chain seven amino acids longer than the normal goat beta C-globin. In addition, the sequence following this termination codon is very AT-rich, unlike that of other globin genes. The recombinants described contain extensive regions of DNA surrounding the globin genes, making them useful for identifying regulatory sequences as well as determining the sequence organization of the goat globin genes.

Animals↗
PubMed1980

Unusual sequence homology at the 5-ends of the developmentally regulated beta A-, beta C-, and gamma-globin genes of the goat.

We have constructed a recombinant library of goat DNA and have isolated clones containing the beta A-, beta C-, and gamma-globin genes and a presumptive embryonic globin gene. These genes are switched on and off during development and, thus, provide a model system for the study of gene regulation. To identify regions that may be involved in this switch, we determined the sequence of the areas surrounding the 5' ends of the beta A-, beta C- and gamma-globin genes. Surprisingly, the sequences of the beta A-, and beta C-globin genes are identical, beginning with the translation initiation codon and extending 131 base pairs (bp) in the 5' direction. At this point, two nucleotide differences are seen and only six additional differences are encountered when the sequences are extended 144 bp further in the 5' direction. Furthermore, the nucleotide sequence of the 5' end of the gamma-globin gene is very similar to those of the beta A-0, and beta C-globin genes. Only three nucleotide differences are found in the gamma-globin gene within the 131-bp region in which the beta A- and beta C-globin genes are identical. We conclude that this identical region cannot contain regulatory signals that mediate the switch from beta C to beta A expression.

Animals↗