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Biomedical subjects

J Hanes

Publications and source records attributed to J Hanes.

At least 19 recordsLinked to original sources
PubMed1998

Relating the phagocytosis of microparticles by alveolar macrophages to surface chemistry: the effect of 1,2-dipalmitoylphosphatidylcholine.

This study examines the potential of 1,2-dipalmitoylphosphatidylcholine (DPPC), a major component of lung surfactant, to reduce the phagocytosis of microspheres by altering the cellular interactions occurring in the alveoli. These microspheres could be designed to act as a controlled delivery system for small molecules, peptides or proteins for pulmonary administration. Microspheres were prepared using poly (lactic-co-glycolic acid) (PLGA, 50/50 and encapsulated peroxidase as a model protein. DPPC was included in some formulations. The interaction of PLGA and DPPC-PLGA microspheres with phagocytic cells was evaluated using lung macrophages in culture. X-ray Photoelectron Spectra (XPS) results indicate that the inclusion of DPPC in the microspheres alters the microsphere surface chemistry, with the DPPC covering a large portion of the microsphere surface. The dominance of DPPC on the microsphere surface is highly beneficial in moderating the interaction occurring between the microspheres and phagocytic cells in the lung. Fluorescent confocal microscopy indicates that only 25% of cells internalized DPPC-coated particles, whereas 70% of those cells exposed to particles without the DPPC coating internalized particles after one hour of incubation.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1997

Large porous particles for pulmonary drug delivery.

A new type of inhalation aerosol, characterized by particles of small mass density and large size, permitted the highly efficient delivery of inhaled therapeutics into the systemic circulation. Particles with mass densities less than 0.4 gram per cubic centimeter and mean diameters exceeding 5 micrometers were inspired deep into the lungs and escaped the lungs' natural clearance mechanisms until the inhaled particles delivered their therapeutic payload. Inhalation of large porous insulin particles resulted in elevated systemic levels of insulin and suppressed systemic glucose levels for 96 hours, whereas small nonporous insulin particles had this effect for only 4 hours. High systemic bioavailability of testosterone was also achieved by inhalation delivery of porous particles with a mean diameter (20 micrometers) approximately 10 times that of conventional inhaled therapeutic particles.

Administration, Inhalation↗
PubMed1997

In vitro selection and evolution of functional proteins by using ribosome display.

We report here a system with which a correctly folded complete protein and its encoding mRNA both remain attached to the ribosome and can be enriched for the ligand-binding properties of the native protein. We have selected a single-chain fragment (scFv) of an antibody 10(8)-fold by five cycles of transcription, translation, antigen-affinity selection, and PCR. The selected scFv fragments all mutated in vitro by acquiring up to four unrelated amino acid exchanges over the five generations, but they remained fully compatible with antigen binding. Libraries of native folded proteins can now be screened and made to evolve in a cell-free system without any transformation or constraints imposed by the host cell.

Amino Acid Sequence↗
PubMed1997

Controlled protein delivery from biodegradable tyrosine-containing poly(anhydride-co-imide) microspheres.

Polymer microspheres capable of the controlled release of macromolecules for periods ranging from days to over a month were developed. The microspheres were made using a new family of anhydride polymers: tyrosine-containing poly(anhydride-co-imides), specifically poly[trimellitylimido-L-tyrosine-co-sebacic acid-co-1,3-bis(carboxphenoxy)propane] anhydrides [poly(TMA-Tyr:SA:CPP)]. These polymers may be of particular interest for controlled delivery of vaccine antigens due to the incorporation of an immunological adjuvant, L-tyrosine, into their backbone. Microspheres were produced from a variety of polymer compositions using a double-emulsion solvent-evaporation technique, and tested for their ability to provide controlled release of a model protein, bovine serum albumin, in vitro. The microspheres are spherical with smooth surfaces and encapsulate greater than 70% of the protein. Protein release rates from polymers of identical composition could be varied from 0.3 to over 125 micrograms per mg spheres per month by changing the amount of protein encapsulated. This effect can be magnified by using polymers with various monomer ratios. A close correlation between protein release and polymer weight loss was observed, suggesting a release mechanism controlled mainly by polymer erosion. Bovine serum albumin release from poly(TMA-Tyr:SA:CPP) microspheres is also pH sensitive, being enhanced at high pH and depressed under acidic conditions.

Anhydrides↗
PubMed1997

A theoretical model of erosion and macromolecular drug release from biodegrading microspheres.

A theoretical model is outlined for predicting the time evolution of total mass, mean molecular weight, and drug release for the case of a spherical bulk-eroding microsphere, prepared by a double emulsification procedure and containing a hydrophilic drug, such as a protein or peptide. Explicit analytical formulae are derived for calculating the time evolution of measurable macroscopic characteristics, such as drug release or mean molecular weight. Microsphere hydration, polymer erosion, and drug release phases are each described. Results indicate that polymer degradation by only random-chain scission or only end scission (or unzipping) cannot explain experimentally observed kinetics of particle mass loss and molecular weight change; thus, a combined model (incorporating both random and end scission) is proposed. A general methodology for determining the microscopic transport coefficients (such as polymer degradation rate constant or drug diffusion coefficient) from erosion and release data is outlined. This paradigm is applied to the specific case of 50:50 poly(D,L-lactic-co-glycolic acid (PLGA) microspheres encapsulating glycoprotein 120 (gp 120), a candidate AIDS vaccine. Predictions permit comparisons with experimental data for mean weight- and number-averaged molecular weights, as well as for mass loss and protein release. Other comparisons are made with data appearing in the literature for release of tetanus toxoid from PLA and PLGA microspheres of variable molecular weight. Agreement between theory and experiment is observed.

AIDS Vaccines↗
PubMed1995

A monoclonal antibody generated against a recombinant peptide fragment of the B3 domain of carcinoembryonic antigen reacts with intact carcinoembryonic antigen.

The chemical synthesis of a gene coding for a polypeptide of 77 amino acid residues (designated ceaB3) representing a fragment of the CEA-B3 domain of carcinoembryonic antigen (CEA) was achieved. The ceaB3 fragment was cloned into the plasmid pLZPWB1 at the C-terminus of a derivative lacZMF of the lacZ gene, devoid of methionine and cysteine amino acid residues. The fusion protein lacZMF-ceaB3 represented approx. 30% of total proteins expressed after induction. The fusion protein was formed as inclusion bodies. Simple washing steps led to an insoluble fusion protein which was of approx. 80% purity. Another fusion gene was generated by inserting ceaB3 between the malE gene encoding maltose binding protein (mbp) and lacZ alpha of the pmal-c2 vector. Expression of the resulting pmal-c2-ceaB3-lacZ alpha yielded the fusion protein mbp-ceaB3-lacZ alpha with a molecular mass of 57.94 kDa, which was obtained as a soluble protein in almost homogeneous form after affinity chromatography employing amylopectin. Polyclonal sheep anti-CEA antiserum specifically reacted with fusion proteins lacZMF-ceaB3 and mbp-ceaB3-lacZ alpha. A monoclonal antibody CEA/HK2 was generated employing lacZMF-ceaB3 for immunization and CEA for screening purposes. The mAB CEA/HK2 specifically recognized CEA in immunoblots. The described experimental strategy should be generally applicable for generation of fusion proteins. These fusion proteins are suitable for epitope characterization of existing antibodies, production of regiospecific polyclonal or monoclonal antibodies.

ATP-Binding Cassette Transporters↗
PubMed1995

The gene for the major protein (SVSP109) of bovine semen: structure and promoter analysis.

We have characterized the gene of SVSP109, specifying the bovine secretory protein SVSP109, which is synthesized in a tissue- as well as species-specific manner. Approximately 1.3 kb upstream of the SVSP109 gene, the 3'-end of another gene designated HG5 was identified. The HG5 gene fragment comprises exon (n-1) and exon (n), separated by an intron. Exon (n) contains the complete 3'-untranslated region, whereas exon (n-1) encodes the C-terminal part of a hitherto unknown protein with high homology to SVSP109. The sequenced region of 6956 bp of a bovine genomic clone comprised the complete SVSP109 gene, which is made up of five exons and four introns. Primer extension analysis and RTPCR of poly(A+)RNA from seminal vesicle revealed that the first exon 1 extends to a position 34 bp downstream of the TATA sequence. Employing a CAT assay, a definitive but weak promoter activity was detected in pCATeSVSP15 (base pairs -639 to +574) and pCATeSVSP10 (base pairs -639 to +198); pCATeSVSP6 (base pairs -262 to +65) displayed promoter activity similar to the positive control. We conclude from these results that the TATA sequence located at position -34 is part of the functional promoter of the SVSP109 gene.

Amino Acid Sequence↗
PubMed1994

Characterization by cDNA cloning of two new human protein kinases. Evidence by sequence comparison of a new family of mammalian protein kinases.

Two new human cDNAs, designated phclk2 and phclk3, which have a high identity to the cDNA of the human protein kinase clk, were characterized. Typical features of hclk2 and hclk3 proteins are non-homologous N-terminal regions and the presence of the C-terminal protein kinase domain, which is characteristic for serine/threonine-type kinases. We also identified the differentially spliced forms phclk2(139) and phclk3(152) with deletions of 88 and 97 nt, respectively, which lead to changes in the open reading frames. hclk2(139) and hclk3(152) proteins do not possess a protein kinase domain and are nearly identical to the N-terminal regions of the above-mentioned protein kinases. We verified that differentially spliced variants also exist for hclk1 as well as for a mouse clk protein kinase. It was shown that shorter and longer alternatively spliced mRNAs co-exist in different human tissues. According to Southern analysis, hclk2 and hclk3 appear to be specified by single copy genes. The genes for hclk2 as well as for hclk3 were localized to human chromosomes 1 and 15, respectively.

Alternative Splicing↗
PubMed1994

The gene for the amyloid precursor-like protein APLP2 is assigned to human chromosome 11q23-q25.

The human amyloid precursor-like protein APLP2 is a highly conserved homologue of a sequence-specific DNA-binding mouse protein with a predicted function in the cell cycle. Somatic cell hybrids segregating human chromosomes were used to assign the APLP2 gene to chromosome 11. Fluorescence in situ hybridization confirmed this assignment and further localized the gene to q23-q25.

Amyloid beta-Protein Precursor↗
PubMed1994

Cloning of the gene for bovine monocyte chemoattractant protein-2.

Bovine monocyte chemoattractant protein-1 (bovine MCP-1) cDNA has recently been characterized and shown to be highly expressed in bovine seminal vesicles secretory epithelium as well as in phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear leukocytes (PMNLs). In an attempt to isolate the MCP-1 gene, we screened a bovine genomic cosmid library with a MCP-1-specific probe pH42. A positive clone, c11/1, was subjected to restriction analysis and fragments probed with pH42 by southern blotting. pH42-positive fragments were subcloned and sequenced. The sequence revealed three exon-like regions that coded for a protein displaying an identity of 51% with bovine MCP-1. Employing this sequence information from c11/1, the c11/1-specific cDNA was generated from poly(A)+RNA of bovine PMNLs by reverse transcription and a combination of polymerase chain reaction (PCR) methods. The assembled c11/1 cDNA comprised a 5' UTR coding region as well as 3' UTR for the gene product c11/1. Amino acid sequence comparison of the bovine c11/1 gene product with human monocyte chemotactic proteins yielded the highest sequence identity with human MCP-2, and it is assumed that the c11/1 gene product represents the bovine MCP-2. The exon/intron structure of the bovine MCP-2 gene was found to be similar to the human MCP-1 gene. The bovine MCP-2 gene consists of three exons separated by two introns. In the 5'-flanking region of the 3.3-kb gene, a TATA box as well as an AP-1 sequence motif were identified. The bovine MCP-2 is specified by a single-copy gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

A human amyloid precursor-like protein is highly homologous to a mouse sequence-specific DNA-binding protein.

From a cDNA sequence, we have deduced the amino acid sequence for a human amyloid precursor-like protein (APPH) with > 92% identity to the CDEI binding protein (CDEBP) of the mouse and the fragmentary rat protein YWK-II of unknown function. Expression of APPH was found in all tissues examined. A striking homology of APPH to human amyloid precursor protein (APP) was observed. Overall identity accounts for 52.7%. However, there are three domains of APPH with remarkably higher similarities, corresponding to amino acid sequence positions 47-204 (76.6%), 308-567 (67.7%), and 694-763 (69.9%). Using an APPH antiserum, we localized APPH in nuclei of human interphase cells and found an increased synthesis of APPH in mitotic cells. Our results indicate that the highly conserved proteins human APPH, mouse CDEBP, and rat YWK-II are apparently homologues of a CDEI binding protein with indispensible function in mammalian genome segregation.

Amino Acid Sequence↗
PubMed1993

Characterization by cDNA cloning of the mRNA of human ribosomal protein L8.

From a lambda UNI-ZAP XR cDNA library derived from poly(A)+RNA of human ovarian granulosa cells a cDNA clone pHG51 was isolated. Sequence analysis showed significant homology to the C-terminal region of rat ribosomal protein L8 cDNA. The 5'-end of the cDNA was completed by PCR with cloned total cDNA. Aligning of DNA sequences from PCR clones with the sequence of the pHG51 insert yielded the full-length cDNA. From the open reading frame of the cDNA an amino acid sequence for a polypeptide of 257 residues was derived, which was identical with rat ribosomal protein L8 and also possessed a high degree of identity to ribosomal proteins L8 of other species. It is therefore assumed that the characterized cDNA represents the mRNA of the human ribosomal protein L8. Southern analysis revealed that human ribosomal protein L8 is specified by multi copy genes.

Amino Acid Sequence↗
PubMed1993

The complete cDNA coding sequence for the mouse CDEI binding protein.

This work describes the cDNA sequence of the mouse CDEI binding protein (CDEBP), comprising the complete coding sequence. The cDNA encodes a protein of 695 amino acid residues. The derived amino acid sequence displays a sequence identity to human amyloid precursor-like protein (APLP) of > 92%.

Amino Acid Sequence↗
PubMed1992

Construction of a plasmid containing the complete coding region of human elongation factor 2.

A plasmid pUChEF-2 containing the coding sequence as well as the complete 3'-untranslated region (3'UTR) of human EF-2 mRNA was constructed. The plasmid construct was assembled from a cDNA insert of pHGR81 (Rapp et al., (1988) Biol. Chem. Hoppe-Seyler 369, 247-250) comprising the C-terminal portion of the coding region and the 3'UTR, as well as a polymer chain reaction PCR fragment (Rapp et al., (1989) Biol. Chem. Hoppe-Seyler 370, 1071-1075) covering the missing part of the coding region from the amino-terminus.

Base Sequence↗
PubMed1989

Cloning of Japanese quail ovalbumin cDNA in E. coli.

Japanese quail oviduct total cDNA was synthesized from total mRNA by the classical method, using AMV reverse transcriptase, the Klenow fragment of DNA polymerase I and S1 nuclease. The results of the synthesis of total ss-cDNA partially differed from those published for the synthesis of chicken total ss-cDNA. The presumed causes of the differences in the complete reverse transcription of mRNAcon and incomplete reverse transcription of mRNAlys and a large part of mRNAov in our case are discussed. An atypical strategy was used for cloning full-length cDNAov. The total cDNA was dC-tailed, then fractionated by analytical agarose electrophoresis and 4 cDNA fractions of different lengths were isolated from the gel using DEAE cellulose membranes. A cDNA fraction about 1500-2500 bp long containing full-length cDNAov was annealed with dG-tailed PstI-linearized plasmid pBR322 and cloned into competent E. coli DHl cells. Seventy-two clones were screened for the presence of full-length cDNAov, initially by insert size and then by means of hybrid-arrested translation. Four clones containing 1900-1980 bp cDNAov were obtained. The cDNA ends in one of these clones were sequenced. Comparison of these sequences with those of chicken mRNAov indicated that almost full-length cDNAov's had been cloned. They lacked a small number of nucleotides at their 5' ends, which had probably been split off during the degradation of the hairpin loop by S1 nuclease. A sequence of 134 bases from the 5' end of mRNAov is presented and compared with the known sequence of chicken mRNAov. The advantages of the cloning strategy employed, in particular, its cloning efficiency and the possibility of simultaneously identifying clones of also other oviduct cDNA species (in this work: cDNAY and, tentatively, cDNAcon), are discussed.

Animals↗