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Biomedical subjects

M Gorecki

Publications and source records attributed to M Gorecki.

At least 55 records · Page 3Linked to original sources
PubMed1981

Cloning of bovine growth hormone gene and its expression in bacteria.

A hybrid plasmid was constructed containing beta-lactamase gene of plasmid pBR322 and cloned coding sequences of bovine growth hormone (BGH). The constructed plasmid contains all DNA sequences required to encode BGH, and when used as a hybridization probe it detects one growth hormone gene in the bovine genome. The cloned DNA sequences are inserted into the beta-lactamase gene in the correct reading frame for BGH synthesis. The hybrid gene is expressed in bacteria and the product, a fused beta-lactamase-bovine growth hormone protein, is specifically immunoprecipitated with anti-serum to BGH. Unlike beta-lactamase, very little growth hormone containing sequences can be detected in the periplasmic space.

Amino Acid Sequence↗
PubMed1980

Peptide inhibitors of sickle hemoglobin aggregation: effect of hydrophobicity.

Thirty-three peptides have been synthesized which have the ability to inhibit aggregation of sickle hemoglobin which occurs upon deoxygenation. Evidence is presented which indicates that the hydrophobicity of the side chains is the predominant factor. A linear correlation exists between the capacity of peptides to inhibit gelation and the additivity of the side-chain hydrophobic contributions. Small di- and tri-peptides are more responsive to changes in their nonpolar content than are larger oligopeptides whose antigelling activity is a slowly varying function of hydrophobicity. With few exceptions, charged substituent groups on the side chain do not appear to contribute to the inhibitory process. Thus, if hydrogen bonding or ionic interactions are involved with these residues, they appear to play only a secondary role in the molecular interactions responsible for inhibiting or delaying the deoxy-HbS gelation.

Calorimetry↗
PubMed1980

Antisickling activity of amino acid benzyl esters.

The sickling of homozygous sickel cells upon deoxygenation is inhibited in the presence of 3 mM L-phenylalanine benzyl ester (Phe-OBzl) or benzyl esters of other aromatic or hydrophobic amino acids. Phe-OBzl was found to permeate into erythrocytes rapidly, and the deoxygenated cells maintained considerable flexibility as measured by their ability to pass through 3-micron pores. The osmotic fragility of the cells was unchanged and the oxygen dissociation curve was shifted slightly from a 50% saturation values of 28.5 mm Hg to 31.0 mm Hg. At lower concentrations of Phe-OBzl some antisickling activity was seen. The Phe-OBzl antisickling activity may involve both binding to deoxyhemoglobin S and modification of the erythrocyte membrane. This class of compounds has considerable potential as therapeutic agents for the treatment of sickle cell disease.

Amino Acids↗
PubMed1980

Cloning of DNA complementary to the measles virus mRNA encoding nucleocapsid protein.

Double-stranded cDNA synthesized from total poly(A)-containing mRNA, extracted from monkey cells infected with measles virus, has been inserted into Pst cleavage site of Escherichia coli plasmid pBR322 and cloned. A clone containing measles virus DNA sequences was identified by hybridization to a measles virus-specific 32P-labeled cDNA probe prepared from the mRNA of measles virus-infected cells. Cellular sequences in the probe were neutralized by prehybridization with an excess of unlabeled mRNA from uninfected monkey cells. The insert of cloned cDNA isolated contans 1420 base pairs, as shown by agarose gel electrophoresis and electron microscopy. The size of the mRNA complementary to this cloned cDNA is 1750 nucleotides, as determined by the reverse Southern technique. The cloned DNA fragment was further identified as the reverse transcript of the mRNA coding for the nucleocapsid protein of measles virus on the basis that the major cell-free translation product of mRNA selected by hybridization to the cloned DNA comigrated with the nucleocapsid protein and was immunoprecipitated by measles virus-specific antibodies. Subsequently, the cloned DNA was used to detect specific measles virus sequences in the poly(A)-RNA extracted from brain autopsy material from a patient with subacute sclerosing panecephalitis. The cloned DNA can thus be used as a probe to study the structure and expression of the measles genome, and in particular, to study diseases of the central nervous system in which persistent infection with measles virus has been implicated.

Animals↗
PubMed1979

Cell-free synthesis of rat parotid preamylase.

Poly(A)-containing RNA from rat parotid gland directs the cell-free synthesis of several products in the reticulocyte lysate translation system including a very prominent 58,000-dalton polypeptide which is immunoreactive with anti-alpha-amylase. Purified alpha-amylase has a molecular weight estimated as 56,000 daltons. The 58,000-dalton, cell-free product and alpha-amylase share common peptides as determined by analysis of their limited proteolysis digests. The cross-reactivity and peptide homology suggest that the cell-free product may be a precursor of mature alpha-amylase. While the NH2 terminus of alpha-amylase is blocked, that of the 58,000-dalton product evidently is not, and automated sequence analysis has yielded its partial sequence as: Met-X-Phe-Phe-Leu-Leu-Leu-X-Leu-Ile-X-Leu-X-X-X-X-X-X-X-X-X-Phe-X-X-X-X-X-Ile-X-X-Leu-Phe. The highly hydrophobic nature of the NH2 terminus of the 58,000-dalton, cell-free product suggests that, like other secreted polypeptides, the extra piece may play a role in the transport and secretion of the mature alpha-amylase.

Amino Acid Sequence↗
PubMed1979

Characterization of measles virus-specific proteins synthesized in vivo and in vitro from acutely and persistently infected cells.

Measles virus protein synthesis has been analyzed in acutely and persistently infected cells. To assess the role of measles in subacute sclerosing panencephalitis (SSPE), measles viral proteins synthesized in vivo or in vitro were tested for reactivity with serum from a guinea pig(s) immunized with measles virus and sera from patients with SSPE. Guinea pig antimeasles virus serum immunoprecipitates the viral polypeptides of 78,000 molecular weight (glycosylated [G]), 70,000 molecular weight (phosphorylated [P]), 60,000 molecular weight (nucleocapsid [N]), and 35,000 molecular weight (matrix [M]) from cells acutely infected with measles virus as well as from chronically infected cells, but in the latter case, immunoprecipitated M protein has a reduced electrophoretic migration. Sera of SSPE patients immunoprecipitated all but the G protein in acutely infected cells and only the P and N proteins from chronically infected cells. In immunoprecipitates of viral polypeptides synthesized in a reticulocyte cell-free translation system, in response to mRNA from acutely or persistently infected cells, the 78,000-molecular-weight form of the G protein was not detected among the cell-free products of either mRNA. Guinea pig antimeasles virus serum immunoprecipitated P, N, and M polypeptides from the products of either form of mRNA, whereas SSPE serum immunoprecipitated the P and N polypeptides but not the M polypeptide. The differences in immunoreactivity of the antimeasles virus antiserum and the SSPE serum are discussed in terms of possible modifications of measles virus proteins in SSPE.

Cell Line↗
PubMed1978

Modulation of the catalytic properties of alpha-chymotrypsin by chemical modification at Tyr 146.

alpha--Chymotrypsin was modified with three different diazonium salts derived from D--phenylalanine. All three reagents react selectively with tyrosine 146 on the surface of the enzyme. The spectral and enzymatic properties of the azochymotrypsins are characteristic for each of the proteins. Dependent on the structure of the reagent employed for modification, the activity towards rho-nitroanilide substrate can be higher or about the same as that of the native enzyme.

Amino Acid Sequence↗
PubMed1978

Discrimination of DNA polymerase and RNase H activities in reverse transcriptase of avian myeloblastosis virus.

The active sites in reverse transcriptase of avian myeloblastosis virus have been selectively modified by various chemical reagents. The DNA polymerase activity is very sensitive to hydrophobic sulfhydryl reagents such as 5,5'-dithiobis(2-nitrobenzoic acid) and p-hydroxymercuribenzoate but resistant to sulfhydryl reagents with hydrophilic properties. The RNase H activity, on the other hand, is resistant to both hydrophobic and hydrophilic sulfhydryl reagents, indicating the absence of cysteinyl residues essential for RNase H activity. N-Ethylmaleimide (NEM), an amino and sulfhydryl group specific reagent, inactivates both DNA polymerase and RNase H, the later activity being fourfold more stable. Polynucleotides, but not nucleotide triphosphates, protect the two enzymatic activites of reverse transcriptase against NEM. Since pretreatment of the enzyme with 5,5' -dithiobis(2-nitrobenzoic acid) does not prevent N-ethylmaleimide from reacting with a residue necessary for DNA polymerase activity, two different reactive groups are probably involved with this enzymatic activity. The pH profile of reverse transcriptase inhibition by N-ethylmaleimide also suggests the involvement of two reactive groups essential for the DNA polymerase activity with apparent pKas of 5.5 and 6.5. Only one reactive group with a pKa of 7.5 is found associated with the RNase H activity.

Avian Leukosis Virus↗
PubMed1978

Modification of a specific tyrosine residue of ribonuclease A with a diazonium inhibitor analog.

The diazonium salt of 5'-(4-aminophenyl phosphoryl)-uridine 2'(3')-phosphate reacts stoichiometrically with pancreatic ribonuclease and modifies only one tyrosyl residue, which was identified as Tyr 73 in the amino acid sequence. The modification does not inhibit the biological activity of RNAase on ribonucleic acid, although a large change in the binding constant towards cytidine cyclic phosphate was observed. The modification can be inhibited by addition of the competitive inhibitor cytidine 2'(3')5'-diphosphate and may indicate that Tyr 73 is the most exposed residue or has a unique reactivity towards this reactive substrate analog.

Amino Acids↗
PubMed1978

Electron microscopic evidence for splicing of Moloney murine leukemia virus RNAs.

Poly (A) containing RNA extracted from Moloney murine leukemia virus infected mouse cells was hybridized with long single-stranded complementary DNA, prepared in detergent disrupted virions. Visualization of the hybrids in the electron microscope revealed among the structures, circles and circles with tails. Measurements performed on the circular molecules revealed two major species with circumferences corresponding to 3 and 8.2 kilobases. The latter structures had identical size to circles obtained after annealing of cDNA with the viral genome, 35S RNA. Circularization of a small viral RNA (3 kb) from infected cells in the RNA-cDNA hybrids is a direct evidence that like the 35S RNA it shares similar nucleotide sequences at both the 5' and 3' ends. The presence of 5' end sequences common to the two RNA species indicates the existence of a spliced viral RNA. Furthermore, based on the circularization of viral RNA in the hybrids, we suggest a new way to quantitate and determine the lengths of spliced RNA in retrovirus infected cells.

Base Sequence↗
PubMed1977

Sickle hemoglobin aggregation: a new class of inhibitors.

A number of tri- and tetrapeptides have been found to inhibit aggregation and gelation of deoxygenated sickle cell hemoglobin. These inhibitors have hydrophobic phenylalanine residues at one end and hydrogen bonding lysine or arginine side chains at the other end. The backbone is not very specific. The inhibitors do not modify the oxygen carrying properties of hemoglobin. When the inhibitor and sickle hemoglobin are put inside reconstituted cells, the erythrocytes do not sickle upon deoxygenation. Compounds of this type may develop useful agents in the therapy of sickle cell anemia.

Antisickling Agents↗
PubMed1976

Direct biochemical mapping of eukaryotic viral DNA by means of a linked transcription-translation cell-free system.

A method is described for mapping regions of eukaryotic viral DNA coding for specific proteins, utilizing a linked transcription-translation cell-free system primed with DNA fragments generated by restriction endonucleases. Three simian virus 40 (SV40) DNA fragments derived from that region of the DNA expressed late in lytic infection were purified. They were: Hpa I-A (0.76-0.175 map units), Bgl I-EcoRI-B (0.672-0), and Hpa II-EcoRI-B (0.735-0). (Fragments are named from the cleaving restriction endonuclease and electrophoretic mobility. End positions on the conventional map are in clockwise order.) These fragments efficiently stimulated the incorporation of [3H]UTP and [35S]methionine into trichloroacetic-acid-insoluble material in the linkec system. The location of the region of DNA coding for the viral structural proteins VPI, VP2 and VP3 was determined from the spectrum of polypeptide synthesis directed by the individual intact fragments and their specific endonucleolytic digests. The polypeptides synthesized in the cell-free system were characterized on urea-sodium dodecyl sulfate polyacrylamide gradient gels and by two-dimensional tryptic peptide analysis. ..

Cell-Free System↗
PubMed1975

Purification of D-alanine carboxypeptidase from Escherichia coli B on a penicillin-Sepharose column.

1. A soluble D-alanine carboxypeptidase from Escherichia coli strain B was purified on a p-aminobenzylpenicillin-Sepharose column. This one-step chromatography followed by an (NH4)2SO4 precipitation yielded an enzyme purified 1200-fold and some of its properties are reported. 2. The pure D-alanine carboxypeptidase was devoid of D-alanine carboxypeptidase II activity and migrated as a single protein band on analytical disc gel electrophoresis. 3. Triton X-100 in the purification procedure is an absolute requirement for obtaining a stable enzyme. 4. The enzymic activity of D-alanine carboxypeptidase was greatly affected in solution of high salt concentrations and varied somewhat with the nature of the cation tested.

Alanine↗
PubMed1975

Simian virus 40 DNA directs synthesis of authentic viral polypeptides in a linked transcription-translation cell-free system.

A linked cell-free system has been developed which is capable of transcribing and translating mamalian viral DNA, and its characteristics and requirements are outlined. In this system, simian virus 40 (SV40) DNA Form I (supercoiled) directed the synthesis of discrete polypeptides up to 85,000 daltons in size. One of these products was indistingusihable from authentic major virus capsid protein VPI, as judged by mobility on sodium dodecyl sulfate/polyacrylamide gels, antibody predipitation, and peptide analyses. The cell-free products larger than VPI comprised a number of polypeptides ranging in molecular weight from 50,000 to 85,000. These polypeptides demonstrated no immunological relationship whatsoever to the structural protein VPI. However, two of these products, along with one of approximately 25,000 dlatons, were precipitated with antiserum to SV40 tumor antigen. Linear SV40 DNA generated by the cleavage of Form I DNA with the restriction endonuclease EcoR1 was an efficient template in this system and also directed the synthesis of a polypeptide migrating with VPI on polyacrylamide gels. The potential of this system for defining a functional map of a DNA genome is discussed.

Animals↗