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Biomedical subjects

R Frank

Publications and source records attributed to R Frank.

At least 361 records · Page 20Linked to original sources
PubMed1988

Adipocyte plasma membranes contain two Gi subtypes but are devoid of Go.

Antisera generated against synthetic peptides were used to identify G-protein alpha-subunits in plasma membranes from rat adipocytes. Applying the immunoblot technique, we detected two Gs alpha-subunits of 42 and 43 kDa, corresponding to the two cholera toxin substrates, and two Gi alpha-subunits of 40 and 41 kDa, corresponding to the two pertussis toxin substrates present in these membranes. The 40 kDa protein was tentatively identified as the Gi2 alpha-subunit. A serum specific for the Go alpha-subunit failed to detect any immunoreactive protein. Thus plasma membranes of adipocytes possess two forms of Gi but not Go.

Adipose Tissue↗
PubMed1988

Human pancreatic secretory trypsin inhibitor (PSTI) produced in active form and secreted from Escherichia coli.

As a basis for a protein design project, we decided to produce the human pancreatic secretory trypsin inhibitor (PSTI) in its active form. Total gene synthesis was carried out efficiently by (i) computer design of the gene fragments, (ii) synthesis of the oligodeoxynucleotides by the segmental support method, and (iii) assembly of double strands under optimized ligation conditions. Fusion to the ompA gene signal peptide led to secretion of processed PSTI in various constructions, with or without additional amino acids (aa) at the N-terminus. The secreted proteins (56 to 63 aa) were biologically active, suggesting that the three cysteine bridges were correctly formed. Surprisingly, after induction the product was found almost exclusively in the culture medium. Variants of PSTI with Asp or Asn at aa positions 21 and 29 [sequences published by Greene et al., Methods Enzymol. (1976) 813-825, and by Yamamoto et al., Biochem. Biophys. Res. Commun. (1985) 605-612] showed the same Ki for both human and porcine trypsin.

Amino Acid Sequence↗
PubMed1988

Three-dimensional structure of d(GGGATCCC) in the crystalline state.

The structure of the self-complementary octamer d(GGGATCCC) has been analysed by single crystal X-ray diffraction methods at a nominal resolution of 2.5 A. With acceptable stereochemistry of the model the crystallographic R factor was 16.6% after restrained least-squares refinement. In the crystal, d(GGGATCCC) forms an A-DNA double helix with slightly varying conformation of the two strands. The average displacement of the base pairs from the helix axis is unusually large and is accompanied by pronounced sliding of the base pairs along their long axes at all dinucleotide steps except for the central AT. With 12 base pairs per complete turn the helix is considerably underwound. As observed with most oligodeoxyribonucleotides analysed by X-ray crystallography so far, the octamer displays reduced base pair tilt, increased rise per base pair and a more open major groove compared with canonical A-DNA. We propose that, based on these parameters, three A-helical sub-families may be defined; d(GGGATCCC) then is a representative of the class with intermediate tilt, rise, and major groove width.

DNA↗
PubMed1988

A human brain glycoprotein related to the mouse cell adhesion molecule L1.

We have employed monoclonal antibody 5G3, an antibody used to label human tumor cells of neural origin (Mujoo, K., Spiro, R.C., and Reisfeld, R. A. (1986) J. Biol. Chem. 261, 10299-10305), to isolate and characterize a large glycoprotein from normal adult human brain. This protein was compared to mouse L1 (Rathjen, F. G., and Schachner, M. (1984) EMBO J. 3, 1-10), a neural cell surface glycoprotein implicated predominantly in neurite-neurite interactions. On the basis of the following results the 5G3 antigen is considered to be the human homologue of mouse L1. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, both proteins share similar molecular masses of their carbohydrate-depleted or undepleted components. In tryptic fingerprint analyses of the iodinated L1 and 5G3 components, 65% of the resolved peptides comigrated. Comparison of NH2-terminal amino acid sequences revealed a high degree of homology between human 5G3 and mouse L1, with 11 of 15 residues being identical. Furthermore, polyclonal antibodies to human 5G3 antigen were found to be cross-reactive with mouse L1 antigen and vice versa. All components of 5G3 and L1 antigens show considerable charge heterogeneity with partial overlapping of regions in isoelectric focusing followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. These findings provide a basis for studying the role of the human L1 homologue in human diseases.

Amino Acid Sequence↗
PubMed1988

Staphylococcal phosphoenolpyruvate-dependent phosphotransferase system: purification and characterization of the mannitol-specific enzyme IIImtl of Staphylococcus aureus and Staphylococcus carnosus and homology with the enzyme IImtl of Escherichia coli.

Enzyme IIImtl is part of the mannitol phosphotransferase system of Staphylococcus aureus and Staphylococcus carnosus and is phosphorylated by phosphoenolpyruvate in a reaction sequence requiring enzyme I (phosphoenolpyruvate-protein phosphotransferase) and the histidine-containing protein HPr. In this paper, we report the isolation of IIImtl from both S. aureus and S. carnosus and the characterization of the active center. After phosphorylation of IIImtl with [32P]PEP, enzyme I, and HPr, the phosphorylated protein was cleaved with endoproteinase Glu(C). The amino acid sequence of the S. aureus peptide carrying the phosphoryl group was found to be Gln-Val-Val-Ser-Thr-Phe-Met-Gly-Asn-Gly-Leu-Ala-Ile-Pro-His-Gly-Thr-Asp- Asp. The corresponding peptide from S. carnosus shows an equal sequence except that the first residue is Ala instead of Gln. These peptides both contain a single histidyl residue which we assume to carry the phosphoryl group. All proteins of the PTS so far investigated indeed carry the phosphoryl group attached to a histidyl residue. According to sodium dodecyl sulfate gels, the molecular weight of the IIImtl proteins was found to be 15,000. We have also determined the N-terminal sequence of both proteins. Comparison of the IIImtl peptide sequences and the C-terminal part of the enzyme IImtl of Escherichia coli reveals considerable sequence homology, which supports the suggestion that IImtl of E. coli is a fusion protein of a soluble III protein with a membrane-bound enzyme II. In particular, the homology of the active-center peptide of IIImtl of S. aureus and S. carnosus with the enzyme IImtl of E. coli allows one to predict the N-3 histidine phosphorylation site within the E. coli enzyme.

Amino Acid Sequence↗
PubMed1988

Production of specific monoclonal antibodies against the active sites of human pancreatic secretory trypsin inhibitor variants by in vitro immunization with synthetic peptides.

Specific monoclonal antibodies against the active sites of two genetically engineered pancreatic secretory trypsin inhibitor (PSTI) variants (PSTI 0 and PSTI 4) were produced. The protease inhibitors PSTI 0 and PSTI 4 differ only by three amino acid substitution at their active sites. PSTI 0 inhibits trypsin, whereas PSTI 4 inhibits human granulocyte elastase and chymotrypsin. Immunization was performed in vitro with a synthetic heptapeptide that covers the mutated region of the protein. For this purpose in vitro culture conditions for the production of specific monoclonal antibodies against synthetic peptides were improved. The monoclonal antibodies obtained react specifically with the corresponding protease inhibitor variant. Competition experiments with trypsin and human elastase demonstrate that the protease displace the monoclonal antibody from the active site of PSTI 0 and PSTI 4 respectively.

Amino Acid Sequence↗
PubMed1988

Identification of the human papillomavirus type 18 E6 and E6 proteins in nuclear protein fractions from human cervical carcinoma cells grown in the nude mouse or in vitro.

We recently reported the transcription patterns of human papillomavirus (HPV) type 18 sequences in human cervical carcinoma cell lines. The open reading frames (ORFs) E6* and E6 represent the 5'-terminal cistrons in HPV18 mRNAs. ORF E6* was assumed to be specific for HPV types associated with genital carcinomas. To identify the predicted gene product, ORF E6* from a HeLa cDNA clone was expressed as an MS2 fusion protein in Escherichia coli. The C-terminal 23 amino acid residues were chemically synthesized. A panel of monoclonal antibodies was generated, recognizing E6* and E6* plus E6, respectively. In human cervical carcinoma cell lines grown in vitro these monoclonal antibodies specifically immunoprecipitate the putative Mr 17,000 and 18,000 HPV18 E6 proteins in nuclear protein fractions. In a HPV18 DNA containing human cervical carcinoma established in nude mice, these monoclonal antibodies specifically immunoprecipitate a polypeptide with a molecular weight of 6500 as predicted for the HPV18 ORF E6* gene product in a nuclear protein fraction.

Animals↗
PubMed1988

Expression of the chemically synthesized gene for ribonuclease T1 in Escherichia coli using a secretion cloning vector.

The gene for ribonuclease T1 from Aspergillus oryzae has been chemically synthesized using the segmental support technique. An Escherichia coli clone producing the ribonuclease at high levels was constructed by linking the gene downstream to the region coding for the signal peptide of the OmpA protein (a major outer membrane protein of E. coli), using the secretion cloning vector pIN-III-ompA2. This strategy was employed in order to circumvent a possible toxic effect of the gene product on the host cell. Active ribonuclease containing four additional amino acids at the N-terminus could be isolated from the periplasmic fraction of the host. The final yield after purification was 20 mg enzyme/l liquid culture. With respect to immunological, catalytic and specific behaviour, no qualitative differences could be detected between the enzyme from the over-producing E. coli strain and ribonuclease T1 isolated from A. oryzae.

Aspergillus↗
PubMed1988

Complete synthesis and transcription in vitro of a gene coding for human ribosomal 5S RNA.

The gene coding for the major human ribosomal 5S RNA was chemically synthesized and cloned into a pUC13 vector. This approach was taken, because attempts to isolate the human 5S gene have thus far yielded either pseudogenes or variant 5S genes of unknown function. The synthetic human gene was transcribed by RNA polymerase III either in a crude HeLa cell extract or in a system reconstituted from partially purified transcription factors. Comparative studies with the Xenopus laevis somatic 5S gene show that the human gene is transcribed with similar fidelity and an efficiency of about 80% under optimal conditions. The time-course of transcription and optimal concentrations of template and transcription factors were found to be similar for both genes studied. The synthetic gene described may prove useful to study its interaction with human transcription factors in a homologous system.

Animals↗
PubMed1988

Polypeptide elongation factor Tu from Halobacterium marismortui.

A GDP-binding protein of 60 kDa from Halobacterium marismortui has been purified to homogeneity. The purification has been carried out in high-salt buffers or in 50% glycerol buffers to protect the halophilic protein from denaturation. Evidence that this protein is the halophilic elongation factor Tu (hEF-Tu) is provided by the high homology of its N terminus with the corresponding sequences of other EF-Tus, and by immunological studies. Like some other EF-Tus the native protein can be cleaved with trypsin without concomitant loss of GDP-binding ability. The molecular mass of this hEF-Tu is higher than that for the corresponding factors from other sources including the halobacterium Halobacterium cutirubrum. The protein possesses typical halophilic characteristics, in that it is stable and active in 3 M KCl or 2 M (NH4)2SO4. Some other properties, like autofragmentation under sample treatment before SDS-PAGE, are described.

Amino Acid Sequence↗
PubMed1988

Site-specific recombination in bacteriophage Mu: characterization of binding sites for the DNA invertase Gin.

Site-specific DNA inversion in phage Mu is catalysed by the phage-encoded DNA invertase Gin and a host factor FIS. We demonstrate that purified Gin protein binds specifically to 34-bp sequences that flank the G segment as inverted repeats. Each inverted repeat (IR) contains two binding sites for Gin which have to be arranged in a specific configuration to constitute a recombinogenic site. While one of these sites is bound when present alone, the other site is bound only in conjunction with the first one, suggesting cooperative binding. In addition to the sites within the IR, Gin binds with lower affinity to AT-rich sequences adjacent to the IR. We demonstrate that these sites do not participate in the inversion reaction. The IR itself can be shortened to 25 bp without effect on inversion frequency. Using gel mobility shift experiments on circular permuted fragments containing the IR we show that Gin bends DNA upon binding. We discuss the possibility that DNA bending is related to the formation of a productive synaptic complex.

Base Sequence↗
PubMed1988

Purification of a NF1-like DNA-binding protein from rat liver and cloning of the corresponding cDNA.

NF1-like proteins play a role in transcription of liver-specific genes. A DNA-binding protein, recognizing half of the canonical NF1 binding site (TGGCA) present on the human albumin and retinol-binding protein genes, has been purified from rat liver. Several peptides deriving from a tryptic digest of the purified protein were sequenced and the sequence was used to synthesize specific oligonucleotides. Two overlapping cDNA clones were obtained from a rat-liver cDNA library; their sequence reveals an open reading frame coding for 505 amino acids, including all the peptides sequenced from the purified protein. The DNA-binding domain, most likely located within the first 250 amino acids, is highly homologous to the sequence of CTF/NF1 purified from HeLa cells. Northern analysis reveals several mRNA species present in different combinations in various rat tissues.

Amino Acid Sequence↗
PubMed1988

Cadmium levels in Ontario moose and deer in relation to soil sensitivity to acid precipitation.

This study examines the influence of buffering capacity of the soil on the levels of cadmium in the kidney, liver and muscle of moose and white-tailed deer from nine sampling sites (four buffered and five non-buffered) in Ontario, Canada. Tissues collected from hunter-killed moose and deer during 1984 and 1985 were analysed for cadmium. Tissue from moose in the non-buffered Algonquin Park site (21.9 +/- 1.1 mg/kg wet weight) and the buffered St. Joseph Island site (12.7 +/- 3.2 mg kg-1) had the highest mean levels of kidney cadmium compared with other sites sampled in Ontario. The highest mean levels of kidney cadmium in deer were found in the non-buffered Loring site (15.1 +/- 0.8 mg kg-1) adjacent to Algonquin. From all sites, the level of cadmium was highest in kidney, lower in liver and was often undetectable in muscle. Cadmium level increased with animal age (p less than 0.05). Levels of cadmium in Ontario moose from some regions are comparable to those found in Quebec and Manitoba and are considerably higher than those of Maine and Scandinavia. Levels of cadmium in kidneys and livers of white-tailed deer in parts of Ontario are considerably lower than those in Pennsylvania. As a result of this study, the Ontario Ministry of Natural Resources, in consultation with the Ontario Ministry of Health, has recommended that the public not consume kidneys or livers of Ontario moose and deer.

Age Factors↗
PubMed1988

Testing of the abiotic degradation of chemicals in the atmosphere: the smog chamber approach.

Methods for measuring the hydroxyl-, ozone-, and direct photochemical reactivity of a substance in one specially designed medium size smog chamber are described. Rate coefficients for the reaction of OH with n-hexane, n-heptane, ethene, ethyne, chloroform, trichloroethene, methanol, 2-propanol, benzene, o-xylene, 1,4-dichlorobenzene, 1,2,4-trichlorobenzene, p-chloroaniline, naphthalene, acenaphthene, 1,4-dichloronaphthalene, biphenyl, and fluorenone are given and discussed. An upper limit of 5 X 10(-13) cm3/sec is given for the sum penta- and hexa-chlorobiphenyls (PCB). Rate coefficients for the ozone reaction are given for beta-pinene, limonene, delta 3-carene, cineol, vinyl chloride and 1,3-butadiene. In cases where the literature data are available for comparison, the rate coefficients (kappa OH and kappa O3) reported here compare favorably with the best data reported. The direct photochemical reactivity has been shown to be measurable if the chamber is cleaned carefully. Preliminary results on benzophenone are reported. The methods described here, except that of direct photochemical reactivity, are in agreement with those proposed to OECD. Moreover, part of the Draft OECD Test Guideline (Berlin, 1987) on "Photochemical-Oxidative Degradation in the Atmosphere" is based on work described here and on closely related work in other laboratories (Becker et al., 1984).

Benzophenones↗