Gary V. Paddock

Expression of the hepatocyte growth factor-like protein gene in human hepatocellular carcinoma and interleukin-6-induced increased expression in hepatoma cells.

Human hepatocellular carcinoma is one of the most frequent malignant tumors. It may occur following exposure to various agents, including viruses and chemical carcinogens; however, the underlying mechanisms of the hepatocarcinogenesis are not known. The present study is the result of our search for genes which may be abundantly expressed in human primary liver carcinoma. One of these genes was found to encode the human hepatocyte growth factor-like protein (HGFLP), also known as macrophage-stimulating protein. HGFLP is structurally homologous to hepatocyte growth factor, a potent growth factor for liver. HGFLP mRNA was also found to be overexpressed in a hepatoblastoma sample and in a sample of subacute fulminant hepatic necrosis. In a study on the effects of cytokines on the expression of HGFLP, we found that IL-6 increased expression of HGFLP mRNA in Hep G2 cells, but IL-1alpha, IL-1beta and TNF-alpha had no effect. An increase in HGFLP could be the result of inflammation and/or tissue injury and its overexpression may prove to be useful as an indicator of hepatoma.

Strategies for constructing complementary DNA for cloning

Abstract We have examined alternative approaches to existing methods for synthesizing complementary DNA suitable for molecular cloning. One model of construction is presented in which ribonucleotides are added to the 3′ end of complementary DNA prior to synthesis of the second DNA strand. The hairpin structure at one end of the molecule is then opened by treatment with RNaase or alkali. This method would eliminate the normal requirement for single-strand specific nucleases and thus shows promise as a means for preserving the 5′ end sequences of mRNA in recombinant complementary DNA studies. Another technique for constructing complementary DNA is proposed in which no cleavage step is required. Instead, a hairpin, double-stranded DNA is extended with a homopolymer at the 3′ end, and displacement or “third-strand” synthesis by the Klenow fragment of DNA polymerase I is primed by an oligonucleotide hybridized to the homopolymer. The end result should be an inverted repeat with two-fold rotational symmetry. The mRNA 5′ end sequences represent the center of symmetry. Cloned, symmetrical DNA should facilitate subsequent nucleotide sequence analysis. Furthermore, the symmetrical molecule may serve as an intermediate in continued DNA synthesis provided the homopolymer chain is sufficiently longer than the primer, thus leading to mRNA sequence amplification in vitro . Alternative options with attendant advantages and disadvantages are given at each stage in the construction schemes. These strategies, along with established procedures, offer a repertoire from which researchers may select in order to fill their specific needs.

Construction of recombinant cDNA via a ribosubstituted hairpin

Abstract A new technique for recombinant cDNA construction is presented in which ribonucleotides are added to the 3′ end of cDNA via ribosubstitution prior to synthesis of the second DNA strand. The hairpin floppy loop is then opened by alkaline hydrolysis. This method eliminates the requirement for S1 nuclease and thus shows promise as a means for preservation of mRNA 5′-end sequences in recombinant cDNAs and for eliminating a potential source of error in those sequences.

Contribution of hydrolyzed nucleic acids and their constituents to the apparent amino acid composition of biological compounds.

Abstract Acid hydrolysis of protein-free mixtures of nucleotides, nucleosides, and nucleic acids yields amino acids, free bases, and possibly other unidentified fragments when analyzed by thin-layer chromatography and by standard amino acid analysis. Glycine is the predominant amino acid detected, which may constitute 47–97% of the apparent amino acid composition, depending on the type of material subjected to hydrolysis. Obviously, hydrolyzed nucleic acids or their constituents can therefore contribute to the apparent amino acid composition of a supposedly pure peptide or of other more complex mixtures of compounds mistakenly believed to contain only protein. To circumvent this problem, we suggest that nucleotides or nucleic acid moieties should be removed from any product for which the amino acid composition is desired, and that whenever a large glycine peak is noted in a hydrolyzed sample, the presence of nucleic acids or their constituents should be suspected.

IMMUNOCHEMICAL AND PHYSICAL-CHEMICAL EVIDENCE FOR THE PRESENCE OF THYMOSIN ALPHA 1 -PEPTIDE IN DIALYZABLE LEUKOCYTE EXTRACTS

Publisher Summary This chapter presents immunochemical and physical–chemical evidence for the presence of thymosin alpha1-peptide in dialyzable leukocyte extracts (DLE). In a study described in the chapter, the collective results obtained from immunologic and physical–chemical evaluations of the E-receptor regeneration promoting activity present in human DLE indicate that it is partly because of the presence of thymosin α1 peptide in DLE. There are, however, additional components in DLE that express a similar activity but do not seen to be intact thymosin α1. Included here might be thymic humoral factor, which has a pi of 5.7 and could be responsible for the activity found in the β region when DLE Sephadex G-25 Fraction IV was fractionated by IEP in a 2.5 to 10 gradient.The documentation of the presence of a variety of T-lymphocyte maturation or differentiation factors in DLE explains why DLE may have nonspecific beneficial effects when it is employed as an immunotherapeutic agent.

Abbreviated 3′ non-coding region in duck alpha D globin messenger RNA defines evolutionarily conserved sequences

Abstract Nucleotide sequence data for the duck alpha D globin gene indicate an extremely short 3′ untranslated region of only 49 nucleotides. Evolutionarily conserved sequences defined short regions of potential functional significance. Comparisons among sequences for duck and chicken alpha globins indicate that this region has importance in the regulation of globin gene expression.

Globin proteins of the normal and anemic duck

The red blood cells of normal adult ducks contain two main hemoglobins. The most abundant type, HbA, comprises approximately 80% of the total, with the remaining 20% being made up of HbD. An attempt was made to determine whether during hemolytic anemia a special alpha globin chain (alpha s) replaces the alpha chain of HbA found in normal animals. This special stress alpha globin, whose existence has been seriously questioned, was originally postulated to explain the sequence discrepancies obtained between alpha chains of normal and anemic chickens and ducks. Using gel electrophoresis, isoelectric focusing, and HPLC peptide mapping techniques no qualitative differences between the alpha A globins of normal and anemic animals were found. The nature of the beta globin chains present in adult ducks has also never been rigorously established. In this work, a variety of techniques, including HPLC, gel electrophoresis, and microcolumn amino acid analysis, were used to examine the beta chains from each hemoglobin. Using these methods, no differences were found between the beta globin chains of the two hemoglobins.

Displacement synthesis and cloning of symmetrical complementary DNA containing duck globin mRNA sequences

Abstract A displacement synthesis procedure was used to construct symmetrical recombinant cDNAs. A double-stranded cDNA containing a hairpin loop was extended by the addition of a homopolymer to the 3′ end. This was followed by displacement, or “third strand” synthesis that was primed by an oligonucleotide hybridized to the homopolymer. Ideally, the product should be a DNA containing an inverted repeat with twofold rotational symmetry about nonsymmetrical sequences representing the hairpin loop in the original double-stranded cDNA. Duck globin cDNAs were synthesized by the displacement mode of construction and cloned in pBR322. An α-globin recombinant, pDGPα-2, was isolated and sequenced. This recombinant was found to have two 3′ half regions (mRNA sequence sense) inverted about a nonsymmetrical 5′ half region and an adjacent oligo(dGṡdC) homopolymer. The sequence arrangement indicates that the cDNA folded back on itself, forming a large loop, to prime synthesis of a second strand. We propose that the internal oligo(dGṡ dC) arose through dynamic shifts in cDNA intrastrand structure during the course of synthesis.

DETECTION OF “DIALYZABLE TRANSFER FACTOR” IN VITRO: STRUCTURAL AND CHEMICAL CHARACTERIZATION OF THE ACTIVITY SPECIFIC FOR TUBERCULIN*

Leukocyte migration inhibition (LMI) and macrophage migration inhibition (MMI) assays have been used by many investigators for in vitro assessments of human cell-mediated immunity (CMI) using either specific antigens (Ag) or mitogens (for review see reference 1 ) such as phytohemagglutinin and concanavalin A.:< The basis for these assays is the determination of responsiveness by measuring the elaboration of a “lymphokine” or mediator of cellular immunity (MCI) . The mediator designated “leukocyte migration inhibition factor” (LIF) inhibits the random migration of polymorphonuclear neutrophils (PMN) :I Recent experimental evidence published by Weisbart et aL5 indicates that the secretion of LIF by sensitized lymphocytes requires stimulation with an appropriate Ag or mitogen and collaboration between T-lymphocytes and monocytes or T-lymphocytes and B-lymphocytes.5 The absolute requirement of T-lymphocytes for LIF production makes LMI assays seem more attractive than the more widely used MMI assays as in vitro systems to study human CMI, which is mediated by T-lymphocytes, since there is evidence that macrophage migration inhibition factor (MIF), the mediator measured in MMI assays, may be secreted by B-lymphocytes alone.6. In addition, measurement of LIF secretion in vitro by LMI has another extremely important attribute of direct relevance to the study of transfer factor. It has been demonstrated by several investigators (for review see reference 1 ) that there is a very good qualitative (but not quantitative) correlation between Ag responsiveness as shown by LIF release in vitro (i.e., LMI) and delayed cutaneous hypersensitivity (DCH) in vivo (i-e., skin testing). Transfer factor (TF) is defined as an activity present in dialyzable leukocyte extracts (DLE) obtained from immune donors that is able to transfer CMI to specific Ag into previously nonimmune human recipients, as measured by the acquisition of positive DCH (skin test) when injected intradermally with Ag. During the past few years we have been evaluating the effects of crude DLE

MECHANISM(S) OF ACTION OF HUMAN TRANSFER FACTOR: INSIGHTS OBTAINED FROM STUDYING “ANTIGEN-LIBERATED TRANSFER FACTCR” SPECIFIC FOR TUBERCULIN

Publisher Summary This chapter discusses the form(s) of transfer factor (TF) that are released by viable leukocytes from tuberculin responsive human donors when their leukocytes are incubated with PPD in vitro . Data obtained from studying this antigen-liberated (or released) has provided further insight into the roles of TF–H5, TF–H7, and dephosphorylated TF–H5 in the induction of antigen specific responsiveness. The chapter concludes that when immune lymphocytes are incubated with specific antigen they release only dephosphorylated TF–H5. Dephosphorylated TP–H5 is apparently derived from the cell surface (or secreted after it is derived from TF–H5) and also is apparently released nonspecifically by lymphocytes incubated for prolonged periods at 37°C as part of a normal turnover process of cellular membrane constituents. It is speculated, however, that cell-free supernatants derived from lymphocytes merely incubated at 37°C for 4 h without specific antigen added would contain a family of TFs representing the range of antigen sensitivities found in the donor.