Sequence analysis of an immunogenic and neutralizing domain of the human T-cell lymphoma/leukemia virus type I gp46 surface membrane protein among various primate T-cell lymphoma/leukemia virus isolates including those from a patient with both HTLV-I-associated myelopathy and adult T-cell leukemia.

Human T-cell lymphoma/leukemia virus type I (HTLV-I) causes adult T-cell leukemia/lymphoma and HTLV-I-associated myelopathy. Specific regions within the outer envelope proteins of other retroviruses, e.g., human immunodeficiency virus type 1, are highly immunogenic and, because of the selective pressure of the host immune system, quite variable. Mutations in the external envelope protein gene of murine retroviruses and human immunodeficiency virus type 1 influence cellular tropism and disease pathogenesis. By contrast, no disease-specific viral mutations have been identified in HTLV-I-infected patients. However, all isolates studied thus far have originated from leukemic cell lines, peripheral blood mononuclear cells, or cerebrospinal fluid lymphocytes from patients with HTLV-I-associated myelopathy and adult T-cell leukemia/lymphoma and, therefore, may not truly reflect tissue-associated variation. The midregion of the HTLV-I gp46 external envelope glycoprotein (amino acids 190-209) induces an antibody response in 90% of infected individuals, and a hexapeptide in this region (amino acids 191-196) elicits antibodies in rabbits which inhibit syncytia formation and infection of target lymphocytes. Because of the above, we expected the neutralizing domain of the gp46 env gene of HTLV-I to possess disease or organ-associated mutations selected by the infected host's immune system. Hence, we amplified, cloned, and sequenced HTLV-I DNA directly from in vivo central nervous system, spleen, and kidney specimens, and a leukemic cell line from a patient (M. J.) with both HTLV-I-associated myelopathy and adult T-cell leukemia/lymphoma to discern the possibility of tissue- and/or disease-specific variants. In addition, we sequenced several HTLV-I isolates from different regions of the world, including Papua New Guinea, Bellona, and Liberia, and compared them to other previously published HTLV-I and related retroviral sequences. The 239-base pair sequence corresponding to amino acids 178 to 256 in gp46 displayed minor tissue-specific variation in clones derived from central nervous system tissues from patient M. J., but overall was highly conserved at both the DNA and amino acid levels. Variation was observed in this region among the other HTLV-I, simian T-cell lymphoma virus type I, and HTLV-II isolates in a pattern that was consistent with their known phylogenetic relationship. No consistent disease-related changes were observed.(ABSTRACT TRUNCATED AT 400 WORDS)

Distinctive properties of mammalian DNA polymerases.

DNA polymerase-alpha and -beta can be distinguished from one another by the differential effects of N-ethylmaleimide, KCl, ara-CTP and temperature, as well as on the basis of sedimentation. The sensitivity of DNA polymerase-beta to elevated temperatures as compared to DNA polymerase-alpha provides a new means of distinguishing between these two enzymes even in crude extracts and a possible probe for determining their function. DNA polymerase-alpha and -beta share several properties in common, including the ability to readily incorporate dUTP in place of dTTP. The Km for dUTP varies from 10 to 30 micron with different preparations of DNA polymerase-alpha and -beta. Thus, in mammalian cells, dUMP could be incorporated into DNA, and if excised by an endonuclease, would lead to discontinuities. Initial analyses of fidelity in direct comparative studies indicate that beta-class DNA polymerases are highly accurate in base selection when copying poly[d(A-T)]. Less than one molecule of dGMP is incorporated for every 12 000-45 000 molecules of dAMP and dTMP polymerized. DNA polymerase-alpha is somewhat less accurate, making one mistake for every 4000-10 000 correct nucleotides incorporated. Since both polymerases lack an exonucleolytic activity, this accuracy must be the result of selectivity for the complementary nucleotide by the polymerase.

Detection and characterization of DNA polymerase from Trypanosoma brucei.

The predominant DNA polymerase activity has been isolated from the parasitic flagellated protozoan, Trypanosoma brucei. Like mammalian DNA polymerase-alpha the trypanosome DNA polymerase is of large molecular weight (S, 6--8), is resistant to thermal denaturation, is sensitive to N-ethylmaleimide, and is inhibited by high ionic strength. However, specific antisera that cross-react with mammalian DNA polymerase-alpha from different species fail to cross-react with the trypanosome polymerase.

Cloning and sequencing of the cDNA for an RNA-binding protein from the Mexican axolotl: binding affinity of the in vitro synthesized protein.

A full length cDNA for an RNA-binding protein (axolotl RBP) with consensus sequence (RNP-CS) from the Mexican axolotl, Ambystoma mexicanum, has been cloned from a subtraction library. In vitro translation with synthetic mRNA and subsequent hybrid-arrested translation with a specific antisense oligonucleotide confirms that the axolotl RBP cDNA encodes an approx. 16 kDa polypeptide. Computer-assisted analyses revealed amino acid similarities of 58-60% to various RNA-binding proteins and a 90 amino acid region at the amino-terminal end constituting the putative RNA-binding domain (RNP-CS) with two highly conserved motifs, RNP2 and RNP1. Phylogenetic analysis suggests that the putative RNA-binding protein from axolotl is unique. A binding assay with radiolabeled axolotl RBP showed that this RNA-binding protein bound strongly with poly(A) and to a lesser degree with poly(U), but not at all with poly(G), poly(C), or DNA.

Identification and expression of a homologue of the murine HoxA5 gene in the Mexican axolotl (Ambystoma mexicanum).

An excellent model for studying heart development in vertebrates is the cardiac non-function lethal mutant (gene c) Mexican axolotl, Ambystoma mexicanum. In order to facilitate our analyses of the mutant system, we have undertaken a search for stage-specific molecular markers during embryonic development of the axolotl. We have concentrated on homeobox genes as suitable candidates for monitoring molecular changes during development. A 270-bp probe encoding a portion of the axolotl homeobox gene Ahox-1 was generated by PCR from a stage-18 axolotl embryonic cDNA library. 32P-labelled PCR-amplified Ahox-1 DNA was used as the probe for screening a lambda AM18 cDNA library using moderately stringent conditions. We isolated six clones and determined their partial nucleotide (nt) sequences. One of the clones, which has very high homology to human, mouse and rat Hox A5 (83 and 99% at the nt and amino-acid levels, respectively, in the homeodomain region), was analyzed further. RT-PCR analyses show that the level of expression of HoxA5 is very low at stage 11 of embryonic development (gastrula). The level of expression reaches maximum at stage 25 (tailbud) and then plateaus at stages 30 and 35 (heartbeat onset). Although the expression of Ahox-1 was also found to start at stage 11, it reaches a maximum level at stage 25 and declines at stage 35. We have also studied, using RT-PCR, the tissue-specific expression of HoxA5 and Ahox-1 in juvenile axolotl.

Cloning, sequencing and expression of a novel homeobox gene AxNox-1 from the Mexican axolotl.

We have cloned and sequenced a cDNA containing a homeobox gene, AxNox-1, from a stage 18 axolotl embryonic cDNA library which shows only moderate levels of similarity to other known homeobox genes. The nucleotide sequence of the cDNA has an open reading frame for 335 amino acids and besides the homeodomain, there is an acidic domain and a proline-rich domain present in the protein. The transcripts for this gene are detectable at stage 4 of embryonic development and, hence, there is a good possibility that the transcripts are maternally contributed. Expression levels for AxNox-1 reach maximum levels by stage 12 of development and thereafter decline to very low levels by stage 25. High levels of the transcript for AxNox-1 are later found in the brains of both neotenous and metamorphosing adult axolotls. Low amounts of the message are also found to be present in a number of other organs that were tested. In situ hybridization studies on whole mounts and sections suggest that this gene is expressed predominantly in neural tissue during development.

The association of thymidine kinase activity and thymidine transport in Escherichia coli.

We have constructed a series of mutants within the putative nucleoside-binding site of the herpes simplex type-1 virus (HSV-1) thymidine kinase (TK)-encoding gene (tk), contained within an expression vector. While most mutations within this sequence produce an inactive protein, we find no absolute requirement for the wild-type Ile166 and Ala167. The uptake of thymidine (dT) into Escherichia coli tdk-, lacking functional endogenous TK activity, is proportional to the amount of TK activity expressed from the heterologous HSV-1 tk gene. In contrast, there is no enhancement in deoxycytidine uptake into E. coli producing (HSV-1) TK. These results imply a specific role for TK in the active transport of dT into E. coli.

Selection of new biologically active molecules from random nucleotide sequences.

Genetic diversity can be achieved in vitro by inserting random nucleotide (nt) sequences into cloned genes. In the case of enzymes, subsequent genetic complementation can be used to select for new mutants that exhibit different substrate specificities, altered catalytic activities, or altered temperature sensitivities. Using this technique, one can also analyze the contribution of different amino acid residues to the structure and function of enzyme. Selecting biologically active DNA sequences from large random populations provides a new method for identifying nt sequences with unique functions. Analogous random sequence selection techniques have been applied to determine the consensus sequence of the Escherichia coli promoters, DNA and RNA sequences that bind specific protein(s), DNA regulatory sequences, ribozyme(s) and ligand-specific RNA(s). In this manuscript, we will consider recent data obtained in our laboratory as a result of inserting random sequences into the putative nucleoside-binding site of herpes simplex virus type 1 (HSV-1) thymidine kinase (TK). We have obtained over 2000 new mutant HSV-1 TKs, some of which are stable at higher temperatures or have altered substrate specificity and/or catalytic rates when compared to those of the wild-type enzyme.

Differential expression of a novel isoform of alpha-tropomyosin in cardiac and skeletal muscle of the Mexican axolotl (Ambystoma mexicanum).

Alternative mRNA splicing is a fundamental process in eukaryotes that contributes to tissue-specific and developmentally regulated patterns of tropomyosin (TM) gene expression. Northern blot analyses suggest the presence of multiple transcripts of tropomyosin in skeletal and cardiac muscle of adult Mexican axolotls. We have cloned and sequenced two tropomyosin cDNAs designated ATmC-1 and ATmC-2 from axolotl heart tissue and one TM cDNA from skeletal muscle, designated ATmS-1. Nucleotide sequence analyses suggest that ATmC-1 and ATmC-2 are the products of the same alpha-TM gene produced via alternate splicing, whereas ATmC-1 and ATmS-1 are the identical isoforms generated from the alpha-gene. RT-PCR analysis using isoform-specific primer pairs and detector oligonucleotides suggests that ATmC-2 is expressed predominantly in adult axolotl hearts. ATmC-2 is a novel isoform, which unlike ATmC-1 and other known striated muscle isoforms expresses exon 2a instead of exon 2b.

Studies on the biosynthesis of the variant surface glycoproteins of Trypanosoma brucei: sequence of glycosylation.

The variant surface glycoprotein (VSG) of several Trypanosoma brucei clones has been metabolically labeled with [35S]methionine in parasites grown in the presence or in the absence of tunicamycin. Pulse and chase experiments followed by immunoprecipiation with anti-VSG sera and polyacrylamide gel electrophoresis of the immunoprecipitates, have helped to elucidate two steps of the sequence of glycosylation of VSG. Immediately after, or concomitantly with the synthesis of the protein, a first type of oligosaccharide side chain is also synthesized. Tunicamycin, a specific inhibitor of asparagine glycosylation, completely inhibits this reaction. The total amount of VSG found in trypanosomes grown in the presence of tunicamycin is less than in controls. The unglycosylated molecule has an apparent molecular weight 5-10% smaller than the mature form. A subsequent step, occurring after translation, is the synthesis of carbohydrate side chains which contain a cross-reacting antigenic determinant detected in all VSGs so far studied by immunoprecipitations with heterologous antisera. The percentage of total VSG bearing sugars involved in cross-reactions is variable in different clones, increases with time and subsequently decreases, suggesting that some of the carbohydrate might undergo trimming. Polyacrylamide gel electrophoresis of immunoprecipitates suggests that the absence or the incomplete synthesis of this oligosaccharide does not alter the apparent molecular weight of VSG. In four out of the five clones studied, the sugar(s) responsible for cross-reactions seem to be located within oligosaccharides linked to the protein through both N-glycosidic and other unidentified types of linkages. This is suggested by the partial effect of tunicamycin on the extent of VSGs cross-reactivities measured by immunoprecipitations with heterologous antiserum.

Antitrypanosomal activity of sinefungin.

Sinefungin, a naturally occurring antifungal antibiotic nucleoside containing an ornithine residue, linked by a C-C bond to C-5' of adenosine, cures mice infected with Trypanosoma brucei brucei, T. congolense, or T. vivax; the effect of the drug is more pronounced towards T. congolense. Anti-trypanosomal activity of sinefungin could be the result of the inhibition of transmethylation reactions or of polyamine biosynthesis--or both--in parasites.

Expression of HoxA5 in the heart is upregulated during thyroxin-induced metamorphosis of the Mexican axolotl (Ambystoma mexicanum).

Widespread external and internal changes in body morphology have long been known to be hallmarks of the process of metamorphosis. However, more subtle changes, particularly at the molecular level, are only now beginning to be understood. A number of transcription factors have recently been shown to alter expression either in levels of message or in isoforms expressed. In this article, we describe a dramatic increase in the expression of the homeobox gene HoxA5 in the heart and aorta of the Mexican axolotl Ambystoma mexicanum during the process of thyroxin-induced metamorphosis. Immunohistochemical analysis with anti-HoxA5 antibody in thyroxin-induced metamorphosing animals showed a pattern of expression of HoxA5 comparable to that in spontaneously metamorphosing animals. Further, by in situ hybridization, we were able to show significant qualitative differences in the expression of this gene within the heart. Maximum HoxA5 expression occurred at the midpoint of metamorphosis in the myocardium, whereas the hearts of completely metamorphosed animals had the highest levels of expression in the epicardium and endocardium. In the aorta, smooth-muscle cells of the tunica media as well as cells of the tunica adventitia had an increase in expression of HoxA5 with thyroxin-induced metamorphosis. HoxA5 expression significantly changed in cells of the aorta and ventricle with treatment by thyroid hormone. HoxA5, a positive regulator of p53, may be involved with the apoptotic pathway in heart remodeling during amphibian metamorphosis.

Alteration of cardiac myofibrillogenesis by liposome-mediated delivery of exogenous proteins and nucleic acids into whole embryonic hearts.

A precise organization of contractile proteins is essential for contraction of heart muscle. Without a necessary stoichiometry of proteins, beating is not possible. Disruption of this organization can be seen in diseases such as familial hypertrophic cardiomyopathy and also in acquired diseases. In addition, isoform diversity may affect contractile properties in such functional adaptations as cardiac hypertrophy. The Mexican axolotl provides an uncommon model in which to examine specific proteins involved with myofibril formation in the heart. Cardiac mutant embryos lack organized myofibrils and have altered expression of contractile proteins. In order to replicate the disruption of myofibril formation seen in mutant hearts, we have developed procedures for the introduction of contractile protein antibodies into normal hearts. Oligonucleotides specific to axolotl tropomyosin isoforms (ATmC-1 and ATmC-3), were also successfully introduced into the normal hearts. The antisense ATmC-3 oligonucleotide disrupted myofibril formation and beating, while the sense strands did not. A fluorescein-tagged sense oligonucleotide clearly showed that the oligonucleotide is introduced within the cells of the intact hearts. In contrast, ATmC-1 anti-sense oligonucleotide did not cause a disruption of the myofibrillar organization. Specifically, tropomyosin expression can be disrupted in normal hearts with a lack of organized myofibrils. In a broader approach, these procedures for whole hearts are important for studying myofibril formation in normal hearts at the DNA, RNA, and/or protein levels and can complement the studies of the cardiac mutant phenotype. All of these tools taken together present a powerful approach to the elucidation of myofibrillogenesis and show that embryonic heart cells can incorporate a wide variety of molecules with cationic liposomes.

Creation of chimeric mutant axolotls: a model to study early embryonic heart development in Mexican axolotls.

The Mexican axolotl (Ambystoma mexicanum) provides an excellent model for studying heart development since it carries a cardiac lethal mutation in gene c that results in failure of contraction of mutant embryonic myocardium. In cardiac mutant axolotls (c/c) the hearts do not beat, apparently because of an absence of organized myofibrils. To date, there has been no way to analyze the genotypes of embryos from heterozygous spawnings (+/c x +/c) until stage 35 when the normal (+/c or +/+) embryos first begin to have beating hearts; mutant (c/c) embryos fail to develop normal heartbeats. In the present study, we created chimeric axolotls by using microsurgical techniques. The general approach was to transect tailbud embryos and join the anterior and posterior halves of two different individuals. The chimeric axolotl is composed of a normal head and heart region (+/+), permitting survival and a mutant body containing mutant gonads (c/c) that permits the production of c/c mutant offspring: 100% c/c offspring were obtained by mating c/c chimeras (c/c x c/c). The mutant phenotypes were confirmed by the absence of beating hearts and death at stage 41 in 100% of the embryos. Examination of the mutant hearts with electron microscopy and comfocal microscopy after immunofluorescent staining for tropomyosin showed identical images to those described previously in naturally-occurring c/c mutant axolotls (i.e., lacking organized sarcomeric myofibrils). These "c/c chimeric" axolotls provide a useful and unique way to investigate early embryonic heart development in cardiac mutant Mexican axolotls.

Downregulation of N1 gene expression inhibits the initial heartbeating and heart development in axolotls.

Recessive mutant gene c in the axolotl results in a failure of affected embryos to develop contracting hearts. This abnormality can be corrected by treating the mutant heart with RNA isolated from normal anterior endoderm or from endoderm conditioned medium. A cDNA library was constructed from the total conditioned medium RNA using a random priming technique in a pcDNAII vector. We have previously identified a clone (designated as N1) from the constructed axolotl cDNA library, which has a unique nucleotide sequence. We have also discovered that the N1 gene product is related to heart development in the Mexican axolotl [Cell Mol. Biol. Res. 41 (1995) 117]. In the present studies, we further investigate the role of N1 on heartbeating and heart development in axolotls. N1 mRNA expression has been determined by using semi-quantitative RT-PCR with specifically designed primers. Normal embryonic hearts (at stages 30-31) have been transfected with anti-sense oligonucleotides against N1 to determine if downregulation of N1 gene expression has any effect on normal heart development. Our results show that cardiac N1 mRNA expression is partially blocked in the hearts transfected with anti-sense nucleotides and the downregulation of N1 gene expression results in a decrease of heartbeating in normal embryos, although the hearts remain alive as indicated by calcium spike movement throughout the hearts. Confocal microscopy data indicate some myofibril disorganization in the hearts transfected with the anti-sense N1 oligonucleotides. Interestingly, we also find that N1 gene expression is significantly decreased in the mutant axolotl hearts. Our results suggest that N1 is a novel gene in Mexican axolotls and it probably plays an important role in myofibrillogenesis and in the initiation of heartbeating during heart development.

Relationship between cardiac protein tyrosine phosphorylation and myofibrillogenesis during axolotl heart development.

The axolotl, Ambystoma mexicanum, is a useful system for studying embryogenesis and cardiogenesis. To understand the role of protein tyrosine phosphorylation during heart development in normal and cardiac mutant axolotl embryonic hearts, we have investigated the state of protein tyrosine residues (phosphotyrosine, P-Tyr) and the relationship between P-Tyr and the development of organized sarcomeric myofibrils by using confocal microscopy, two-dimensional isoelectric focusing (IEF)/SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotting analyses. Western blot analyses of normal embryonic hearts indicate that several proteins were significantly tyrosine phosphorylated after the initial heartbeat stage (stage 35). Mutant hearts at stages 40-41 showed less tyrosine phosphorylated staining as compared to the normal group. Two-dimensional gel electrophoresis revealed that most of the proteins from mutant hearts had a lower content of phosphorylated amino acids. Confocal microscopy of stage 35 normal hearts using phosphotyrosine monoclonal antibodies demonstrated that P-Tyr staining gradually increased being localized primarily at cell-cell boundaries and cell-extracellular matrix boundaries. In contrast, mutant embryonic hearts showed a marked decrease in the level of P-Tyr staining, especially at sites of cell-cell and cell-matrix junctions. We also delivered an anti-phosphotyrosine antibody (PY 20) into normal hearts by using a liposome-mediated delivery method, which resulted in a disruption of the existing cardiac myofibrils and reduced heartbeat rates. Our results suggest that protein tyrosine phosphorylation is critical during myofibrillogenesis and embryonic heart development in axolotls.

Evidence for a chromosomally determined bacteriocin production by Klebsiella pneumoniae.

Location of the genes responsible for pneumocin production in Klebsiella pneumoniae was examined by classical procedures. Conjugal intrageneric transfers, elimination experiments with various curing agents, high temperature and plasmid isolation procedures showed that this strain did not harbour any plasmid. Hence chromosomal location of the genetic determinants is suggested.

Induction of carboxymethylcellulase in Macrophomina phaseolina.

Macrophomina phaseolina, the well-known jute pathogenic fungus produces very low levels of both extra- and intracellular carboxymethylcellulase even in the absence of any cellulose as carbon source in the medium. However, the production of these enzymes is greatly induced by soluble carboxymethylcellulose. The carboxymethylcellulase in M. phaseolina is repressed by glucose.

Role of liver and brain lysosomal and mitochondrial enzymes in development of delayed neuropathy in hens by 0,0-diisopropyl phosphorofluoridate.

Subacute dose of 0,0-diisopropyl phosphorofluoridate (DFP), a potent organophosphorus ester capable of producing delayed neurotoxicity (OPIDN), did not produce any significant change in the levels of lysosomal and mitochondrial marker enzymes of brain, liver and serum at any time after treatment in hens protected with atropine. The results suggest the absence of any involvement of mitochondrial and lysosomal enzymes at any stage in the development of OPIDN in susceptible species by treating with DFP.

Does Nebulin Make Tropomyosin Less Dynamic in Mature Myofibrils in Cross-Striated Myocytes?

Myofibrils in vertebrate cardiac and skeletal muscles are characterized by groups of proteins arranged in contractile units or sarcomeres, which consist of four major components - thin filaments, thick filaments, titin and Z-bands. The thin actin/tropomyosin-containing filaments are embedded in the Z-bands and interdigitate with the myosin-containing thick filaments aligned in A-bands. Titin is attached to the Z-band and extends upto the middle of the A-Band. In this mini review, we have addressed the mechanism of myofibril assembly as well as the dynamics and maintenance of the myofibrils in cardiac and skeletal muscle cells. Evidence from our research as well as from other laboratories favors the premyofibril model of myofibrillogenesis. This three-step model (premyofibril to nascent myofibril to mature myofibril) not only provides a reasonable mechanism for sequential interaction of various proteins during assembly of myofibrils, but also suggests why the dynamics of a thin filament protein like tropomyosin is higher in cardiac muscle than in skeletal muscles. The dynamics of tropomyosin not only varies in different muscle types (cardiac vs. skeletal), but also varies during myofibrillogenesis, for example, premyofibril versus mature myofibrils in skeletal muscle. One of the major differences in protein composition between cardiac and skeletal muscle is nebulin localized along the thin filaments (two nebulins/thin filament) of mature myofibrils in skeletal muscle cells, but which is expressed in a minimal quantity (one nebulin/50 actin filaments) in ventricular cardiomyocytes. Interestingly, nebulin is not associated with premyofibrils in skeletal muscle. Our FRAP(Fluorescence Recovery After Photobleaching) results suggest that tropomyosin is more dynamic in premyofibrils than in mature myofibrils in skeletal muscle, and also, the dynamics of tropomyosin in mature myofibrils is significantly higher in cardiac muscle compared to skeletal muscle. Our working hypothesis is that the association of nebulin in mature myofibrils renders tropomyosin less dynamic in skeletal muscle.