Demethylation of the progesterone receptor CpG island is not required for progesterone receptor gene expression

In a previous study we have shown that an intravenous infusion of pramlintide (an analogue of human amylin) delayed gastric emptying, but the dose of pramlintide was supraphysiological in relation to the amylin response to food in non-diabetic subjects. The purpose of this study was to examine the dose response relationship of subcutaneous injections of pramlintide on gastric emptying and to determine whether administration of the drug before one meal has an impact on the subsequent meal. Eleven men with insulin-dependent diabetes mellitus were studied in a double-blind, randomised, four-way crossover design. None had autonomic neuropathy. Euglycaemia was maintained overnight before the study day. At -30 min the patients self-injected their usual morning insulin and at -15 min they injected the study drug (either placebo or 30, 60 or 90 microg pramlintide) subcutaneously. At 0 min they ate a standard meal consisting of a pancake, labelled with 99mTc, and a milkshake containing 3-ortho-methylglucose (3-OMG). Gastric emptying images were obtained for the next 8 h. At 240 min the subjects ate a similar meal, but on this occasion the pancake was labelled with (111)In. All three doses of pramlintide delayed emptying of the solid component of the first meal (p < 0.004) with no significant difference between the drug doses. There were no differences between placebo and pramlintide after the second meal. All three doses of pramlintide resulted in a prolongation in the time to peak plasma 3-OMG level (p < 0.0001) after the first meal but there was no difference after the second meal.     

Gene A protein of bacteriophage S13 is required for singel-stranded DNA synthesis

The product of gene A of the small icosahedral DNA phage S13 has been found to be needed for single-stranded DNA synthesis in vivo in addition to its previously known role in progeny replicative-form DNA synthesis.     

Abrogation of TGF-beta activity during retroviral transduction improves murine hematopoietic progenitor and repopulating cell gene transfer efficiency

Transforming growth factor-beta has complex activities on hematopoietic cells. We have previously shown that murine long-term repopulating activity is compromised by ex vivo culture in TGF-beta 1 and conversely is increased by abrogating endogenous TGF-beta activity with a neutralizing antibody. In the current study, we investigated the effect of abrogation of autocrine or paracrine TGF-beta present during retroviral transduction on gene transfer efficiency to primitive hematopoietic cells. Murine marrow cells were cultured and retrovirally transduced for 4 days in the presence of interleukin-3, interleukin-6 and stem cell factor, and either a neutralizing anti-TGF-beta antibody or an isotype control. Committed progenitor cells were analyzed for gene transfer efficiency, and cells were also injected into W/Wv recipient mice for analysis of transduction of long-term repopulating cells. The progenitor (CFU-C) transduction efficiency in the presence of anti-TGF-beta was significantly greater. Semiquantitative PCR analysis and Southern blot analysis for the retroviral marker gene in the blood and bone marrow of recipient mice revealed a significant increase in the transduction efficiency of long-term repopulating cells after culture and transduction in the presence of the anti-TGF-beta. Thus neutralization of TGF-beta activity during retroviral transduction allows more efficient gene transfer into primitive murine hematopoietic cells and may prove beneficial in future clinical gene transfer or therapy trials.     

The gene encoding the elongation factor P protein is essential for viability and is required for protein synthesis

Elongation factor P (EFP) is a protein that stimulates the peptidyltransferase activity of fully assembled 70 S prokaryotic ribosomes and enhances the synthesis of certain dipeptides initiated by N-formylmethionine. This reaction appears conserved throughout species and is promoted in eukaryotic cells by a homologous protein, eIF5A. Here we ask whether the Escherichia coli gene encoding EFP is essential for cell viability. A kanamycin resistance (KanR) gene was inserted near the N-terminal end of the efp gene and was cloned into a plasmid, pMAK705, that has a temperature-sensitive origin of replication. After transformation into a recA+ E. coli strain, temperature-sensitive mutants were isolated, and their chromosomal DNA was sequenced. Mutants containing the efp-KanR gene in the chromosome grew at 33 degrees C only in the presence of the wild-type copy of the efp gene in the pMAK705 plasmid and were unable to grow at 44 degrees C. Incorporation of various isotopes in vivo suggests that translation is impaired in the efp mutant at 44 degrees C. At 44 degrees C, mutant cells are severely defective in peptide-bond formation. We conclude that the efp gene is essential for cell viability and is required for protein synthesis.     

The product of the herpes simplex virus type 1 UL25 gene is required for encapsidation but not for cleavage of replicated viral DNA

The herpes simplex virus type 1 (HSV-1) UL25 gene contains a 580-amino-acid open reading frame that codes for an essential protein. Previous studies have shown that the UL25 gene product is a virion component (M. A. Ali et al., Virology 216:278-283, 1996) involved in virus penetration and capsid assembly (C. Addison et al., Virology 138:246-259, 1984). In this study, we describe the isolation of a UL25 mutant (KUL25NS) that was constructed by insertion of an in-frame stop codon in the UL25 open reading frame and propagated on a complementing cell line. Although the mutant was capable of synthesis of viral DNA, it did not form plaques or produce infectious virus in noncomplementing cells. Antibodies specific for the UL25 protein were used to demonstrate that KUL25NS-infected Vero cells did not express the UL25 protein. Western immunoblotting showed that the UL25 protein was associated with purified, wild-type HSV A, B, and C capsids. Transmission electron microscopy indicated that the nucleus of Vero cells infected with KUL25NS contained large numbers of both A and B capsids but no C capsids. Analysis of infected cells by sucrose gradient sedimentation analysis confirmed that the ratio of A to B capsids was elevated in KUL25NS-infected Vero cells. Following restriction enzyme digestion, specific terminal fragments were observed in DNA isolated from KUL25NS-infected Vero cells, indicating that the UL25 gene was not required for cleavage of replicated viral DNA. The latter result was confirmed by pulsed-field gel electrophoresis (PFGE), which showed the presence of genome-size viral DNA in KUL25NS-infected Vero cells. DNase I treatment prior to PFGE demonstrated that monomeric HSV DNA was not packaged in the absence of the UL25 protein. Our results indicate that the product of the UL25 gene is required for packaging but not cleavage of replicated viral DNA.     

Dnmt2 is not required for de novo and maintenance methylation of viral DNA in embryonic stem cells

We have shown previously that de novo methylation activities persist in mouse embryonic stem (ES) cells homozygous for a null mutation of Dnmt1 that encodes the major DNA cytosine methyltransferase. In this study, we have cloned a putative mammalian DNA methyltransferase gene, termed Dnmt2 , that is homologous to pmt1 of fission yeast. Different from pmt1 in which the catalytic Pro-Pro-Cys (PPC) motif is 'mutated' to Pro-Ser-Cys, Dnmt2 contains all the conserved methyltransferase motifs, thus likely encoding a functional cytosine methyltransferase. However, baculovirus-expressed Dnmt2 protein failed to methylate DNA in vitro . To investigate whether Dnmt2 functions as a DNA methyltransferase in vivo , we inactivated the Dnmt2 gene by targeted deletion of the putative catalytic PPC motif in ES cells. We showed that endogenous virus was fully methylated in Dnmt2 -deficient mutant ES cells. Furthermore, newly integrated retrovirus DNA was methylated de novo in infected mutant ES cells as efficiently as in wild-type cells. These results indicate that Dnmt2 is not essential for global de novo or maintenance methylation of DNA in ES cells.     

Herpes simplex virus type 1 alkaline nuclease is required for efficient processing of viral DNA replication intermediates

Mutations in the alkaline nuclease gene of herpes simplex type 1 (HSV-1) (nuc mutations) induce almost wild-type levels of viral DNA; however, mutant viral yields are 0.1 to 1% of wild-type yields (L. Shao, L. Rapp, and S. Weller, Virology 195:146-162, 1993; R. Martinez, L. Shao, J.C. Bronstein, P.C. Weber, and S. Weller, Virology 215:152-164, 1996). nuc mutants are defective in one or more stages of genome maturation and appear to package DNA into aberrant or defective capsids which fail to egress from the nucleus of infected cells. In this study, we used pulsed-field gel electrophoresis to test the hypothesis that the defects in nuc mutants are due to the failure of the newly replicated viral DNA to be processed properly during DNA replication and/or recombination. Replicative intermediates of HSV-1 DNA from both wild-type- and mutant-infected cells remain in the wells of pulsed-field gels, while free linear monomers are readily resolved. Digestion of this well DNA with restriction enzymes that cleave once in the viral genome releases discrete monomer DNA from wild-type virus-infected cells but not from nuc mutant-infected cells. We conclude that both wild-type and mutant DNAs exist in a complex, nonlinear form (possibly branched) during replication. The fact that discrete monomer-length DNA cannot be released from nuc DNA by a single-cutting enzyme suggests that this DNA is more branched than DNA which accumulates in cells infected with wild-type virus. The well DNA from cells infected with wild-type and nuc mutants contains XbaI fragments which result from genomic inversions, indicating that alkaline nuclease is not required for mediating recombination events within HSV DNA. Furthermore, nuc mutants are able to carry out DNA replication-mediated homologous recombination events between inverted repeats on plasmids as evaluated by using a quantitative transient recombination assay. Well DNA from both wild-type- and mutant-infected cells contains free U(L) termini but not free U(S) termini. Various models to explain the structure of replicating DNA are considered.     

Subtyping analysis of Fanconi anemia by immunoblotting and retroviral gene transfer

Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome with at least eight complementation groups (A-H). Two of the FA genes (FAA and FAC) have been cloned, and mutations in these genes account for approximately 80% of FA patients. Subtyping of FA patients is an important first step toward identifying candidates for FA gene therapy. In the current study, we analyzed a reference group of 26 FA patients of known subtype. Most of the patients (18/26) were confirmed as either type A or type C by immunoblot analysis with anti-FAA and anti-FAC antisera. In order to resolve the subtype of the remaining patients, we generated retroviral constructs expressing FAA and FAC for transduction of FA cell lines (pMMP-FAA and pMMP-FAC). The pMMP-FAA construct specifically complemented the abnormal phenotype of cell lines from FA-A patients, while pMMP-FAC complemented FA-C cells. In summary, the combination of immunoblot analysis and retroviral-mediated phenotypic correction of FA cells allows a rapid method of FA subtyping.     

Bacteriophage phi29 early protein p17 is conditionally required for the first rounds of viral DNA replication

Taurine is a very important organic osmolyte in most adult cells. Because of this property it has been proposed that large changes in the intracellular content of taurine can osmotically stress the cell, causing changes in its size and shape. This hypothesis was examined by measuring cell dimensions of taurine deficient cardiomyocytes using confocal microscopy. Incubation of isolated neonatal rat myocytes with medium containing 5 mM beta-alanine led to a 55% decrease in intracellular taurine content. Associated with the loss of taurine was a reduction in cell size. Two factors contributed to the change in cell size. First, there was a shift in cell shape, favoring the smaller of the two cellular configurations commonly found in the myocyte cell culture. Second, the size of the polyhedral configuration was reduced after beta-alanine treatment. These same two events also contributed to size reduction in cardiomyocytes incubated with medium containing 30 mM mannitol. Nonetheless, some qualitative differences exist between cells osmotically stressed by increasing the osmolality of the incubation medium and decreasing intracellular osmolality. The results support a role for taurine in the regulation of osmotic balance in the neonatal cardiomyocyte.     

Use of amphotropic retroviral vectors for gene transfer in human colon carcinoma cells

Previous studies in rodent models have demonstrated the feasibility of gene transfer to the stem cells of the intestinal epithelium using ecotropic retroviral vectors delivered luminally. This report represents a next step toward targeting the human intestine as a site for somatic gene therapy. The first experiment assessed the viability of amphotropic retroviral vectors in the luminal environment. It was found that after 4 hr at 37 degrees C in luminal effluent, the loss of titer was no greater than when incubated in control media. Likewise, neither the vector nor the target cells were adversely affected by N-acetylcysteine, which is likely to be used as a preparatory agent for mucus removal. To determine whether human intestinal cells are transducible by these vectors, three colon carcinoma cell lines were studied: HT-29, T84, and Caco-2. All were transduced; however, the expression of the reporter gene was highest in the HT-29 cells. Subsequent studies using these cells showed that with regular stocks of vector, gene transfer peaked at a stock dilution of 1/10 and declined at full strength. This problem could be partially overcome by centrifugal concentration of the retroviral stocks. With this approach, gene transfer increased with increasing particles up to 10x regular stock titers but was inefficient at 100x. Overall, these findings provide encouraging evidence that amphotropic retroviral vectors may eventually be used for in vivo gene transfer into human intestinal epithelium. However, they also point to the need for improved methods of concentrating retroviral vectors.     

Transcription factor AP2 is required for expression of the rat transforming growth factor-alpha gene

DNase I footprinting of the rat TGF alpha promoter in the presence of crude cell nuclear extract revealed three sites of protein-DNA interaction (Fp-A, Fp-B, Fp-C) in the region from -222 to +73. Mutation of specific sites within the Fp-A and Fp-B regions reduced expression of a TGF alpha promoter-reporter gene (TGF alphaLUC) from 50-90% in transiently transfected CHO cells, indicating the importance of protein/DNA interactions at these sites. Since Fp-A contained a perfect AP2 consensus sequence (5'-GCCNNNGGC-3') as its center, we investigated the possibility that AP2 binding is important for TGF alpha promoter activity. A double-stranded oligonucleotide spanning Fp-A displayed a distinct mobility shift in the presence of nuclear extract that was inhibited by an excess of known functional AP2-binding sequence. Moreover, a similar mobility shift occurred in the presence of purified AP2 protein, and the further addition of AP2 antibody produced a supershifted complex. More refined DNase I footprinting of a smaller, oligonucleotide probe in the presence of purified AP2 protein revealed a protected region that included the putative AP2 binding site. Additionally, co-transfection of an AP2 expression vector increased TGF alphaLUC expression 25-fold in Drosophila Schneider cells. These various findings corroborate a role for AP2 in TGF alpha promoter activity. The Fp-B region contains a T5 motif that has been previously suggested to function as an atypical TATA box. An Fp-B oligonucleotide displayed a specific gel mobility shift in the presence of a TATA binding protein (TBP)-TFIIA complex, and the further addition of TBP antibody produced a supershift. These results confirm that protein binding within Fp-B is functionally important, and they also indicate that the T5 motif functions as a TBP binding site.     

p38 MAPK is required for CD40-induced gene expression and proliferation in B lymphocytes

We have investigated the activation of the p38 MAPK pathway in response to CD40 engagement in multiple B cell lines and in human tonsillar B cells to define the role of p38 MAPK in proliferation, NF-kappaB activation and gene expression. Cross-linking CD40 rapidly stimulates both p38 MAPK and its downstream effector, MAPKAPK-2. Inhibition of p38 MAPK activity in vivo with the specific cell-permeable inhibitor, SB203580, under conditions that completely prevented MAPKAPK-2 activation, strongly perturbed CD40-induced tonsillar B cell proliferation while potentiating the B cell receptor (BCR)-driven proliferative response. SB203580 also significantly reduced expression of a reporter gene driven by a minimal promoter containing four NF-kappaB elements, indicating a requirement for the p38 MAPK pathway in CD40-induced NF-kappaB activation. However, CD40-mediated NF-kappaB binding was not affected by SB203580, suggesting that NF-kappaB may not be a direct target for the CD40-induced p38 MAPK pathway. In addition, SB203580 selectively reduced CD40-induced CD54/ICAM-1 expression, whereas CD40-dependent expression of CD40 and CD95/Fas and four newly defined CD40-responsive genes cIAP2, TRAF1, TRAF4/CART and DR3 were unaffected. Our observations show that the p38 MAPK pathway is required for CD40-induced proliferation and that CD40 induces gene expression via both p38 MAPK-dependent and -independent pathways.     

Noninfectious gene transfer and expression systems for cancer gene therapy

Gene therapy provides a significant opportunity to devise novel strategies for the control or cure of cancer. Success of this modality will ultimately depend on the ability to express a therapeutic gene of interest at high levels, and specific gene delivery to targeted tumor cells will minimize toxicities. Although current gene therapy trials typically use viral-based, infectious vectors to express suitable target genes in human cancer cells, these vectors have significant limitations in their expression characteristics, lack of specificity in targeting tumor cells for gene transfer, and safety concerns regarding induction of secondary malignancies and recombination to form replication-competent virus. These limitations have refocused efforts to develop noninfectious gene transfer technologies for in vivo gene delivery of plasmid-based expression vectors. This article reviews recent developments in non-infectious gene transfer techniques, including liposome and receptor-mediated methods, which can efficiently deliver plasmid vectors into tumor cells in vivo. Additionally, strategies are reviewed for efficiently expressing target genes in tumor cells, including use of tissue-specific promoters, inducible promoters, and replication-control sequences to regulate extrachromosomal amplification of vector DNA in human tumor cells. Optimal coupling of these noninfectious gene transfer and expression technologies have the potential to yield safe and effective gene therapies for patients with cancer.     

Replication-competent retroviral vectors encoding alkaline phosphatase reveal spatial restriction of viral gene expression/transduction in the chick embryo

Replication-competent avian retroviruses, capable of transducing and expressing up to 2 kb of nonviral sequences, are now available to effect widespread gene transfer in chicken (chick) embryos (S. H. Hughes, J. J. Greenhouse, C. J. Petropoulos, and P. Sutrave, J. Virol. 61:3004-3012, 1987). We have constructed novel avian retroviral vectors that encode human placental alkaline phosphatase as a marker whose expression can be histochemically monitored. These vectors have been tested for expression by introducing them into the embryonic chick nervous system. They have revealed that the expression of retrovirally transduced genes can be spatially and temporally limited without the need for tissue-specific promoters. By varying the site and time of infection, targeted gene transfer can be confined to selected populations of neural cells over the course of several days, a time window that is sufficient for many key developmental processes. The capability of differentially infecting specific target populations may avoid confounding variables such as detrimental effects of a transduced gene on processes unrelated to the cells or tissue of interest. These vectors and methods thus should be useful in studies of the effect of transduced genes on the development of various organs and tissues during avian embryogenesis. In addition, the vectors will facilitate studies aimed at an understanding of viral infection and expression patterns.     

Cytoplasmic gene expression system enhances the efficiency of cationic liposome-mediated in vivo gene transfer into mouse brain

The use of in vivo electroporation in combination with a chemotherapeutic agent (electrochemotherapy) for the treatment of liver tumors was examined. Induced rat hepatomas were treated with a 0.5 unit intratumor bleomycin dose followed by rectangular direct current pulses. Six pulses were administered during treatment using a needle array electrode that rotated the applied electric field around the tumors. A 84.6% objective response rate resulted for tumors that received both bleomycin and electric pulses. Control groups that received partial or no treatment showed less than 10% objective response rates. These results indicate that electrochemotherapy can be translated from the treatment of cutaneous cancers to the treatment of liver tumors and other internal tumors.     

A SecY homologue is required for the elaboration of the chloroplast thylakoid membrane and for normal chloroplast gene expression

Results of in vitro and genetic studies have provided evidence for four pathways by which proteins are targeted to the chloroplast thylakoid membrane. Although these pathways are initially engaged by distinct substrates and involve some distinct components, an unresolved issue has been whether multiple pathways converge on a common translocation pore in the membrane. A homologue of eubacterial SecY called cpSecY is localized to the thylakoid membrane. Since SecY is a component of a protein-translocating pore in bacteria, cpSecY likely plays an analogous role. To explore the role of cpSecY, we obtained maize mutants with transposon insertions in the corresponding gene. Null cpSecY mutants exhibit a severe loss of thylakoid membrane, differing in this regard from mutants lacking cpSecA. Therefore, cpSecY function is not limited to a translocation step downstream of cpSecA. The phenotype of cpSecY mutants is also much more pleiotropic than that of double mutants in which both the cpSecA- and DeltapH-dependent thylakoid-targeting pathways are disrupted. Therefore, cpSecY function is likely to extend beyond any role it might play in these targeting pathways. CpSecY mutants also exhibit a defect in chloroplast translation, revealing a link between chloroplast membrane biogenesis and chloroplast gene expression.