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A truncated human xeroderma pigmentosum complementation group A protein expressed from an adenovirus sensitizes human tumor cells to ultraviolet light and cisplatin.

Individuals with the genetic disease xeroderma pigmentosum (XP) have impaired nucleotide excision repair (NER). Group A XP cells are defective in the XPA protein essential for NER and serve, together with other NER proteins, as a nucleation factor for the demarcation of bulky DNA damage. Because XPA cells are extremely sensitive to UV and drugs that cause bulky DNA damage, the XPA protein is an attractive target for manipulating cellular sensitivity to certain cancer therapeutics, a concept that perhaps can be applied toward developing more effective cancer treatments. We have made a replication-defective adenovirus, AdCMV-FlagXPA(59-114), that expresses a truncated form of XPA encompassing amino acids 59-114 sufficient for binding to the excision repair cross-complementing protein 1 (ERCC1)/xeroderma pigmentosum complementation group F (XPF) nuclease essential for making an incision 5' of the damage. On the basis of previous work, it was expected that this truncated XPA protein would work as a decoy and impair NER and, thus, sensitize cells to UV and drugs that produce bulky DNA lesions. Because the truncated XPA protein is "tagged" with the Flag epitope, an anti-Flag antibody can be used to detect protein expression and to isolate proteins associated with the XPA complex. We show that relatively large quantities of truncated XPA protein are present in infected human lung carcinoma A549 cells 2-4 days postinfection. Moreover, in a pull-down assay using anti-Flag antibody, we show that ERCC1 is present in the FlagXPA complex but not in a complex isolated from cells infected with a control virus. Most importantly, cells infected with AdCMV-FlagXPA(59-114) are significantly more sensitive than control cells to UV-induced damage as determined by host-cell reactivation of UV-irradiated AdLacZ adenovirus and in a cytotoxicity assay that appears to be the result of aberrant processing of 6-4 photoproducts. Infected cells were also more sensitive to treatment with cisplatin, an important cancer drug. These results suggest that NER, and the XPA protein in particular, can be a direct target for sensitizing tumor cells to UV and cisplatin and perhaps also certain other clinically important drugs.

Adenoviridae↗

Dominant-negative synthesis suppression of voltage-gated calcium channel Cav2.2 induced by truncated constructs.

Voltage-gated calcium channel alpha1 subunits consist of four domains (I-IV), each with six transmembrane segments. A number of truncated isoforms have been identified to occur as a result of alternative splicing or mutation. We have examined the functional consequences for expression of full-length Ca(v)2.2 (alpha1B) of its coexpression with truncated constructs of Ca(v)2.2. Domains I-II or domains III-IV, when expressed individually, together with the accessory subunits beta1b and alpha2delta-1, did not form functional channels. When they were coexpressed, low-density whole-cell currents and functional channels with properties similar to wild-type channels were observed. However, when domain I-II, domain III-IV, or domain I alone were coexpressed with full-length Ca(v)2.2, they markedly suppressed its functional expression, although at the single channel level, when channels were recorded, there were no differences in their biophysical properties. Furthermore, when it was coexpressed with either domain I-II or domain I, the fluorescence of green fluorescent protein (GFP)-Ca(v)2.2 and expression of Ca(v)2.2 protein was almost abolished. Suppression does not involve sequestration of the Ca(v)beta subunit, because loss of GFP-Ca(v)2.2 expression also occurred in the absence of beta subunit, and the effect of domain I-II or domain I could not be mimicked by the cytoplasmic I-II loop of Ca(v)2.2. It requires transmembrane segments, because the isolated Ca(v)2.2 N terminus did not have any effect. Our results indicate that the mechanism of suppression of Ca(v)2.2 by truncated constructs containing domain I involves inhibition of channel synthesis, which may represent a role of endogenously expressed truncated Ca(v) isoforms.

Animals↗

[Overexpression of truncated type II transforming growth factor-beta receptor in dermal fibroblasts].

OBJECTIVE: To study the effect of overexpression of truncated type II TGF-beta receptor on transforming growth factor-beta 1(TGF-beta 1) autoproduction in normal dermal fibroblasts. METHODS: In vitro cultured dermal fibroblasts were treated with recombinant human TGF-beta 1(rhTGF-beta 1) (5 ng/ml) or recombinant adenovirus containing truncated type II TGF-beta receptor gene (50 pfu/cell). Their effects on regulating gene expression of TGF-beta 1 were observed with Northern blotting. RESULTS: rhTGF-beta 1 up-regulated the gene expression of TGF-beta 1 and type I procollagen. Overexpression of truncated receptor II down-regulated the gene expression of TGF-beta 1. CONCLUSION: Overexpression of the truncated TGF-beta receptor II decreases TGF-beta 1 autoproduction via blocking TGF-beta receptor signal. The results may provided a new strategy for scar gene therapy.

Cell Division↗

[Efficient soluble expression, purification and identification of the truncated SAG1 gene of Toxoplasma gondii in Escherichia coli].

OBJECTIVE: To express truncated SAG1 gene in Escherichia coli to obtain purified recombinant SAG1, and identify the immunoreactivity of the product. METHODS: The plasmid pET-30a(+)-trSAG1 was constructed, which was cut by Nco I and HindIII to obtain truncated SAG1 gene and inserted into pET-32a(+) cut by the same two restriction enzymes. After identification by restriction enzymes, the plasmid pET-32a(+)-trSAG1 was transformed into E.coli BL21, and the soluble product induced by 0.1 mmol/L IPTG for 4 hour before purification with Ni-NTA agarose, followed by identification of the purified recombinant protein with SDS-PAGE, Western-blotting and ELISA. RESULTS: Recombinant pET-32a(+)-trSAG1 was successfully constructed and high level of truncated SAG1 expression achieved in E.coli. SDS-PAGE showed that the expressed protein was approximately 40,000 in size and expressed in a soluble form that could be easily purified by Ni-NTA agarose. Western-blotting demonstrated that the purified product could be specifically recognized by sera from rabbits immunized with native antigen of T.gondii, and could also be recognized, as shown by ELISA, by sera from human with T.gondii infection. CONCLUSIONS: The truncated SAG1 gene was successfully expressed in E.coli in a soluble form, and the recombinant protein may be of value in the diagnosis of toxoplasmosis.

Animals↗

[The anti-HBV effect and mechanism of C gene truncated mutant in vitro].

OBJECTIVE: To explore the effect and mechanism on HBV replication in C gene truncated mutant. METHODS: Protein expression of C gene truncated vector and wild C gene vector were assay by SDS-PAGE Western blot. Constructed C gene truncated expression vector was cotransfected with wild HBV genome; virus load was detected by PCR in the culture medium and the cell. The formation of core particle was assay by Native western blot. RESULTS: The recombinant vectors can efficiently express. Virus load of the cotransfected group by pcDNA3-deltaC and adwR9 was lower than that of control group in the culture medium and the cell. Protein band of the co-expressed group by pcDNA3-deltaC and pcDNA3-C showed slightly weaker than that of the co-expressed group by pcDNA3 and pcDNA3-C. CONCLUSION: C gene truncated mutant could interfere with the formation of core particle and reduce of HBV replication

Cell Line↗

[Construction and expression of recombinant antibody/granzyme B containing truncated translocating peptide].

AIM: To construct an eukaryotic expression vector for recombinant antibody/granzyme B gene containing truncated translocating peptide and express it in HER-2(+) SKBR-3 cells and HER-2(-) Hela cells. METHODS: PCR amplication was used to obtain recombinant DNA encoding antibody/granzyme B containing truncated translocating peptide DNA, and then the DNA fragment was cloned into eukaryotic expression vector pCMV-e23sFv-PE40. After being transfected into SKBR-3 cells and Hela cells, the expression of target gene and its effect on cellular morphology were detected by immunocytochemical staining. RESULTS: The eukaryotic expression vector encoding recombinant antibody/granzyme B containing truncated translocating peptide was successfully constructed. e23sFv-FSD-GrB protein was expressed in most of Hela cells and had no effect on Hela cells, but the transfected SKBR-3 cells which also express e23sFv-FSD-GrB protein exhibited condensed nucleus and cytoplasm. CONCLUSION: The construction of eukaryotic expression vector of recombinant antibody/granzyme B gene containing truncated translocating peptide lays the foundation for further determination of minimal fragment responsible for translocation.

ADP Ribose Transferases↗

[Carboxyl terminus truncated HPV58 virus L1 protein expressed with baculovirus system and its bioactivity].

To prepare carboxyl terminus truncated human papillomavirus type 58L1 protein, and study on its in vitro bioactivity. PCR was used to amplify carboxyl terminus truncated HPV 58L1 gene, the product was inserted into the PUCMT cloning vector, preparing recombinant PFastBacHTb containing carboxyl terminus truncated HPV58L1 gene. Further more, the recombinant plasmid PfastbacHTb was used to transform DH10Bac cells, constructing recombinant Baculovirus, then the recombinant virus was successfully used to infect Sf-9 insect cells. After incubating at 27 degrees C for 72 hours, the infected cells were collected and total cellular proteins were extracted. The target protein with MW 58KD was revealed by SDS-PAGE and confirmed by Western blot. The interested protein was purified by ProBond purification system. The purified interested protein was identified to self-assemble into VLPs by Transmission electron microscope, and induce murine erythrocyte hemagglutination, indicating that the given proteins had the conformation of VLPs, collecting, HPV58L1 proteins with carboxyl terminus truncation could be efficiently expressed in baculovirus Sf-9 cells expression system, it has identical in vitro bioactivity to the wild type HPV58L1, The present study is fundmental for preparing HPV58L1 prophylactic vaccine.

Animals↗

[Cloning and prokaryotic expression of truncated muramidase-released protein gene of human Streptococcus suis type 2].

AIM: To clone and express the truncated Habb mrp gene of human Streptococcus suis type 2 (S.suis 2) and detect its activity. METHODS: A pair of primers based on S.suis 2 mrp gene were schemed out. The turncated mrp (tmrp)gene of S.suis 2 strain Habb isolated from diseased person in Haian, Jiangsu province was cloned and analyzed. The prokaryotic expression plasmid pGEX4T-2-tmrp was constructed.The expression of recombinant protein with glutathione S-transferase (GST) was induced in E.coli TG1. The fusion protein (tMRP-GST) was purified by affinity chromatography, and the GST was cut from tMRP-GST with thrombin protease to gain the truncated MRP (tMRP) antigen. The activity of recombinant protein was analyzed by Western blot. RESULTS: Sequence analysis showed that the length of the truncated mrp was 957 bp. The prokaryotic expressed production was a fusion protein, whose molecular weight was about 61 kD, and the molecular weight of the purified tMRP protein was about 35 kD. Western blot analysis showed that tMRP-GST and tMRP were detected specifically by MRP antiserum on nitrocellulose membrane. CONCLUSION: The truncated mrp gene of human S.suis 2 strain Habb is successfully cloned, and the high expression of the functional recombinant protein is achieved in the prokaryotic system, which facilitates the further studies on the bio-function and immunology.

Animals↗

Expression of carboxyl-terminally truncated forms of human apolipoprotein B in rat hepatoma cells. Evidence that the length of apolipoprotein B has a major effect on the buoyant density of the secreted lipoproteins.

We examined the relationship between the size of human apolipoprotein (apo) B and the formation and secretion of apoB-containing lipoprotein particles. Stable transformants of the rat hepatoma cell line McA-RH7777 harboring a variety of human apoB cDNA constructs were established, and these produced carboxyl-terminally truncated apoB proteins (apoB18, -B23, -B28, -B31, -B48, and -B53). Immunoblotting of apoB proteins secreted into the culture medium and fractionated by equilibrium density ultracentrifugation revealed that each of the truncated apoB species was secreted from the cells. The peak densities of the apoB-containing particles decreased as the length of the apoB proteins increased. Apolipoproteins B18 and B23 appeared at the bottom of the salt gradient (d = 1.23 g/ml), whereas particles containing apoB28, -B31, -B37, -B48, and -B53 exhibited progressive decreases in density. The density distribution of secreted apolipoproteins was not affected by the expression or secretion of these recombinant apoB species. As determined by nondenaturing gel electrophoresis, apoB28, -B31, -B37, -B48, and -B53 formed their own discrete particles, and there was a direct correlation between the size of the particles and the length of the apoB species. The efficiency and rate of secretion of these truncated forms of apoB were studied by measuring the decrease of immunoprecipitated 35S-labeled apoB proteins in the cells and their accumulation in the medium. Proteins corresponding to apoB28 or larger were rapidly and efficiently secreted, whereas apoB18 and apoB23 were secreted much more slowly. These data imply that the size of these truncated apoB forms governs the lipid content of the apoB-containing lipoproteins formed as well as the kinetics of secretion.

Animals↗

A 3' truncation of MYC caused by chromosomal translocation in a human T-cell leukemia increases mRNA stability.

The proto-oncogene MYC is rearranged at its 3' end in the human T-cell leukemia line Hut 78 as a result of a translocation between the long arms of chromosomes 8 and 2. The nucleotide sequence at the breakpoint shows that the rearranged allele of MYC is truncated 24 nucleotides before the first poly(A)-addition signal. The 3' truncated MYC lacks a 61 nucleotide AT-rich sequence that has been reported to mediate selective mRNA degradation. We show that the truncation results in prolonged stability of MYC mRNA: the half life of the MYC mRNA in Hut 78, as well as in Rat 1A cells transfected with the truncated allele of MYC is increased by at least 5-fold. Our results document yet another mechanism by which MYC may be rendered pathogenic and dramatize the importance of mRNA stability in the regulation of MYC activity.

Base Sequence↗

The molecular basis of truncated forms of apolipoprotein B in a kindred with compound heterozygous hypobetalipoproteinemia.

Krul et al. (1) have identified two truncated species of apolipoprotein B-100 in a kindred with familial hypobetalipoproteinemia. Five family members were identified who produce either one or both of two truncated apolipoprotein B-100 proteins estimated to be 40% and 90% the amino-terminal end of apolipoprotein B-100. Low density lipoprotein with the apolipoprotein B-90 binds more strongly to the low density lipoprotein-receptor on cultured fibroblasts. In this present study, we have identified the DNA mutations leading to these truncated apolipoprotein B-100 variants in this kindred. Sequencing of amplified DNA from the proband revealed that deletions of one or two nucleotide bases produced frameshift mutations and generated premature stop codons in both cases. Apolipoprotein B-40 (Val1829----Cys-TERM) is the result of a dinucleotide (TG) deletion in exon 26 that generates a stop codon at position 1830 and produces a protein with a predicted molecular mass of 207.14 kDa. The other truncated apolipoprotein B Glu4034----Arg-Gln-Leu-Leu-Ala-Cys-TERM) is due to a single nucleotide (G) deletion in exon 29. This results in a protein with 4039 amino acids and a predicted molecular mass of 457.6 kDa that is now designated apolipoprotein B-89. Mechanisms by which the removal of the last 497 amino acids might increase the binding of the apoB-89 to the LDL-receptor are discussed.

Apolipoproteins B↗

Antigen presentation abrogated in cells expressing truncated Ia molecules.

Oligonucleotide site-directed mutagenesis was used to introduce a premature stop codon in wildtype A beta k and A alpha k cDNA clones to create truncated A beta k and A alpha k molecules lacking the cytoplasmic domain. Transfected B lymphoma cells expressing an I-Ak molecule with a truncated beta-chain or with truncated alpha- and beta-chains showed profound defects in two Ia-related functions: Ia-restricted Ag presentation and intracytoplasmic signaling. The ability of these transfected cell lines to activate autoreactive T hybrids was markedly impaired whereas loss of Ag presentation to nominal Ag-specific T hybrids was more subtle. Ia-mediated transmembrane signaling as measured by PKC translocation from cytosol to nucleus after stimulation with anti-Ak antibody was greatly affected by truncation of the A beta and A alpha cytoplasmic domains. These results indicate an important role for the highly conserved cytoplasmic domain in Ia-mediated responses.

Amino Acid Sequence↗

Synthesis, post-translational processing, and autocrine transforming activity of a carboxylterminal truncated form of colony stimulating factor-1.

The mononuclear phagocyte colony stimulating factor encoded by a 1.6 kilobase pair human cDNA is synthesized as a homodimeric transmembrane glycoprotein that is released from the plasma membrane by proteolysis. Premature termination of the CSF-1 coding sequence upstream of its carboxylterminal transmembrane-spanning segment and expression of the truncated CSF-1 cDNA in either bovine papilloma virus or retrovirus vectors led to the synthesis of a soluble, biologically active growth factor that was rapidly secreted from cells. Like the full-length CSF-1 precursor, the truncated polypeptide was rapidly assembled through disulfide bonds immediately after synthesis and acquired asparagine-linked oligosaccharide chains that underwent progressive post-translational modifications during intracellular transport. Soluble CSF-1 encoded by the truncated cDNA stimulated the formation of bone marrow-derived mouse macrophage colonies in semisolid medium and induced transformation of mouse NIH-3T3 cells when coexpressed with the human c-fms proto-oncogene product (CSF-1 receptor). Compared to results obtained with the full-length CSF-1 cDNA, the efficiency of transformation obtained with the truncated CSF-1 gene was reduced, in spite of the fact that transfected cultures produced similar levels of the extracellular growth factor. The results indicate that CSF-1 amino acid residues 1-158 (together with the aminoterminal signal peptide at residues -32 to -1) are sufficient for biological activity and that CSF-1 cDNAs encoding either membrane-bound or soluble precursors are active in autocrine transformation.

Animals↗

Constitutive expression of a truncated INT3 gene in mouse mammary epithelium impairs differentiation and functional development.

INT3 is interrupted by retroviral DNA insertion in approximately 18% of primary Czech mouse mammary tumors induced by mouse mammary tumor virus. One consequence of these insertions is the production of a 2.4-kilobase, tumor-specific RNA transcript encoding the entire intracellular domain of the Int3 protein which is initiated from the 3' long terminal repeat promoter of the inserted viral genome. Female mice (FVB-3) transgenic for a genomic fragment comprised of this truncated region of INT3 express the 2.4-kilobase truncated INT3 transcript and exhibit focal mammary tumors at 100% penetrance. INT3 is a member of a family of genes, highly conserved through evolution and characterized by Drosophila melanogaster Notch and Caenorhabditis elegans lin-12, the function of which relates to cell fate determination. Upon transfection into the appropriate hosts, expression vectors of truncated Notch and lin-12, representing their respective cytoplasmic domains, have been demonstrated to effect their complete gene function with respect to cell fate determination. This suggests that the extracellular portion of these proteins function only to regulate activity. Reciprocal transplantation of transgenic FVB-3 and normal mammary tissue to the epithelium-divested fat pads of the respective donor females demonstrates that FVB-3 mammary epithelium is unable to grow and/or to functionally differentiate. However, normal epithelium grows and fully differentiates in transgenic FVB-3 fat pads, indicating that the dysfunction of FVB-3 mammary glands is due to a deficiency inherent in their epithelium. Electron microscopy reveals that transgenic INT3 epithelial cells do not form intercellular junctional complexes in the developing subadult mammary gland. The hormonal stimulation of pregnancy overcomes the deficiency for ductal growth so apparent in the virgin gland such that pregnant FVB-3 glands produce complete ductal systems. Nevertheless, during pregnancy, FVB-3 mammary cells fail to form secretory lobules and to produce milk. Examination of INT3 expression by immunocytochemistry and reverse transcriptase-PCR show that INT3 is expressed constitutively in mammary stroma and epithelia at all stages of postpubertal mammary evolution. These results indicate that deregulated expression of a truncated Int3 in mammary epithelial cells limits their capacity to perform the cell fate decisions required for morphogenesis and functional differentiation.

Amino Acid Sequence↗

Truncated logistic regression.

Truncated binary data occurs when a group of individuals, who each have a binary response, are observed only if one or more of the individuals has a positive response. In this paper the group will be taken to be a motor vehicle accident and the binary response taken to be survival or death. We compare two regression techniques that can be used for truncated binary data. The first procedure, conditional logistic regression (Breslow and Day, 1980, Statistical Methods in Cancer Research. 1: The Analysis of Case-Control Studies. No. 32. Lyon: IARC) conditions on the actual number of deaths, and has been previously used for this type of data. The second procedure, truncated logistic regression, conditions on there being at least one death. It is computationally simpler than conditional logistic for groups of size greater than two and can be considerably more efficient. A major difference between the two methods is that only truncated logistic regression requires a knowledge of group level covariates and allows estimation of group level effects.

Accidents, Traffic↗

Two truncated forms of rat insulin receptor-related receptor.

The insulin receptor-related receptor (IRR) (1271 amino acids) is expected to have unique functions as a novel member of the insulin receptor family. In this paper, we report two alternatively spliced variants of rat IRR mRNA, which are predicted to encode two truncated forms of IRR, sIRR-1 (410 amino acids) and sIRR-2 (469 amino acids). The amino acid sequence of sIRR-1 is identical to the N-terminal 410-amino acid sequence of IRR. sIRR-2 has an additional 59-amino acid insertion in the C-terminal region. Both truncated forms retain the N-terminal and cysteine-rich domains but lack the transmembrane and intracellular tyrosine kinase domains, indicating that the truncated forms are the secreted forms. The translation products of the truncated form mRNAs were detected in the stomach and kidney by Western analysis. However, the physiological significance of the secreted forms remains to be elucidated.

Alternative Splicing↗

Truncated forms of mannose-binding protein multimerize and bind to mannose-rich Salmonella montevideo but fail to activate complement in vitro.

Human serum (MBP) and human recombinant (rMBP) mannose-binding protein bind to mannose-rich, serum-resistant Salmonella montevideo (SH5770), enhance C3 deposition, and render the organisms serum-sensitive. We investigated structural features of MBP necessary for this effect. MBP has a cysteine-rich amino-terminal region, a collagen-like region, and a carboxyl-terminal carbohydrate-recognition domain. We prepared carbohydrate-recognition domains lacking the other two domains either by deletion mutagenesis (delta MBP, 16 kDa) or by collagenase digestion of whole rMBP (cdMBP, 16-18 kDa). Whole and truncated MBP were detected on immunoblot by specific monoclonal antibodies that recognize both bound and free MBP. rMBP enhanced C3 deposition on SH5770 8-fold, while cdMBP and delta MBP did not increase C3 deposition over control levels. All forms of MBP bound to SH5770 by enzyme-linked immunosorbent assay and by measuring binding of radiolabeled whole and truncated MBP. Binding by 125I-delta MBP was inhibited by mannan and by MBP. Thus failure of truncated MBPs to enhance C3 deposition was not due to failure to specifically bind carbohydrate residues. To determine the molecular form of truncated MBP in nondenaturing conditions, 125I-delta MBP was centrifuged through a 5-20% sucrose-density gradient. The peak of 125I-delta MBP sedimented to estimated S20,w 2.01, but larger multimers also were present. Multimers bound SH5770 with higher affinity than monomers. We conclude that carbohydrate-recognition regions of MBP produced by collagenase digestion or by deletion mutagenesis are sufficient for ligand binding. However, the collagen-like region is necessary for MBP enhancement of C3 deposition on SH5770.

Base Sequence↗

Expression of native and truncated forms of the human integrin alpha 1 subunit.

We report here the molecular cloning of cDNAs encoding for the human integrin alpha 1 subunit. The sequence is characteristic of an I domain containing integrin alpha subunit, with a high degree of homology to the rat integrin alpha 1 subunit, including complete identity of the transmembrane and cytoplasmic domains between the two species. The human cDNA directs the expression in mouse NIH 3T3 cells of authentic human alpha 1 protein as demonstrated by the reactivity of this subunit with two human-specific anti-alpha 1 monoclonal antibodies. This exogenous integrin specifically binds to type IV collagen in a Mg(2+)-dependent fashion. We have expressed in both transient systems and in stable cell lines truncated, soluble forms of the human alpha 1 subunit combined with truncated, soluble forms of beta 1 subunits. Although soluble beta 1 subunit was found in the media when the corresponding cDNA was used, the secretion of the soluble alpha 1 subunit was found to be dependent on dimerization with soluble beta 1. Co-transfection of truncated human alpha 1 cDNA with truncated forms of either the human or avian beta 1 cDNA led to efficient secretion of alpha 1.beta 1 heterodimers. These soluble heterodimers specifically bind to collagen IV in a manner similar to their full-length counterparts. Biosynthetic studies using stably expressing cell lines demonstrate that the soluble heterodimers and the native heterodimers are formed independently, strongly suggesting that the transmembrane or cytoplasmic domains of alpha and beta subunits are involved in the assembly of native heterodimers.

3T3 Cells↗