Differential in vitro phenotype pattern, transforming growth factor-β1 activity and mRNA expression of transforming growth factor-β1 in Apert osteoblasts

The phenotype of Apert osteoblasts differs from that of normal osteoblasts in the accumulation of macromolecules in the extracellular matrix. Apert osteoblasts increase type I collagen, fibronectin and glycosaminoglycans secretion compared with normal osteoblasts. Because the extracellular matrix macromolecule accumulation is greatly modulated by transforming growth factor-beta(1), we examined the ability of normal and Apert osteoblasts to secrete transforming growth factor-beta(1) by CCL-64 assay and to produce transforming growth factor-beta(1 )by analysis of the mRNA expression of transforming growth factor-beta(1). Northern blot analysis revealed an increased amount of transforming growth factor-beta(1) mRNA expression in Apert osteoblasts compared with normal ones. Moreover, the level of the active transforming growth factor-beta(1) isoform was higher in Apert than in normal media. In pathologic cells, the increase in transforming growth factor-beta(1) gene expression was associated with a parallel increase in the factor secreted into the medium. The level of transforming growth factor-beta(1) was decreased by the addition of basic fibroblast growth factor. Transforming growth factor-beta(1) is controlled temporally and spatially during skeletal tissue development and produces complex stimulatory and inhibitory changes in osteoblast functions. We hypothesise that in vitro differences between normal and Apert osteoblasts may be correlated to different transforming growth factor-beta(1) cascade patterns, probably due to an altered balance between transforming growth factor-beta(1) and basic fibroblast growth factor.     

Release of transforming plasmid DNA from actively growing genetically engineered Escherichia coli

We studied the transforming ability of the extracellular plasmid DNA released from a genetically engineered Escherichia coli pEGFP and the culturing conditions for the release of transforming DNA. The transforming ability was evaluated by transformation of competent cells with filtrates of E. coli pEGFP cultures. The number of transformants increased with time when E. coli pEGFP cells grew exponentially in rich medium, but not in stationary phase or when inoculated in freshwater. These results suggested that crude extracellular plasmid DNA had transforming ability and this transforming DNA was mainly released by actively growing bacteria.     

Two autonomous myc oncogenes in avian carcinoma virus OK10.

The oncogenic avian retrovirus OK10 has the genetic structure gag-delta pol-myc-delta-env. The myc sequence is transduced from a cellular gene, proto-myc, while gag, pol, and env are essential retrovirus genes. By analogy with other directly oncogenic retroviruses, the specific myc sequence of OK10 is thought to be essential for transforming function. However, unlike the specific sequences of all other transforming retroviruses that encode unique transforming proteins, the myc sequence of OK10 encodes two potential transforming proteins, p58 and p200. p200 is translated from the gag-delta pol-myc region of genomic RNA, while p58 is thought to be translated from the gag leader and the open reading frame of myc via a subgenomic mRNA. In this paper, we ask whether both myc genes of OK10 are autonomous transforming genes. By differentially inactivating the p200 myc gene of OK10 provirus in vitro and analyzing transforming function in quail embryo cells, it was found that mutants expressing only p58 transformed like wild-type OK10. Further, it was shown that p58 with and without the gag leader had transforming function and that p58 of wild-type OK10 is initiated from the gag leader. Mutants expressing only p200 were also transforming but less efficiently than mutants that express only p58. A mutant OK10 virus in which the native frameshift of retroviruses between gag and pol was deleted expressed a shortened p200 (delta p200). Although this virus expressed more delta p200 than wild-type OK10 did, it transformed cells less efficiently. It follows that OK10 expresses two autonomous transforming genes, which is unique among retroviruses with onc genes.     

Distinct tyrosine autophosphorylation sites negatively and positively modulate neu-mediated transformation.

A number of cytoplasmic signaling molecules are thought to mediate mitogenic signaling from the activated Neu receptor tyrosine kinase through binding specific phosphotyrosine residues located within the intracellular portion of Neu/c-ErbB-2. An activated neu oncogene containing tyrosine-to-phenylalanine substitutions at each of the known autophosphorylation sites was generated and assessed for its specific transforming potential in Rat1 and NIH 3T3 fibroblasts. Mutation of these sites resulted in a dramatic impairment of the transforming potential of neu. To assess the role of these tyrosine phosphorylation sites in cellular transformation, the transforming potential of a series of mutants in which individual tyrosine residues were restored to this transformation-debilitated neu mutant was evaluated. Reversion of any one of four mutated sites to tyrosine residues restored wild-type transforming activity. While each of these transforming mutants displayed Ras-dependent signaling, the transforming activity of two of these mutants was correlated with their ability to bind either the GRB2 or SHC adapter molecules that couple receptor tyrosine kinases to the Ras signaling pathway. By contrast, restoration of a tyrosine residue located at position 1028 completely suppressed the basal transforming activity of this mutated neu molecule or other transforming neu molecules which possessed single tyrosine residues. These data argue that the transforming potential of activated neu is mediated both by positive and negative regulatory tyrosine phosphorylation sites.     

瘦素和转化生长因子β1在大鼠肝纤维化过程中的作用

目的探讨瘦素和转化生长因子β1（transforming growthβ1,TGF-β1）在肝纤维化发生、发展过程中的作用。方法雌性Wistar大鼠随机分为正常对照组和纤维化模型1周组、2周组、4周组、6周组。纤维化模型各组以CCl4造成化学性肝损伤。常规HE染色观察肝脏病变;检测血浆和肝组织瘦素及TGF-β1水平;天狼猩红胶原染色和肝组织羟脯氨酸（Hyp）含量测定观察肝纤维化程度,并对肝组织瘦素水平、TGF-β1水平及肝纤维化指数（FI）进行相关性分析;赖氏法测定血浆丙氨酸氨基转移酶（ALT）。结果与正常对照组比较,纤维化模型各组血清和肝组织瘦素和TGF-β1水平显著升高（P〈0.05）,且纤维化模型各组肝组织瘦素水平、TGF-β1水平及FI之间两两均呈显著正相关。结论肝组织瘦素在肝纤维化发病过程中具有独立的致病作用,并可能通过激活TGF-β1通路导致肝维化的形成。     

Biological and biochemical properties of human rasH genes mutated at codon 61

Using site-directed mutagenesis, we have introduced mutations encoding 17 different amino acids at codon 61 of the human rasH gene. Fifteen of these substitutions increased rasH transforming activity. The remaining two mutants, encoding proline and glutamic acid, displayed transforming activities similar to the normal gene. Overall, these mutants vary over 1000-fold in transforming potency. Increased levels of p21 expression were required for transformation by weakly transforming mutants. The mutant proteins were unaltered in guanine nucleotide binding properties. However, all 17 different mutant proteins displayed equivalently reduced rates of GTP hydrolysis, 8- to 10-fold lower than the normal protein. There was no quantitative correlation between reduction in GTPase activity and transformation, indicating that reduced GTP hydrolysis is not sufficient to activate ras transforming potential.     

The human cytomegalovirus mtrII colinear region in strain Tanaka is transformation defective.

The morphological transforming region II (mtrII) of human cytomegalovirus (HCMV) strain Towne has been localized to a 980-base-pair fragment containing three putative open reading frames (ORFs) of 79, 83, and 34 amino acids (aa). In addition, noncoding DNA sequence elements which have the potential to form stem-loop structures were also observed within mtrII. To determine what elements within HCMV Towne mtrII are important in transformation, colinear regions in other HCMV strains (AD169 and Tanaka) were isolated and a comparison of transforming potential was performed. The results indicated that the 2.2-kilobase colinear region in strain AD169 was transforming, whereas the colinear mtrII region in strain Tanaka showed significantly reduced transforming potential. Analysis of the nucleotide sequence data of these colinear regions revealed the presence of the 79-aa ORF in strains Towne and AD169 and its absence in strain Tanaka. In addition, BglII-digested Towne mtrII, which was cleaved within the 79-aa ORF, was shown to display significantly reduced transforming potential. Since the 83- and 34-aa coding sequences were interrupted in both the transforming AD169 colinear region and the nontransforming Tanaka strains, these ORFs were thought not to be important in transformation. Analysis of the stem-loop structures within each of the mtrII colinear regions did not reveal significant changes among the transforming and nontransforming colinear fragments. Thus, the comparative data indicate an important role for the 79-aa ORF in transformation.     

In vitro methylation of specific regions of the cloned Moloney sarcoma virus genome inhibits its transforming activity.

The transforming activity of cloned Moloney sarcoma virus (MSV) proviral DNA was inhibited by in vitro methylation of the DNA at cytosine residues, using HpaII and HhaI methylases before transfection into NIH 3T3 cells. The inhibition of transforming activity due to HpaII methylation was reversed by treatment of the transfected cells with 5-azacytidine, a specific inhibitor of methylation. Analysis of the genomic DNA from the transformed cells which resulted from the transfection of methylated MSV DNA revealed that the integrated MSV proviral DNA was sensitive to HpaII digestion in all cell lines examined, suggesting that loss of methyl groups was necessary for transformation. When cells were infected with Moloney murine leukemia virus at various times after transfection with methylated MSV DNA, the amount of transforming virus produced indicated that the loss of methyl groups occurred within 24 h. Methylation of MSV DNA at HhaI sites was as inhibitory to transforming activity as methylation at HpaII sites. In addition, methylation at both HpaII and HhaI sites did not further reduce the transforming activity of the DNA. These results suggested that; whereas methylation of specific sites on the provirus may not be essential for inhibiting the transforming activity of MSV DNA, methylation of specific regions may be necessary. Thus, by cotransfection of plasmids containing only specific regions of the MSV provirus, it was determined that methylation of the v-mos gene was more inhibitory to transformation than methylation of the viral long terminal repeat.     

Delineation of functional determinants in the transforming protein of Fujinami sarcoma virus.

We analyzed linker insertion mutations throughout the 3' region of the v-fps gene of Fujinami sarcoma virus to identify tyrosine kinase transforming protein (P130gag-fps) determinants that are important for catalysis and transforming activity and, in particular, to define residues that participate in substrate selection. Mutations that encode kinase-active, transformation-defective v-fps alleles were recovered, defining sites in the transforming protein that may normally facilitate kinase-substrate interaction. Additionally, one region within the catalytic domain of the transforming protein (amino acid residues 1012 to 1020) that tolerates peptide insertions without loss of transforming activity was discovered, although the insertion mutations in this region of v-fps exhibited qualitatively abnormal transforming function. Transformed rat cell lines that express these mutations displayed unusual phenotypes, including giant cells and cells with an extremely fusiform shape. Furthermore, the insertion mutations in this region were temperature sensitive, transformed cells assumed a flat morphology, cellular protein phosphotyrosine was reduced, and the kinase activity of the transforming protein was decreased when cells were incubated at 40.5 degrees C. Point mutations that specify the ancestral chicken c-fps sequence in the insertion-tolerant region were also introduced into v-fps. These back mutations led to a modest decrease in kinase activity, decreased tumorigenic potential in chickens, and an unexpected increase in transforming activity in rat cells. These results indicate that the insertion-tolerant region of P130gag-fps influences the biologic activity and thermostability of the kinase.     

Repair of Deoxyribonucleic Acid in Haemophilus influenzae I. X-Ray Sensitivity of Ultraviolet-sensitive Mutants and Their Behavior as Hosts to Ultraviolet-irradiated Bacteriophage and Transforming Deoxyribonucleic Acid

Seven mutants of Haemophilus influenzae were isolated by the criterion of sensitivity to ultraviolet (UV) inactivation of colony formation. These mutants and the wild type were characterized with regard to X-ray inactivation of colony formation, UV induction of division inhibition, the ability of the eight strains to act as recipients to UV-irradiated H. influenzae phage and transforming deoxyribonucleic acid (DNA), and the influence of acriflavine on the survival of UV-irradiated transforming DNA with these strains as recipients. The photoreactivable sector of transforming DNA with yeast photoreactivating enzyme was measured for the most UV-sensitive mutant and was found to be greater than that of wild type. Judged by the above criteria, the order of the strains' sensitivities shows some, but by no means complete, correlation from one type of sensitivity characterization to another, indicating that a minimum of two variables is needed to explain the differences in the strains. Acriflavine increases the UV sensitivity of transforming DNA except in the most sensitive mutant. This effect is usually, but not always, more pronounced in the case of the more UV-resistant marker. The acriflavine effect is postulated to be the result of at least two factors: (i) interference with repair of transforming DNA in the host cell, and (ii) interference with the probability of recombination between transforming DNA and host DNA.     

Effect of palladium and platinum salts on bacteriophage T4 and its isolated DNA]

The effect of palladium and platinum salts (K2PdCl4, K2PtCl4) on bacteriophage F4 and its isolated DNA in genetic transformation is investigated. Both compounds efficiently inactivated the phage and decreased the transforming activity of donor DNA. The palladium salt exhibited the higher activity. The palladium compounds inhibited the transforming activity of native donor DNA to a greater degree. No difference was observed in the degree of inactivation of the transforming activity of native and denatured DNA under the effect of platinum salt. It is suggested that the difference in the transforming activity of donor DNA treated with the tested compounds reflect the pattern of their interactions with nucleic acids.     

Ultraviolet-Induced Decrease in Integration of Haemophilus influenzae Transforming Deoxyribonucleic Acid in Sensitive and Resistant Cells

The decrease in integration of transforming deoxyribonucleic acid (DNA) caused by ultraviolet irradiation of the DNA was found to be independent of the presence or absence of excision repair in the recipient cell. Much of the ultraviolet-induced inhibition of integration resulted from the presence in the transforming DNA of pyrimidine dimers, as judged by the photoreactivability of the inhibition with yeast photoreactivating enzyme. The inhibition of integration made only a small contribution to the inactivation of transforming ability of the DNA by ultraviolet radiation.     

Simian adenovirus genome. II. Identification of specific fragments of viral DNA possessing transforming activity

The antigenic variant of simian adenovirus 7 (SA7) DNA was cleaved by restriction endonucleases EcoRI, XbaI, BamHI, SalI. The resulted digests of viral DNA were tested for transforming activity using the "calcium" technique. It was shown that BamHI. XbaI and SalI digests transformed primary baby rat kidney cells as well as native viral DNA. The transforming activity of separated BamHI and SalI fragments was tested also. The viral DNA fragments with transforming activity (BamHI-B and SalI-B) were localised on the left of the physical map of the viral genome. It was also shown that fragment-transformed cell lines were able to form colonies in 0.33% agarose medium.     

Biological activity of thiophosphamide-alkylated DNA in the transformation system of Bacillus subtilis

DNA treated with thiophosphamide was studied for changes in its biological activity in transformation system of Bac. subtilis. DNA alkylation by this three-functional alkylating agent is shown to be followed by the reduction of transforming activity, changes in the competitive ability as well as by a decrease of the cotransfer coefficient for linked markers of transforming DNA. The observed changes in the transforming and cotransforming activities may be explained by variations in the DNA structure.     

The transforming activity of sonicated Haemophilus influenzae DNA

Summary The inactivation of transforming Haemophilus influenzae DNA by sonication in aqueous solution was investigated. The molecular weight decrease of the molecules is the major factor in DNA inactivation. It impairs strongly the uptake of the DNA by the recipient bacteria, especially when the molecular weight is lower than 3x10⁶ daltons. The uptake of sonicated DNA by the bacterial cells seems not to be further reduced when molecules of about 0.5x10⁶ daltons are submitted to further depolymerisation. However the transforming activity of these molecules is still sensitive to further sonication. The transforming activity of the sonicated DNA is related in the last resort to the intact length of the DNA molecules, at the level of their single-strand structure, available for recombination. Rupture by ultrasound was found to be twice as efficient in reducing transforming activity as a nick induced by pancreatic DNAse.     

Action of restriction endonucleases on transforming DNA of Haemophilus influenzae.

Cleavage of DNA from Haemophilus influenzae with restriction endonucleases caused inactivation of transforming ability to an extent that depended on the genetic marker and the enzyme. The rate of inactivation, but not the final level of survival, depended on the concentration of enzyme in the restriction digest. In general, the greatest extent of inactivation of transforming activity was obtained with endonucleases that are known to produce the shortest fragments. We electrophoresed restriction digests of H. influenzae DNA in agarose gels and assayed transforming activity of DNA extracted from gel slices. In this way, we determined the lengths of restriction fragments that contain genetic markers of H. influenzae. For the marker that we studied most thoroughly (nov), the shortest restriction fragment that possessed detectable transforming activity was a 0.9-kilobase pair fragment produced by endonuclease R . PstI. The shortest marker-bearing restriction fragment that retained substantial transforming activity (50% of value for undigested DNA) was a 2.1-kilobase pair EcoRI fragment bearing the kan marker. Among marker-bearing restriction fragments 1 to 4 kilobase pairs in length, survival of transforming activity varied 10,000-fold. We relate these observations to the recent findings by Sisco and Smith (Proc. Natl. Acad. Sci. U.S.A. 76:972-976, 1979) that efficient entry of DNA into competent H. influenzae cells appears to require the presence of a recognition sequence that is scattered throughout the Haemophilus genome in many more copies than in unrelated genomes.     

Transforming growth factor beta 1 dysregulation in a human oral carcinoma tumour progression model

A human oral tumour progression model was established that consists of normal epithelial cells and three cell lines representing stages from dysplastic to metastatic cells. To investigate the impact of exogenous transforming growth factor-beta 1 on this model system, we analysed the responsiveness of those cells to transforming growth factor-beta 1 and explored the potential mechanism underlying the transforming growth factor-beta 1 activity. We found that the growth of all cell types, regardless of their stage of tumour progression, is inhibited by transforming growth factor-beta 1, although to different degrees. Transforming growth factor-beta 1 induced the expression of cyclin-dependent kinase inhibitors p15(INK4B), p21WAF1/(CIP1) and p27(KIP1). In contrast, transforming growth factor-beta 1 was found to stimulate the invasive potential of one cell type that represents the most advanced stage of tumour phenotype, suggesting that the impact of transforming growth factor-beta 1 on functional features of tumour cells other than cellular proliferation may play a significant role in the process of oral tumour progression.     

Transformation of Saccharomyces cerevisiae with nonhomologous DNA: illegitimate integration of transforming DNA into yeast chromosomes and in vivo ligation of transforming DNA to mitochondrial DNA sequences.

When the yeast Saccharomyces cerevisiae was transformed with DNA that shares no homology to the genome, three classes of transformants were obtained. In the most common class, the DNA was inserted as the result of a reaction that appears to require base pairing between the target sequence and the terminal few base pairs of the transforming DNA fragment. In the second class, no such homology was detected, and the transforming DNA was integrated next to a CTT or GTT in the target; it is likely that these integration events were mediated by topoisomerase I. The final class involved the in vivo ligation of transforming DNA with nucleus-localized linear fragments of mitochondrial DNA.     

Integration and expression of alpha-amylase and endoglucanase genes in the Lactobacillus plantarum chromosome.

A commercial grass silage starter strain of Lactobacillus plantarum was transformed by high-frequency electroporation with plasmids containing an alpha-amylase gene from Bacillus stearothermophilus and an endoglucanase gene from Clostridium thermocellum. Both genes were expressed from their native regulatory signals, and active enzymes were found in the supernatant. However, the segregational stability of the transforming plasmids was rather low. Therefore, the transforming genes were inserted in the L. plantarum chromosome by means of single homologous recombination. In the majority of the transformants, this led to extremely stable segregation and expression of the transforming genes, without generating secondary mutations in the host. Increased selective pressure led to tandem amplification of the transforming DNA. The transformed strains demonstrated the ability of L. plantarum to express heterologous gene products; they can be used to detect the inoculum in silage ecology studies; and they demonstrate the feasibility of engineering truly cellulolytic silage starter bacteria.