[Pelvic coronal inclination change in adolescent flexible flatfoot surgically treated with arthroereisis].

Objective: To evaluate adolescent pelvic coronal inclination angle change after flatfoot treated with arthroereisis. Method: A case-series study. From June 2018 to September 2020, 25 children with flexible flat foot and pelvic obliquity were included in this retrospective study in Peking University Shenzhen Hospital. There were 17 males and 8 females with a mean age of (11.2±2.2) years (9-15 years). There were 5 cases of unilateral flatfoot and 20 cases of bilateral flatfoot. All of the patients were surgically treated with arthroereisis. Regular follow-up was done in 3 months, 1 and 2 years postoperatively. Weightbearing fluoroscopy of entire lower limb and foot were investigated to measure Meary's angle, calcaneal pitch angle, height difference at ankle and pelvic plane, pelvic inclination and sacrum-iliac distance (F value) on coronal plane. Results: The mean Mearys' angle at 3 month postoperatively was improved when compared with that before the operation (3.1°±1.5° vs 25.9°±4.3°, P<0.001), and it remained at the same level 2 years after the operation (compared with that at 1 year after the operation, P=0.748). The calcaneal pitch angle improved significantly at 3-month follow-up when compared with that before the operation (16.6°±2.4° vs 9.9°±1.5°, P<0.001), and there was no significant change between 1 year and 2 years after operation (P=0.542). The height difference at mortise plane were also reduced at the 3-month follow-up(P<0.001), and it remained at the same level at 1 year and 2 years after the operation (P=0.159). Pelvic height difference decreased dramatically from (12.4±1.7) mm (before operation) to (7.1±1.2) mm(3 month after the operation) (P<0.001), it decreased to (3.6±1.8) mm 1 year after the operation (compared with that at 3 months after the operation, P<0.001), and no further reduction was observed 2 years after the surgery (P=0.483). The pelvic inclination angle and sacrum-iliac distance were also improved at 3-month follow-up when compared with those before the operation (both P<0.001), and they declined further 1 year after the operation(both P<0.05), but the decreasing trend disappeared at the 2-year follow-up (both P>0.05). Conclusion: For adolescent flexible flat foot patients with pelvic obliquity, the coronal inclination and pelvic height discrepancy would partially recovered with correction of flatfoot deformity, but it could not be completely corrected in the mean follow-up period of 2 years after the operation.

Association among interleukin-6 gene polymorphism, diabetes and periodontitis in a Chinese population.

OBJECTIVES: Diabetics significantly increase risk for periodontitis. Interleukin-6 (IL-6) gene polymorphism may play certain roles in the progression of periodontitis with diabetes. The purpose of this study was to assess the association among IL-6 gene polymorphisms, type 2 diabetes mellitus (T2DM) and chronic periodontitis (CP) in a Chinese population. MATERIAL AND METHODS: DNA was obtained from 159 patients with CP, 88 patients with T2DM, 110 patients with CP&T2DM and 135 control subjects. The -174/-572/-597 polymorphisms of IL-6 gene were investigated by restriction fragment length polymorphism of polymerase chain reaction products. The results were further confirmed by sequencing. Significance was set at P < 0.008 after Bonferroni correction. RESULTS: Among four groups, CP&T2DM group showed the lowest IL-6-572 CC genotype and C-allele frequencies (54.5% and 74.1%). In this regard, there were significant differences between CP&T2DM group and the control group [P = 0.006, odds ratio (OR) = 0.475, 95% CI: 0.279-0.808 and P = 0.002, OR = 0.502, 95% CI: 0.319-0.788 respectively]. Logistic regression with adjustment for age, gender, body mass index, smoking and stress showed no significant difference in terms of IL-6-572 genotypes (P = 0.058, OR= 0.523, 95% CI: 0.268-1.022). CONCLUSIONS: The IL-6-572 genotype and allele distributions are unique to subjects with CP&T2DM in a Chinese population.

DNA immunization against very virulent infectious bursal disease virus with VP2-4-3 gene and chicken IL-6 gene.

The present study was to investigate the feasibility and efficiency of the DNA vaccine to protect chickens against very virulent infectious bursal disease virus (vvIBDV) infection. A plasmid DNA carrying VP2-4-3 genes of vvIBDV SH95 and a plasmid DNA carrying chicken interleukin-6 (ChIL-6) genes were constructed and designated as pALTER-MAX-VP2-4-3 and pALTER-MAX-ChIL-6 respectively. Several DNA vaccination experiments were performed: 1-week-old chickens were intramuscularly injected with only plasmid pcDNA3-VP2, pALTER-MAX-VP2-4-3 or mixture with pALTER-MAX-ChIL-6. The chickens at 4 weeks old were orally inoculated with vvIBDV SH95. The results showed that immunization with the mixture of pALTER-MAX-VP2-4-3 and pALTER-MAX-ChIL-6 three times conferred protection for 90% of chickens. Enzyme-linked immunosorbent assay (ELISA) antibody titres in chickens immunized together with pALTER-MAX-ChIL-6 were higher than those immunized simply with plasmid pcDNA3-VP2 or pALTER-MAX-VP2-4-3. IBDV was not detected in the bursa of the protected chickens at 8 days after challenge by RT-PCR. The results indicate that protection against vvIBDV can be achieved by using the VP2-4-3 gene of vvIBDV as a DNA vaccine. Furthermore, the simultaneous injection of ChIL-6 plasmid significantly increased the protection after challenge with the very virulent strain.

Sequence and analysis of genomic segment A and B of very virulent infectious bursal disease virus isolated from China.

A very virulent infectious bursal disease virus (vvIBDV) field strain, named SH95, was identified and characterized from flocks with vaccination failure in Shanghai. The use of random primer and a reverse transcriptase lacking RNase-H activity produced full-length cDNA copies of the viral genomic A and B segments of SH95. The 3259 base pairs (bp) of segment A and 2827 bp of segment B were amplified by long and accurate PCR in a single step, then successfully cloned and sequenced. There were five to 27 amino acid substitutions compared with other IBDV strains within the segment A polyprotein (of these, three are unique) and about nine to 38 amino acid substitutions within VP1 (of which, four are unique). The comparison of sequences encoding the polyprotein showed that vvIBDV SH95 was most closely related to Asiatic vvIBDVs, which formed a closely related group clearly distinguishable from other classical strains of IBDV. Phylogenetic analysis suggested that vvIBDV SH95 and some other Asiatic vvIBDVs were derived from similar origin. But the topology tree performed on segment B was quite different from that performed on segment A, indicating that a genetic reassortment had played an important role in the emergence of vvIBDV SH95.

Transforming growth factor-alpha enhances cyclin D1 transcription through the binding of early growth response protein to a cis-regulatory element in the cyclin D1 promoter.

Cyclin D1 is a critical oncogene involved in the regulation of progression through the G1 phase of the cell cycle, thereby contributing to cell proliferation. This is mediated through interaction of cyclin D1 with its catalytic partners, the cyclin-dependent kinases, and the subsequent phosphorylation of the retinoblastoma protein. Cyclin D1, in turn, is regulated by mitogenic stimuli. We demonstrate that transforming growth factor-alpha (TGFalpha) induces cyclin D1 mRNA in esophageal squamous epithelial cells, and this appears to correlate with increased cyclin D1 protein expression and cyclin-dependent kinase 6 activity. The induction of cyclin D1 transcription by TGFalpha is mediated in part through the induction of the early growth response protein (Egr-1) and its subsequent binding of Egr-1 to a cis-regulatory region spanning nucleotides -144 to -104 of the cyclin D1 promoter. The Egr-1 binding activity to the cyclin D1 promoter appears to require de novo protein synthesis and is not influenced by Sp1 binding to overlapping Sp1 motifs. Taken together, these data provide evidence that TGFalpha enhances cyclin D1 transcription through the induction of Egr-1 binding to a cis-regulatory region in the cyclin D1 promoter. This has important mechanistic implications into the transcriptional regulation of cyclin D1 by an essential proproliferative growth factor and cell cycle progression.

Ki-ras and p53 mutations in pancreatic ductal adenocarcinoma.

Pancreatic adenocarcinoma involves activation of the Ki-ras oncogene, inactivation of the p53 tumor suppressor gene, and dysregulation of growth factors and perhaps metastasis genes. Ki-ras oncogene point mutations are known to be involved in pancreatic oncogenesis. The p53 tumor suppressor gene product plays a critical role in cell cycle regulation and also functions as a nuclear transcription factor. Point mutations in the p53 gene have been observed in a variety of malignancies. We determined the frequency of p53 protein overexpression and p53 point mutations in the conserved and nonconserved domains in pancreatic cancers as well as the coincidence of Ki-ras mutation in pancreatic ductal adenocarcinoma. Genomic DNA was isolated from 20 frozen pancreatic adenocarcinomas (14 primary, six metastases) along with six specimens of control pancreatic tissue and screened by single-strand conformation polymorphism (SSCP) analysis followed by direct genomic sequencing of SSCP variants. SSCP analysis was accomplished by incorporating 32P-dCTP in 12 separate polymerase chain (PCR) amplifications covering the p53 coding exons 2-11. All mobility shifts on SSCP were subjected to direct genomic sequencing by the modified dideoxy method. Immunoperoxidase (IP) staining was also done with a p53 monoclonal antibody. Ki-ras codon 12 mutational analysis was accomplished by incorporating 32P-dCTP by polymerase chain reaction amplification utilizing mismatched primers, which create a BstN1 restriction endonuclease site spanning codon 12; the products were digested by BstN1. Polyacrylamide gel electrophoresis allowed distinction between wild-type and mutant Ki-ras. p53 mutations were found in 5 of 20 pancreatic cancers (three of 14 primary tumors, two of six metastatic tumors). Point mutations were observed in three of 14 primary tumors, and one of six metastases, while a 2-base pair duplication resulting in a premature stop codon in exon 5 was found in one metastatic tumor. Point mutations were noted in conserved domains (exons 4, 5, 8) and in the nonconserved domain (exon 10). IP staining revealed that eight of 14 of the primary tumors and two of six metastases exhibited moderate to strong nuclear staining (> 30%), while no nuclear staining was evident in the controls. Ki-ras codon 12 mutations were found in 14 of 20 (70%) pancreatic cancers (nine of 14 primary tumors, five of six metastatic tumors) and none of the six controls. Fifty percent of the primary pancreatic tumors demonstrated moderate to strong nuclear staining. Extensive genetic analysis demonstrated mutations in 30% of the pancreatic cancers. One cancer had a nonsense mutation not detected by IP. Seven of 19 (37%) pancreatic cancers exhibited both Ki-ras point mutation and p53 protein overexpression or mutation. Both genetic analysis and IP are required to characterize all p53 mutations in pancreatic cancer. Ki-ras codon 12 mutations and p53 protein overexpression are important steps in pancreatic oncogenesis.

WT1 induces expression of insulin-like growth factor 2 in Wilms' tumor cells.

The Wilms' tumor suppressor gene WT1 encodes a zinc finger transcription factor, whose expression inhibits the growth of the RM1 Wilms' tumor cell line. Transient transfection of WT1 constructs into 3T3 or 293 cells results in transcriptional repression of a number of cotransfected promoters containing the early growth response gene 1 consensus sequence. We now show that WT1 has properties of a transcriptional activator in RM1 cells, an effect that may be associated with the presence of a mutated p53 gene in these cells. Stable transfection of wild-type WT1 into RM1 cells results in induction of endogenous insulin-like growth factor 2 (IGF2) but not of other previously postulated WT1-target genes. The induction of IGF2 is dramatically enhanced by WT1 mutants encoding an altered transactivation domain. We conclude that IGF2 is a potentially physiological target gene for WT1 and that its induction may contribute to the growth-stimulating effects of WT1 variants.

Mutational analysis of the carboxy-terminal portion of p53 using both yeast and mammalian cell assays in vivo.

Increasing evidence indicates that p53 is a transcriptional trans-activator through its sequence-specific DNA binding domain. Tumor-derived p53 mutations disrupt the trans-activation ability mainly due to loss of its sequence-specific DNA binding. Using both yeast and mammalian cell assays, the effect of p53 mutations in the carboxy terminal portion was investigated in order to address how p53 mutations outside of the DNA binding domain affect p53 function. The p53 cDNA in the carboxy-terminus was randomly mutagenized by error-prone polymerase chain reactions and the amplified cDNA was screened for the ability to trans-activate using a yeast assay. Four p53 mutations, including two missense and two nonsense mutations located in the carboxy-terminal oligomerization domain, were further analysed for trans-activation, cell cycle arrest and colony formation in a human osteosarcoma cell line, Saos-2. These functional properties of p53 were disrupted by the missense mutations. Surprisingly, one of the nonsense mutations disrupts the trans-activation function and the ability to G1 arrest but shows a strong inhibition of colony formation. These results confirm that mutations in the oligomerization domain can inactivate p53 function and also indicate that p53-mediated cell growth inhibition does not necessarily depend on the ability to arrest cell cycle.

Germ-line p53 mutations in 15 families with Li-Fraumeni syndrome.

Germ-line mutations of the tumor-suppressor gene p53 have been observed in some families with the Li-Fraumeni syndrome (LFS), a familial cancer syndrome in which affected relatives develop a diverse set of early-onset malignancies including breast carcinoma, sarcomas, and brain tumors. The analysis of the p53 gene in LFS families has been limited, in most studies to date, to the region between exon 5 and exon 9. In order to determine the frequency and distribution of germ-line p53 mutations in LFS, we sequenced the 10 coding exons of the p53 gene in lymphocytes and fibroblast cell lines derived from 15 families with the syndrome. Germ-line mutations were observed in eight families. Six mutations were missense mutations located between exons 5 and 8. One mutation was a nonsense mutation in exon 6, and one mutation was a splicing mutation in intron 4, generating aberrant shorter p53 RNA(s). In three families, a mutation of the p53 gene was observed in the fibroblast cell line derived from the proband. However, the mutation was not found in affected relatives in two families and in the blood from the one individual, indicating that the mutation probably occurred during cell culture in vitro. In four families, no mutation was observed. This study indicates that germ-line p53 mutations in LFS are mostly located between exons 5 and 8 and that approximately 50% of patients with LFS have no germ-line mutations in the coding region of the p53 gene.(ABSTRACT TRUNCATED AT 250 WORDS)

MTS-1 (CDKN2) tumor suppressor gene deletions are a frequent event in esophagus squamous cancer and pancreatic adenocarcinoma cell lines.

MTS-1 is a candidate tumor suppressor gene on chromosome 9p21-22, a region frequently observed to have loss of heterozygosity in esophagus squamous cell carcinomas and pancreatic ductal adenocarcinomas. In order to determine whether MTS-1 sequences are deleted or mutated in cell lines derived from these cancers, we performed PCR amplification of MTS-1 exons 1 and 2. In this fashion, we found that 67% of esophagus squamous cancer cell lines have deletions of both exons 1 and 2, and 50% of pancreatic cancer cell lines have similar deletions. Furthermore, an additional 30% of pancreatic cancer cell lines harbored point mutations or microdeletions based on DNA sequencing. MTS-1 encodes p16, an inhibitor of cyclin-dependent kinase 4 (cdk4) which complexes with cyclin D1. Our data suggest that MTS-1 deletions and mutations may play an important role in the molecular pathogenesis of esophagus squamous cell and pancreatic cancers.

Identification of a human rasGAP-related protein containing calmodulin-binding motifs.

Conversion of active GTP-bound Ras to its inactive GDP-bound form is catalyzed by GTPase-activating proteins (GAPs). Two mammalian Ras-specific GAPs, p120GAP and neurofibromin, the product of the NF1 tumor suppressor gene, have been previously described. We report here the identification of a new human cDNA clone, IQGAP1, which predicts a 1657-amino acid protein that displays extensive sequence similarity to the catalytic domain of all previously reported RasGAPs. IQGAP1 is most closely related to the Schizosaccharomyces pombe RasGAP-like protein, Sar1. Sequence similarity to IQGAP1 is seen throughout the entire Sar1 protein. The N-terminal half of IQGAP1, which does not overlap with Sar1, contains six copies of a unique amino acid motif, as well as four so-called IQ motifs. The latter motifs are found in several proteins, including conventional and unconventional myosins, and mediate the interaction with calmodulin and calmodulin-related proteins. Thus, IQGAP1 appears to represent a novel RasGAP-like protein that may link Ras signaling to some calmodulin-mediated process.

Germline mutations of the p53 tumor suppressor gene in children with osteosarcoma.

PURPOSE: We investigated the possibility that a significant proportion of children with osteosarcoma harbor germline mutations of the p53 tumor suppressor gene and, therefore, this subgroup of pediatric cancer patients should be considered for large-scale predictive testing. PATIENTS AND METHODS: Genomic DNA extracted from peripheral-blood leukocytes from 235 unselected children with osteosarcoma from 33 institutions were screened for the presence of germline p53 mutations using constant denaturant gel electrophoresis (CDGE). Exons 5 through 8 were evaluated in all patients and exon 2 and exon 9 were analyzed in 59 and 95 patients, respectively. Those samples that showed aberrant migration on CDGE were sequenced or analyzed by restriction enzyme digestion of polymerase chain reaction (PCR) products to confirm the nature of the gene alteration. RESULTS: In 18 samples, CDGE showed fragments of the p53 gene with altered electrophoretic mobilities compared with wild-type p53. DNA sequencing showed that 11 samples had an identical, previously described polymorphism. The other seven contained heterozygous p53 mutations located in exon 5 (n = 3), exon 6 (n = 1), exon 7 (n = 1), and exon 8 (n = 2). Six alterations were missense mutations and one was a nonsense mutation. Three of these patients had first-degree relatives with cancer. One of these three kindreds had a family history consistent with Li-Fraumeni syndrome (LFS). CONCLUSION: We identified germline p53 mutations in seven of 235 (3.0%) children with osteosarcoma. Four of these mutations were found in patients who did not have first-degree relatives with cancer. Although genetic transmission of the altered p53 gene could not be tested in this survey because of how it was designed, it is possible that predictive testing for p53 mutations could identify unaffected relatives of gene carriers who also have a high risk for the development of cancer. This study provides evidence for the importance of considering children with osteosarcoma for predictive testing for germline p53 mutations.

Screening patients for heterozygous p53 mutations using a functional assay in yeast.

Inherited mutations of the p53 gene significantly increase the risk of developing diverse malignancies, and germline p53 mutations can be detected by assaying the transcriptional activity of the p53 protein in mammalian cells. Here we describe a method starting with lymphocytes that allows detection of germline p53 mutations by 'functional' analysis of p53 protein expressed in Saccharomyces cerevisiae. The p53 PCR products are directly cloned into yeast expression vectors in vivo and subsequently tested for transcriptional activity in a simple growth assay. This technique, functional analysis of separated alleles in yeast (FASAY), requires only a few steps, can be automated readily and should permit screening for germline or somatic heterozygous mutations in any gene whose function can be monitored in yeast.