[Clinicopathological and molecular characteristics of esophageal carcinoma with ductal differentiation: analysis of 17 cases].

Objective: To investigate the clinicopathological features and molecular genetic characteristics of esophageal carcinoma with ductal differentiation, and to summarize the experiences in its diagnosis and treatment. Methods: A total of 17 cases of esophageal carcinoma with ductal differentiation diagnosed in Ningbo Clinical Pathological Diagnosis Center, Ningbo, China from June 2011 to December 2022 were collected. The clinical information and pathological diagnosis was reviewed. The tumor histological features and immunohistochemical results were analyzed. The next-generation sequencing was performed to detect and analyze the gene mutations in tumor samples. Results: The 17 patients included in this study were 54-77 years old, with a median age of 66 years. There were 16 males and 1 female. Among them, 9 cases were mainly carcinoma with ductal differentiation. The squamous epithelium on the tumor's surface was accompanied by high-grade intraepithelial neoplasia. The tumor and atypical squamous epithelium were transitional, and the focus was accompanied by various proportions of squamous cell carcinoma component (less than 10%). The other 8 cases were mostly squamous cell carcinoma, basaloid squamous cell carcinoma or sarcomatoid carcinoma with various degrees of tumor specific differentiation and focal area of carcinoma with ductal differentiation (less than 10%). The tumor cells in the area with ductal differentiation were mainly arranged in small tubes, while the tubes showed a double-layer structure, including the inner cells and outer cells of the lumen. Immunohistochemical results showed that the outer cells of the tumorous tubules expressed p63, p40, CK5/6 and CK34ßE12, while the inner cells expressed CK7. Compared with esophageal squamous cell carcinoma reported in the literature, the frequency of gene mutations such as MYC (P=0.002), TP63 (P=0.002), CDKN1C (P=0.002) and NFE2L2 (P=0.045) was significantly lower in this group of cases. At the signaling pathway level, the mutation frequency of NOTCH signaling pathway (P=0.041) was significantly higher, while the mutation frequencies of NRF2 pathway (P=0.013) and PI3K pathway (P=0.009) were significantly lower than that of esophageal squamous cell carcinoma. Conclusion: Esophageal carcinoma with ductal differentiation is a type of esophageal carcinoma with unique morphology, and its molecular changes are also significantly different from those of conventional esophageal squamous cell carcinoma.

The expression of ferroptosis-related genes in osteoporosis based on GEO.

OBJECTIVE: The aim of this study was to screen the differential genes related to ferroptosis in osteoporosis patients. MATERIALS AND METHODS: GEO2R was used to screen the differential genes related to ferroptosis in osteoporosis patients by searching the relevant chips in the GEO database, and Spearman's correlation analysis was used to describe the correlation between quantitative variables without normal distribution. p-values lower than 0.05 were considered statistically significant. Another group of osteoporosis patients was selected in the GEO database to verify the significantly differentially expressed genes. RESULTS: The results showed that 10 samples in chip GSE35956 were identified as research objects, and a total of 5 ferroptosis differential genes were screened out: ATP5MC3, CDKN1A, MT1G, NCOA4, SLC1A5, of which 3 up-regulated genes (CDKN1A, MT1G, SLC1A5), 2 down-regulated genes (ATP5MC3, NCOA4). The above differential genes were placed in 19 samples of chip GSE35959 for verification, and the same expression trend was obtained, but only the MT1G difference was statistically significant. CONCLUSIONS: The gene correlation test found that MT1G and ATP5MC3 had a strong negative correlation.

Microstructure and mechanical properties of dissimilar NiTi and 304 stainless steel joints produced by ultrasonic welding.

Superelastic NiTi alloy and 304 stainless steel (304 SS) were joined with a Cu interlayer by ultrasonic spot welding (USW) using different welding energy inputs. The surface morphology, interfacial microstructure, mechanical properties, and fracture mechanisms of the dissimilar NiTi/304 SS USWed joints were studied. The results showed that the surface oxidation intensified with increasing ultrasonic welding energy due to mutual rubbing between tools and sheets. The weld interface microstructure exhibited voids or unbonded zones at low energy inputs, while an intimate contact was established at the joining interface when applying a higher energy input of 750 J. With increasing energy input to 750 J, the weld interface shows two interfaces due to the behavior of plastic flow of Cu interlayer. The lap-shear load of the joints first increased, achieving a maximum value of ∼690 N at an energy input of 750 J, and then decreased with further increase in welding energy. Interfacial failure was observed at NiTi/Cu interface at all energy inputs, and no intermetallic compounds were found on the fracture surfaces of both the NiTi/Cu and Cu/304 SS interfaces.

The effect of BMP2/Smads pathway mediating platelet-rich fibrin on rat bone mesenchymal stem cells.

OBJECTIVE: We explored the influences on platelet-rich fibrin (PRF) to rat Bone Mesenchymal Stem Cells (BMSCs), as well as the role of bone morphogenetic protein 2 (BMP2)/maternal signal protein homolog (Smads) pathway. MATERIALS AND METHODS: The proposed research is approved by the ethics board of the Second Affiliated Hospital of Harbin Medical University. The BMSCs were isolated and purified. The BMSCs were assigned to a control group arbitrarily, PRF group, BMP activator group and BMP inhibitor group (hereinafter referred to as activator group and inhibitor group). Each group of BMSCs in the logarithmic growth phase was detected for the alkaline phosphatase (ALP) activity since 3 days and 14 days of culture; CCK-8 assay was conducted for detection of the proliferation of BMSCs; Real time PCR was conducted for detection of the osteogenic differentiation marker collagen I (COL-I), BMP2, Runt-related transcription factor 2(RUNX2), osteocalcin (OCN) mRNA relative expression levels; Western-Blot detection of BMP2, OCN, P-SMAD1/5/8, relative expression level of RUNX2 protein. RESULTS: In contrast to the control group, BMSCs' the ALP activity of the PRF group, activator group, as well as inhibitor group increased for 3 days and 14 days, and the activator group>PRF group>inhibitor group (p≤0.05). ALP activity in each group was elevated with the increase in culture time, the ALP activity of the control group, PRF group, activator group and inhibitor group increased (p≤0.05). In comparison to the control group, the relevant expression levels of COL-I, BMP-2, RUNX2 and OCN in the PRF group, activator group, and inhibitor group increased, and the activator group>PRF group>inhibitor group (p≤0.05). The relative expression levels of BMP2, OCN, p-SMAD1/5/8 and RUNX2 protein in each group were statistically different, the activator group>PRF group>control group>inhibitor group (p≤0.05). CONCLUSIONS: PRF can promote the proliferation and osteogenic differentiation of BMSCs by activating the BMP2/Smads signaling pathway.

Regulation of CDC42 GTPase by proline-rich tyrosine kinase 2 interacting with PSGAP, a novel pleckstrin homology and Src homology 3 domain containing rhoGAP protein.

Proline-rich tyrosine kinase 2 (PYK2), a tyrosine kinase structurally related to focal adhesion kinase (FAK), is implicated in regulating cytoskeletal organization. However, mechanisms by which PYK2 participates in and regulates cytoskeletal organization remain largely unknown. Here we report identification of PSGAP, a novel protein that interacts with PYK2 and FAK and contains multiple domains including a pleckstrin homology domain, a rhoGTPase-activating protein domain, and a Src homology 3 domain. PYK2 interacts with PSGAP Src homology 3 domain via the carboxyl-terminal proline-rich sequence. PSGAP is able to increase GTPase activity of CDC42 and RhoA in vitro and in vivo. Remarkably, PYK2, but not FAK, can activate CDC42 via inhibition of PSGAP-mediated GTP hydrolysis of CDC42. Moreover, PSGAP is localized at cell periphery in fibroblasts in a pleckstrin homology domain-dependent manner. Over expression of PSGAP in fibroblasts results in reorganization of cytoskeletal structures and changes of cellular morphology, which requires rhoGTPase-activating activity. Taken together, our results suggest that PSGAP is a signaling protein essential for PYK2 regulation of cytoskeletal organization via Rho family GTPases.

A persistent untranslated sequence within bacteriophage T4 DNA topoisomerase gene 60.

A 50-nucleotide untranslated region is shown to be present within the coding sequence of Escherichia coli bacteriophage T4 gene 60, which encodes one of the subunits for its type II DNA topoisomerase. This interruption is part of the transcribed messenger RNA and appears not to be removed before translation. Thus, the usual colinearity between messenger RNA and the encoded protein sequence apparently does not exist in this case. The interruption is bracketed by a direct repeat of five base pairs. A mechanism is proposed in which folding of the untranslated region brings together codons separated by the interruption so that the elongating ribosome may skip the 50 nucleotides during translation. The alternative possibility, that the protein is efficiently translated from a very minor and undetectable form of processed messenger RNA, seems unlikely, but has not been completely ruled out.

Combining functional and linkage disequilibrium information in the selection of tag SNPs.

UNLABELLED: We have developed an online program, WCLUSTAG, for tag SNP selection that allows the user to specify variable tagging thresholds for different SNPs. Tag SNPs are selected such that a SNP with user-specified tagging threshold C will have a minimum R2 of C with at least one tag SNP. This flexible feature is useful for researchers who wish to prioritize genomic regions or SNPs in an association study. AVAILABILITY: The online WCLUSTAG program is available at http://bioinfo.hku.hk/wclustag/

Human SOX11, an upregulated gene during the neural differentiation, has a long 3' untranslated region.

To obtain essential genes for neuronal development, we have performed a molecular indexing method using a human teratocarcinoma cell line, NTera-2. We isolated a cDNA fragment, designated B18, as an upregulated gene during the neural differentiation. From the complete cDNA sequence of B18 it was revealed that this cDNA was the human SOX11 gene. While a previous report has determined only a approximately 2 kb of the SOX11 cDNA including the entire open reading frame, our full length cDNA was 8743 bp possessing a long 3' untranslated region. Human SOX11 cDNA was mapped to chromosome region 2p25.3 between markers AFMA070WC9 and WI-1412 by radiation hybrid mapping.

Identification and characterization of a novel human cDNA encoding a 21 kDa pRb-associated protein.

The retinoblastoma protein (pRb) functions as a critical master regulator in cell cycle regulation, which is an important cell-regulatory process, through its interaction with various cellular proteins. Using the C-terminus of human pRb and the yeast two-hybrid system, a novel protein named RBP21 that contains 187 amino acid residues with a calculated molecular weight of 21 kDa was identified as a pRb-binding protein. Sequence analysis indicates that RBP21 shares homology with other retinoblastoma-binding proteins in the pRb-binding motif LxCxE at the C-terminal region. In vitro specific interaction between pRb and RBP21 was confirmed using in vitro translation products. When overexpressed in COS-7 cells, RBP21 could co-immunoprecipitate with pRb. This interaction requires the LxCxE motif of RBP21 and the entire pocket region of pRb. Each point mutation of the conserved amino acid residues in pRb-binding motif of RBP21 abolished its specific interaction with pRb. RH mapping result showed that this novel gene was mapped to chromosome region 15q21.1-21.3. Northern blot analysis suggested that RBP21 was widely expressed in various human tissues and cancer cell lines. When expressed in HeLa cells as a green fluorescent protein fusion, RBP21 was distributed throughout the cell.

Identification of a novel gene encoding a p53-associated protein.

p53 exerts important physiological functions in cell-cycle control, gene regulation, cell differentiation, apoptosis and tumor suppression by interacting with many cellular proteins. Using the yeast two-hybrid system, we screened a HeLa cDNA library and identified a novel gene encoding a p53-binding protein (p53BP3). The full-length cDNA of p53BP3 was isolated from a HeLalambdagt10 cDNA library. This predicted protein was composed of 815 amino acids. Sequence analysis indicated that p53BP3 contained two bipartite nuclear localization signals and was confirmed to be a nuclear protein. FISH mapping results showed that this novel gene was located at human chromosome 12, region p11.2-p12.1. Northern blot analysis suggested that p53BP3 was broadly expressed in human tissues. A further study showed that p53BP3 had a homologue in mouse.

Effect of instillation of aldose reductase inhibitor FR74366 on diabetic cataract.

The authors investigate the effect of aldose reductase inhibitor FR74366 on diabetic cataract. Streptozocin (STZ)-induced diabetic rats were treated with eye drops of FR74366 (0.03%, 0.1%, and 0.3%) for 16 weeks. Lenses were examined using a slit lamp, and the score of lens opacity was determined on a scale of from 0 (normal lens) to 4 (matured nuclear cataract). Diabetic placebo control rats developed lens opacity linearly, beginning at 3 weeks and reaching a maximum at 8 weeks after STZ injection. Instillation of FR74366 to diabetic rats delayed the cataract formation and inhibited lens sorbitol accumulation in a dose-dependent manner. At 16 weeks after STZ injection, the score of lens opacity was more than 3 (diffuse central opacities) in diabetic placebo control rats, whereas it was less than 2 (peripheral vesicles and cortical opacities) and the lenses remained clear in animals treated with 0.3% of FR74366. Measurement of tissue drug concentrations indicated that FR74366 penetrated into the lens, where its levels were increased in a dose- and time-dependent manner. These three parameters (score of lens opacity and sorbitol and FR74366 levels) were well correlated with each other. Instillation of FR74366 also reduced the sorbitol levels in the retina. However, the sorbitol levels in the sciatic nerve and renal cortex were not changed by instillation of FR74366. Instillation or oral administration of FR74366 has not shown serious side effects in animal toxicity studies. These results suggested that instillation of FR74366 may be a useful therapeutic agent against diabetic cataract and retinopathy.

Characterization of a novel aldose reductase inhibitor, FR74366, and its effects on diabetic cataract and neuropathy in the rat.

FR74366 (FK366) ([3-(4-bromo-2-fluorobenzyl)-7-chloro-2,4-dioxo-1,2,3,4- tetrahydroquinazolin-1-yl] acetic acid) is a chemically novel aldose reductase (AR) inhibitor. It exhibited a highly potent, reversible, and mixed type inhibition of partially purified AR from the rat sciatic nerve (IC50 = 3.6 nmol/L) and rat lens (IC50 = 4.4 nmol/L). FR74366 inhibited sorbitol accumulation in the isolated human erythrocyte (IC50 = 1.6 mumol/L), rat lens (IC50 = 39 mumol/L), and rat sciatic nerve (IC50 = 17 mumol/L) incubated with high glucose concentrations. The oral administration of FR74366 to streptozotocin (STZ)-induced diabetic rats for 2 weeks decreased sorbitol levels (ED50 = 3.7 mg/kg for sciatic nerve, 23 mg/kg for lens, 52 mg/kg for retina, and 62 mg/kg for renal cortex). Administration of FR74366 to diabetic rats for 17 weeks delayed cataract formation and admixture of 0.028% FR74366 in the diet completely inhibited the cataract formation. Moreover, the recovery of reduced motor nerve conduction velocity by FR74366 in diabetic rats was demonstrated in prevention and reversal experiments. This recovery effect correlated well with reduction of accumulated sorbitol and fructose levels and normalization of decreased myoinositol levels. The duration and tissue specificity of inhibitory effects of FR74366 on sorbitol accumulation also correlated well with the levels of FR74366 in various tissues of diabetic rats. These data indicate that both decreases in tissue sorbitol levels and improvement of functional defects reflect FR74366 levels in tissue rather than plasma in diabetic rats. These results, taken together, suggest that FR74366, which is currently undergoing clinical trials in Japan and the United States, will be a useful therapeutic agent for diabetic complications.

Preparation and a structure-function analysis of human ciliary neurotrophic factor.

Ciliary neurotrophic factor (CNTF) is a trophic protein that promotes survival and/or differentiation of a variety of neuronal cell types including sensory, sympathetic, and motor neurons. CNTF, leukemia inhibitory factor (LIF), interleukin-6 (IL-6) and oncostatin M (OSM) share a predicted common helical framework and partially identical receptor components. In this study, we present the preparation and structure--functional analysis of recombinant human CNTF. The human CNTF gene was expressed under the control of the PL promoter in Escherichia coli, and the mutants were constructed by insertion, deletion and site-directed mutagenesis. The recombinant proteins were purified from bacteria via DEAE A-50 and Sephacryl S-200 chromatography, and their survival promoting activities were determined using cultures of embryonic chick dorsal root ganglion (DRG) neurons. Insertion at position 23 with APGL, or at position 79 with PRGA, or substitution of 162L163Q for PIDG resulted in proteins with no neurotrophic activity. However, insertion at position 186 with PRGI did not alter human CNTF activity. Deletion of the carboxy-terminal amino acid 186-200 did not reduce the biological activity, but elimination of the amino acid 162-186 abolished the activity. The mutant substituting of 17 Cys for Ser was found to display a biological activity equivalent to that of the wild type. Our data provided experimental confirmation for the structural prediction of CNTF.

Changes in tissue metals after cadmium intoxication and intervention with chlorpromazine in male rats.

Cadmium (Cd), one of the most dangerous heavy metals, has a very similar ionic radius to calcium (Ca). The interference of cadmium in calcium homeostasis may play an important role in cadmium toxicity. Recent reports indicate that calmodulin (CaM) inhibitors such as trifluoperazine and chlorpromazine (CPZ) could protect rodents against cadmium toxicity. It was also reported that pretreatment of mice with zinc (Zn) could reduce the adverse effects induced by cadmium. The aim of this study is to determine whether Cd changes the balance of other essential metals such as Zn and copper (Cu) in rat tissues, and whether CPZ can reverse these changes which are induced by cadmium intoxication. Adult male Sprague-Dawley (SD) rats were injected intraperitoneally (i.p.) with cadmium chloride (CdCl2) (0.2, 0.4, 0.8 mg Cd/kg body weight) alone and 0.4 mg Cd/kg in association with CPZ (5 mg/kg) daily for a week. The control animals were injected with normal saline only. The results showed that the cadmium content in the liver, kidney and testis increased significantly with a dose-response relationship. Cadmium treatment markedly increased the Zn and Ca content in some of the tissues. Hepatic and renal metallothionein (MT) increased significantly after cadmium intoxication. CPZ treatment, however, reduced cadmium content in liver, but not blood and kidney. CPZ seemed to decrease the content of MT in liver and significantly increase the amounts of MT in kidney. These data suggest that the intervention of cadmium with tissue essential metals may play a role in cadmium toxicity in rats, and calmodulin inhibitors to some extent can reduce the adverse effect of cadmium by decreasing the cadmium load in tissues and reversing the unbalance of essential metals.

Identification and cloning of the cDNA of a Rb-associated protein RAP140a.

Rb exerts important physiological functions in cell-cycle control, gene expression, cell differentiation, apoptosis, development and tumorigenesis by interacting with many cellular proteins. Using human partial Rb as bait, we screened a human fetal brain cDNA library through yeast two-hybrid system and obtained six novel cDNA fragments. Among them, one cDNA fragment corresponds to two different transcripts, 7 kb and 9 kb in Northern blot analysis. These two transcripts showed uniform distribution in various human tissues. We cloned the full-length cDNA of a 7.2 kb transcript through three times PCR amplifications. It was namedRAP140a and predicted to encode a 1 233 amino acids hydrophilic protein.RAP140a was mapped to chromosome 3p13-p14. 1. RAP140a may be functionally related to the intracellular translocation of Rb or other proteins.

[Studies on the cloning, expression and function of the yeast PHO 80 gene].

Through in situ hybridization, a 4.2 kb Pst I-BamH I fragment was obtained from S. cerevisiae gene library. The cloned fragment contained 1100 bp upstream sequence and 879 bp coding sequence of the PHO80 gene. Coding region of PHO80 gene was substituted with URA3 gene and used as donor to transform YPH499 to URA3. A pho80 mutant resulted from deletion of the chromosomal counterpart in PHO80 was obtained. In vivo functional study of the PHO80 gene indicated that PHO80 was a negative regulator in the Pi-repressible acid phosphatase system including the structural genes PHO5 and PHO11 and the regulatory gene PHO81, whereas the expression of PHO4 or PHO85 was independent of PHO80. The coding region of PHO80 was fused in frame with LacZ and beta-galactosidase activities in various cells was determined. The results Showed that the PHO80 gene was expressed at a low level and suppressed by itself and PHO85.

CLUSTAG: hierarchical clustering and graph methods for selecting tag SNPs.

UNLABELLED: Cluster and set-cover algorithms are developed to obtain a set of tag single nucleotide polymorphisms (SNPs) that can represent all the known SNPs in a chromosomal region, subject to the constraint that all SNPs must have a squared correlation R2>C with at least one tag SNP, where C is specified by the user. AVAILABILITY: http://hkumath.hku.hk/web/link/CLUSTAG/CLUSTAG.html CONTACT: mng@maths.hku.hk.

RBP95, a novel leucine zipper protein, binds to the retinoblastoma protein.

We recently identified a novel cDNA encoding a retinoblastoma protein (pRb)-associated protein. It was named RBP95, which was composed of 838 amino acid residues with a calculated molecular size of 94,789 Da. Northern blot analysis showed a single mRNA of about 4. 5 kb ubiquitously expressed in human tissues. RH mapping results showed that RBP95 is mapped to chromosome region 16p11.2-11.1. Sequence analysis indicated that RBP95 contains a conserved pRb-binding motif LXCXE. Interaction between pRb and RBP95 was confirmed in vivo and in vitro. This interaction requires the LXCXE motif of RBP95 and the entire pocket region of pRb. Each point-mutant of the conserved amino acid residues in pRb-binding motif of RBP95 would destroy its interaction with pRb. RBP95 also contains a basic region leucine zipper and could homodimerize through its leucine zipper region. RBP95 was located in the nucleus with a special pattern when expressed as a GFP fusion in HeLa cells. All these findings suggested that RBP95, a new member of pRb-associated protein, may function as a regulation factor in the process of RNA polymerase II-mediated transcription and/or transcriptional processing.