[Detection and analysis of serum food-specific IgG antibody in Beijing area].

Objective: The level of food specific IgG antibody in serum of patients with suspected food intolerance in Beijing area was detected and analyzed, so as to provide a basis for patients to eliminate the food that causes disease in their diet. Methods: Data were retrospectively analyzed in a total of 2368 suspected chronic food intolerance patients (all resident population in Beijing) admitted to Beijing Jishuitan Hospital from October 2015 to December 2019, including 859 males and 1509 females. The age range ranged from 2 months to 90 years, (40.2±18.6) years, and the median age was 36 years. Serum specific IgG (sIgG) antibody concentrations in 14 kinds of food (beef, chicken, pork, cod, shrimp, crab, egg, milk, corn, wheat, mushroom, soybean, tomato and rice) were determined by enzym-linked immunosorbentassay (ELISA).SPSS V22.0 was used for statistical analysis, and χ2 was used to compare the positive rate of each group. Results: The positive rate of food sIgG in 2 368 patients was 82.7% (1959/2368), the higher sIgG positive rates were found in eggs (54.7%), shrimp (29.0%) and milk (26.0%), and the lowest sIgG positive rates were found in pork (4.1%) and beef (1.2%) (P<0.05).There was significant gender difference in egg (χ²=10.885) and shrimp (χ²=6.607) sIgG, and female was significantly higher than male (P<0.05).The sIgG positive rates of egg (χ²=515.645), shrimp (χ²=74.20.4), milk (χ²=305.65), soybean (χ²=39.413), wheat (χ²=133.222), cod (χ²=25.540), crab (χ²=55.521), corn (χ²=20.645), tomato (χ²=118.108), chicken (χ²=32.821), pork (χ²=26.539), beef (χ²=21.632) and rice (χ²=25.335) were significant in different age groups. The positive rate of shrimp and crab sIgG increased with age (P<0.05).The positive rate of tomato sIgG decreased with age, and then increased slightly after 45 years. However, egg, milk, soybean, wheat, corn, and rice all showed a decreasing positive rate with the increase of age. The increase of chicken, pork and cod was observed in childhood, and then decreased with age, and then increased in middle and old age. Conclusions: There may be a certain correlation between food intolerance and elevated food sIgG levels in Beijing area. It is suggested that patients of different genders and ages should be optimized and adjusted according to their own characteristics, and children patients are still the key population for food intolerance control.

Cloning differentially expressed mRNAs.

Differential gene expression occurs in the process of development, maintenance, injury, and death of unicellular as well as complex organisms. Differentially expressed genes are usually identified by comparing steady-state mRNA concentrations. Electronic subtraction (ES), subtractive hybridization (SH), and differential display (DD) are methods commonly used for this purpose. A rigorous examination has been lacking and therefore quantitative aspects of these methods remain speculative. We compare these methods by identifying a total of 58 unique differentially expressed mRNAs within the same experimental system (HeLa cells treated with interferon-gamma). ES yields digital, reusable data that quantitated steady-state mRNA concentrations but only identified abundant mRNAs (seven were identified), which represent a small fraction of the total number of differentially expressed mRNAs. SH and DD identified abundant and rare mRNAs (33 and 23 unique mRNAs respectively) with redundancy. The redundancy is mRNA abundance-dependent for SH and primer-dependent for DD. We conclude that DD is the method of choice because it identifies mRNAs independent of prevalence, uses small amounts of RNA, identifies increases and decreases of mRNA steady-state levels simultaneously, and has rapid output.

[Regulation of Ruxolitinib on matrix metalloproteinase in JAK2V617F positive myeloroliferative neoplasms cells].

Objective: To investigate the regulation of JAK2 tyrosine kinase inhibitor ruxolitinib on extracellular matrix metalloproteinase (MMP in JAK2V617F positive myeloproliferative neoplasms (MPN) cells. Methods: ①Forty cases of newly diagnosed JAK2V617F positive MPN patients and 15 healthy volunteers as control in Baoding No.1 Hospital between January 2012 and December 2015 were enrolled in this study. JAK2V617F/JAK2 ratio was detected by real-time-PCR; the expression levels of phosphorylation protein tyrosine kinase 2 (p-JAK2) , MMP-2 and MMP-9 in pathological tissues of bone marrow were detected by immunohistochemistry. The bone marrow cells of JAK2V617F positive MPN patients were treated with ruxolitinib, then the migration ability and MMP-2, MMP-9 gene and protein expression levels were detected. ②The human erythroleukemia cell line HEL cells were treated with different concentrations of ruxolitinib (0, 50, 100, 250, 500, 1 000 nmol/L) . The cell viability was detected by CCK-8 test; cell migration ability was tested by transwell chambers. The mRNA expression levels of JAK2, MMP-2 and MMP-9 were detected by real-time-PCR. The protein expression levels of p-JAK2, MMP-2 and MMP-9 were detected by Western blot. Results: ①The expression levels of p-JAK2, MMP-2 and MMP-9 in the newly diagnosed group were significantly higher than control group respectively [ (78.56±24.55) % vs (41.59±17.29) %, P<0.05; (48.25±18.74) % vs (22.79±13.89) %, P<0.05; (53.29±19.28) % vs (15.56±14.96) %, P<0.05]. Spearman correlation analysis showed the positive correlation of MMP-2 and MMP-9 protein expression levels with JAK2V617F mutation (r=0.526, P=0.001; r=0.543, P=0.001) . ②The proliferation of HEL cells was inhibited by different concentrations of ruxolitinib in time and dose dependent manner. ③Cell migration test showed the number of cells leaked to the low chamber in MPN patients bone marrow cells and HEL cells treated with 5 nmol/L of ruxolitinib group were significantly lower than that without ruxolitinib treatment after 24 h [ (154.7±27.5) vs (320.3±67.3) , t=13.47, P<0.05; (70.7±10.5) vs (135.3±16.7) , t=13.89, P<0.05]. The mRNA and protein expression levels of JAK2, MMP-2 and MMP-9 decreased with the increased concentration of ruxolitinib. Conclusion: Ruxolitinib inhibits MPN cell migration and expression of MMP-2 and MMP-9 via JAK2 signal pathway.

Proteolytic processing in a non-lysosomal compartment is required for transcytosis of protein-polylysine conjugates in cultured Madin-Darby canine kidney cells.

The transcytosis of horseradish peroxidase, as well as its poly(L-lys) and poly(D-lys) thioether conjugates, was investigated in Strain I Madin-Darby canine kidney (MDCK) cell monolayers grown on 0.4 microns pore size polycarbonate membranes in Costar Transwells. The 3 types of HRP had almost identical rates of transport during the first 2 hr of incubation. However, a significant increase of basal-to-apical transport was detected beginning at 3 hr only in Transwells containing the poly(L-lys) conjugate. This increase was inhibited by colchicine (2 microM) and by the Bowman-Birk protease inhibitor (0.1 mg/ml), but not by NH4Cl (10 mM) or chloroquine (0.1 mM). The increase was abolished either by prior trypsinization of the conjugate or by incubation at 4 degrees C. Ultrafiltration studies indicated that the transcytosed poly(L-lys) conjugate was smaller in size than the original conjugate. These results indicate that the conjugate was processed during transcytosis in a non-lysosomal proteolytic compartment, where its poly(L-lys) moiety was selectively degraded, allowing active peroxidase to be released into the apical medium.

Yeast histone H3 and H4 N termini function through different GAL1 regulatory elements to repress and activate transcription.

Previous work has shown that N-terminal deletions of yeast histone H3 cause a 2- to 4-fold increase in the induction of GAL1 and a number of other genes involved in galactose metabolism. In contrast, deletions at the H4 N terminus cause a 10- to 20-fold decrease in the induction of these same GAL genes. However, H3 and H4 N-terminal deletions each decrease PHO5 induction only 2- to 4-fold. To define the GAL1 gene regulatory elements through which the histone N termini activate or repress transcription, fusions were made between GAL1 and PHO5 promoter elements attached to a beta-galactosidase reporter gene. We show here that GAL1 hyperactivation caused by the H3 N-terminal deletion delta 4-15 is linked to the upstream activation sequence. Conversely, the relative decrease in GAL1 induction caused by the H4N-terminal deletion delta 4-28 is linked to the downstream promoter which contains the TATA element. These data indicate that the H3 N terminus is required for the repression of the GAL1 upstream element, whereas the H4N terminus is required for the activation of the GAL1 downstream promoter element.

Transcellular processing of disulfide- and thioether-linked peroxidase--polylysine conjugates in cultured MDCK epithelial cells.

Horseradish peroxidase (HRP) was conjugated to nondegradable polycationic poly(D-lysine) (PDL) through either a thioether (HRP-S-PDL) or a disulfide (HRP-SS-PDL) linkage. The binding and transcytosis of these conjugates was studied in Madin-Darby canine kidney (MDCK) cell monolayers grown on 3-microns microporous polycarbonate filters. Conjugation of HRP to PDL with both linkages markedly increased the binding of this protein onto the cell monolayers. However, an enhancement of the transcellular transport of HRP in both apical-to-basal and basal-to-apical directions was observed only in HRP-SS-PDL, but not in HRP-S-PDL. HRP-SS-PDL transport was inhibited by colchicine and by 4 degrees C incubation. The transport of 14C-sucrose was not affected by the presence of conjugates. These results indicate that the transport of the conjugate across the cell monolayers was due to a transcellular process rather than to any leakage of the cell junction caused by polycations. The disulfide linkage between HRP and PDL was cleaved rapidly at the basal and, to a lesser extent, at the apical surface of the cell. Neuraminidase treatment decreased the binding of the conjugates onto the cell surface, but did not decrease the transcellular transport, suggesting that not all surface-bound conjugates were available for transcytosis. These results demonstrate that disulfide linkages can be cleaved during transcytosis in MDCK cells. The cleavage, however, occurs mostly at the binding site on the cell surface, which may prevent the cellular uptake of the intact conjugate.

Screening differentially expressed cDNA clones obtained by differential display using amplified RNA.

The major obstacle of differential display is not the technique itself but rather the post-differential display issueof discriminating between false positives and the truly differentially expressed mRNAs. This process is arduous and requires large amounts of RNA. We present and validate a method which allows one to screen putative positives from differential display analysis using only micrograms of total RNA. More importantly, we demonstrate that cDNA probes generated from amplified RNA are representative of the starting mRNA population and can be used for differential screening of mRNA species at a detectable limit of sensitivity of>/=1/40 000.

DNA microarrays of the complex human cytomegalovirus genome: profiling kinetic class with drug sensitivity of viral gene expression.

We describe, for the first time, the generation of a viral DNA chip for simultaneous expression measurements of nearly all known open reading frames (ORFs) in the largest member of the herpesvirus family, human cytomegalovirus (HCMV). In this study, an HCMV chip was fabricated and used to characterize the temporal class of viral gene expression. The viral chip is composed of microarrays of viral DNA prepared by robotic deposition of oligonucleotides on glass for ORFs in the HCMV genome. Viral gene expression was monitored by hybridization to the oligonucleotide microarrays with fluorescently labelled cDNAs prepared from mock-infected or infected human foreskin fibroblast cells. By using cycloheximide and ganciclovir to block de novo viral protein synthesis and viral DNA replication, respectively, the kinetic classes of array elements were classified. The expression profiles of known ORFs and many previously uncharacterized ORFs provided a temporal map of immediate-early (alpha), early (beta), early-late (gamma1), and late (gamma2) genes in the entire genome of HCMV. Sequence compositional analysis of the 5' noncoding DNA sequences of the temporal classes, performed by using algorithms that automatically search for defined and recurring motifs in unaligned sequences, indicated the presence of potential regulatory motifs for beta, gamma1, and gamma2 genes. In summary, these fabricated microarrays of viral DNA allow rapid and parallel analysis of gene expression at the whole viral genome level. The viral chip approach coupled with global biochemical and genetic strategies should greatly speed the functional analysis of established as well as newly discovered large viral genomes.