[Analysis of the types and functions of CD34+ cells in full-thickness skin defect wounds of normal mice and diabetic mice by single-cell RNA sequencing].

Objective: To analyze the types and functions of CD34+ cells in full-thickness skin defect wounds of normal mice and diabetic mice by single-cell RNA sequencing. Methods: This study was an experimental study. The CD34+ cell lineage tracing mouse was produced, and the visualization of CD34+ cells under the fluorescent condition was realized. Six male CD34+ cell lineage tracing mice aged 7-8 weeks (designated as diabetic group) were intraperitoneally injected with streptozotocin to establish a diabetic model, and full-thickness skin defect wounds were prepared on their backs when they reached 13 weeks old. Another 6 male CD34+ cell lineage tracing mice aged 13 weeks (designated as control group) were also subjected to full-thickness skin defect wounds on their backs. On post-injury day (PID) 4, wound tissue was collected from 3 mice in control group and 2 mice in diabetic group, and digested to prepare single-cell suspensions. CD34+ cells were screened using fluorescence-activated cell sorting, followed by single-cell RNA sequencing. The Seurat 4.0.2 program in the R programming language was utilized for dimensionality reduction, visualization, and cell clustering analysis of CD34+ cell types, and to screen and annotate the marker genes for each CD34+ cell subpopulation. Kyoto encyclopedia of genes and genomes (KEGG) and gene ontology (GO) enrichment analysis was performed to analyze the differentially expressed genes (DEGs) of CD34+ fibroblasts (Fbs), smooth muscle cells (SMCs), keratinocytes (KCs), and chondrocyte-like cells (CLCs) in the wound tissue of two groups of mice for exploring cellular functions. Results: On PID 4, CD34+ cells in the wound tissue of both groups of mice were consisted of 7 cell types, specifically endothelial cells, Fbs, KCs, macrophages, T cells, SMCs, and CLCs. Among these, Fbs were further classified into 5 subpopulations. Compared with those in control group, the proportions of CD34+ endothelial cells, Fbs subpopulation 1, Fbs subpopulation 4, KCs, and CLCs in the wound tissue of mice were increased in diabetic group, while the proportions of CD34+ Fbs subpopulation 2, Fbs subpopulation 3, and SMCs were decreased. The marker genes for annotating CD34+ CLCs, endothelial cells, Fbs subpopulation 1, Fbs subpopulation 2, Fbs subpopulation 3, Fbs subpopulation 4, Fbs subpopulation 5, KCs, macrophages, SMCs, and T cells were respectively metastasis-associated lung adenocarcinoma transcript 1, fatty acid binding protein 4, Gremlin 1, complement component 4B, H19 imprinted maternally expressed transcript, Dickkopf Wnt signaling pathway inhibitor 2, fibromodulin, keratin 5, CD74 molecule, regulator of G protein signaling 5, and inducible T-cell co-stimulator molecule. KEGG and GO enrichment analysis revealed that, compared with those in control group, DEGs with significant differential expression (SDE) in CD34+ Fbs from the wound tissue of mice in diabetic group on PID 4 were significantly enriched in terms related to inflammatory response, extracellular matrix (ECM) organization, regulation of cell proliferation, and aging (with Pvalues all <0.05), DEGs with SDE in CD34+ SMCs were significantly enriched in terms related to cell migration, apoptotic process, positive regulation of transcription, and phagosome (with P values all <0.05), DEGs with SDE in CD34+ KCs were significantly enriched in terms related to mitochondrial function, transcription, and neurodegenerative diseases (with P values all <0.05), and DEGs with SDE in CD34+ CLCs were significantly enriched in terms related to rhythm regulation, ECM, and viral infection (with P values all <0.05). Conclusions: CD34+ cells display high heterogeneity in the healing process of full-thickness skin defect wounds in both normal mice and diabetic mice. The significantly enriched functions of DEGs with SDE in CD34+ cell subpopulations in the wound tissue of the two mouse groups are closely related to the wound healing process.

Clinical efficacy of targeted therapy, immunotherapy combined with hepatic artery infusion chemotherapy (FOLFOX), and lipiodol embolization in the treatment of unresectable hepatocarcinoma.

To evaluate the clinical efficacy of targeted therapy and immunotherapy combined with hepatic arterial infusion chemotherapy (HAIC) of FOLFOX and lipiodol embolization in the treatment of unresectable hepatocellular carcinoma. Patients included in the study were those who received targeted therapy and immunotherapy combined with HAIC of FOLFOX and lipiodol embolization in Zhongshan People's Hospital from December 2020 to June 2021 for unresectable hepatocellular carcinoma. Evaluation indicators included objective response rate (ORR), median progression-free survival (mPFS), median duration of response (mDOR), 1-year overall survival rate (OS), surgical conversion rate, and adverse events. Treatment response was assessed using Response Evaluation Criteria in Solid Tumors (mRECIST and RECIST v1.1). A total of 35 patients were included in this study, 30 of whom completed treatment evaluation. According to mRECIST evaluation criteria, the objective response rate (ORR) was 83.3% (25/30); the complete response (CR) was 60% (18/30); the partial response (PR) was 23.3% (7/30), and stable disease (SD) was 16.7% (5/30). The mDOR was 10.3 months (95% Cl: 8.27-NE), and the mPFS was 13.2 months (95% CI: 10.3-NE); the surgical conversion rate was 30.0% (9/30). The 1-year OS was 96.7%. There were no serious surgical complications and grade 4 or 5 adverse events of targeted therapy, immunotherapy and HAIC. Some patients had grade 3 adverse reactions in gastrointestinal toxicity or hepatotoxicity, and the adverse reactions were improved after corresponding symptomatic treatment. We concluded that HAIC of FOLFOX and lipiodol embolization combined with targeted therapy and immunotherapy had a significant curative effect in the treatment of unresectable hepatocellular carcinoma, with no serious adverse reactions and a high rate of surgical conversion rate.

[Comparison of the curative effect of transjugular intrahepatic portosystemic shunt with expanded polytetrafluoroethylene-covered stent and drug combined with gastroscopy as the secondary prevention of esophageal -gastric variceal bleeding in portal hypertension].

Objective: To compare the clinical efficacy of transjugular intrahepatic portosystemic shunt (TIPS) with expanded polytetrafluoroethylene (ePTFE)-covered stent and drug combined with gastroscopy as the secondary prevention of esophageal-gastric variceal bleeding in portal hypertension. Methods: Patients with esophageal-gastric variceal bleeding who received TIPS treatment (ePTFE covered stent) or gastroscopy for the first time as the secondary prevention for portal hypertension at Nanfang Hospital of Southern Medical University through March to July 2017 were selected. One year after the operation, liver function changes, ascites remission rates, incidence of hepatic encephalopathy, re-bleeding rate, average hospitalization frequency and expenses, survival time, as well as the TIPS patency conditions were analyzed in the two groups of patients. 2 test, Kaplan-Meier method and Mann-Whitney U test were used to analyze the data. Results: There were 74 and 66 cases in the TIPS and the drug combined gastroscopy group and the follow-up duration (14.57 ± 0.79) was 12-16 months. One year after surgery, the remission rate of ascites in the TIPS group was higher 57.1% (32/56) than that of the drug combined gastroscopy group (0), and the difference was statistically significant (χ(2) = 2 = 36.73, P < 0.01). The cumulative incidence of hepatic encephalopathy at 1, 3, 6, and 12 months after surgery in the TIPS group was 32.4% (24/74), 37.8% (28/74), 40.5% (30/74), and 40.5% (30/74), respectively. The cumulative incidence of hepatic encephalopathy in the drug combined gastroscopy group was 3.0% (2/66), 3.0% (2/66), 3.0% (2/66), and 6.1% (4/66), respectively. Kaplan-Meier analysis showed that the cumulative incidence of hepatic encephalopathy in the TIPS group was higher than that of the drug combined gastroscopy group (χ(2) = 11.29, P < 0.01). The incidence of severe hepatic encephalopathy ( grade III to IV) at 1, 3, 6, and 12 months after surgery in the TIPS group was 2.7% (2/74), 0, 0, and 0, respectively. The incidence of severe hepatic encephalopathy in drug combined gastroscopy group was 0, and there was no statistically significant difference in development of hepatic encephalopathy between the two groups (P > 0.05). The re-bleeding rates of TIPS group and drug combined gastroscopy group were 0 and 27.3% (18/66), respectively, and the difference was statistically significant (χ(2) = 22.42, P < 0.01). There was no death reported during the follow-up period between both groups. The hospitalization frequency times (1.45 ± 0.80) in TIPS group was lower than that of the drug combined gastroscopy group times (3.24 ± 1.80), and the difference was statistically significant (U = -4.52, P < 0.01). Conclusion: In the prevention of esophageal-gastric variceal bleeding, TIPS (ePTFE-covered stent) treatment has the advantages of reducing re-bleeding rate, high ascites remission rate and hospitalizations frequency. In addition, patients treated with TIPS have a higher incidence of hepatic encephalopathy than that of drugs combined with gastroscopy. However, TIPS did not exacerbate the incidence of hepatic encephalopathy, and there was no significant difference in the 1-year survival rate after TIPS and drugs combined with gastroscopy treatment.

[Analysis of the medium-term curative effect of transjugular intrahepatic portosystemic shunt through jugular vein with covered Viatorr stent].

Objective: To investigate the medium-term curative effect of transjugular intrahepatic portosystemic shunt (TIPS) through jugular vein with covered Viatorr stent. Methods: Data of 105 consecutive patients with covered Viatorr stent of our hospital was retrospectively analyzed. Follow-up was performed after surgery, and color Doppler was reviewed to evaluate the efficacy of TIPS. Results: Transjugular intrahepatic shunt was successfully established in all patients. The pressure gradients of portal vein before and after operation were 22.33 ± 6.4 mmHg and 9.78 ± 4.9 mmHg, respectively,P< 0.01, and the difference was statistically significant. The follow-up period ranged from 12.7 to 15.6 months, with an average of 13.09 ± 1.4 month. Total bilirubin and coagulation time increased after operation, but there was no significant difference in total bilirubin and coagulation time at 1, 3, 6 and 12 months after operation. The patency rate of shunt was 100%, 99.05%, 99.05% and 99.05% at 1, 3, 6 and 12 months after operation. The incidence of hepatic encephalopathy was 34.2%, 29.5%, 19.1% and 14.3% respectively. The recurrence rate of symptoms was 0%. Conclusion: Patients with cirrhotic portal hypertension who underwent TIPS with covered Viatorr stent had a lower rate of restenosis, improved liver function, and a lower incidence of severe hepatic encephalopathy. However, the long-term efficacy needs further observation.

Microring resonator-assisted Fourier transform spectrometer with enhanced resolution and large bandwidth in single chip solution.

Single chip integrated spectrometers are critical to bring chemical and biological sensing, spectroscopy, and spectral imaging into robust, compact and cost-effective devices. Existing on-chip spectrometer approaches fail to realize both high resolution and broad band. Here we demonstrate a microring resonator-assisted Fourier-transform (RAFT) spectrometer, which is realized using a tunable Mach-Zehnder interferometer (MZI) cascaded with a tunable microring resonator (MRR) to enhance the resolution, integrated with a photodetector onto a single chip. The MRR boosts the resolution to 0.47 nm, far beyond the Rayleigh criterion of the tunable MZI-based Fourier-transform spectrometer. A single channel achieves large bandwidth of ~ 90 nm with low power consumption (35 mW for MRR and 1.8 W for MZI) at the expense of degraded signal-to-noise ratio due to time-multiplexing. Integrating a RAFT element array is envisaged to dramatically extend the bandwidth for spectral analytical applications such as chemical and biological sensing, spectroscopy, image spectrometry, etc.

[Therapeutic effect of low-temperature radiofrequency coblation on early-stage laryngeal cancer].

Objective: To evaluate the safety,efficacy and prognosis of low-temperature plasma radiofrequency coblation for early-stage laryngeal cancer(Tis, T1 and T2). Method: A retrospective analysis of 202 patients with early-stage laryngeal cancer who underwent the low-temperature radiofrequency coblation surgery, including 34 cases of Tis(16.83%), 49 cases of stage T1aN0M0(24.26%), 50 cases of stage T1bN0M0(24.75%) and 69 cases of stage T2N0M0(34.16%). Surgical patients were followed up closely for 6 to 60 months, with a median follow-up of 29 months. Result: Of the 202 patients,165(81.68%) had no recurrence and achieved good surgical results. None of them suffered severe complications such as post-operative hemorrhage and asphyxia. 37 cases(18.32%) had recurrence, including 1 case(0.50%) in stage Tis, 7 cases(3.47%) in stage T1a,7 cases(3.47%) in stage T1b, and 22 cases(10.89%) in stage T2. Thirteen patients who had recurrence underwent total laryngectomy(5 of which had a recurrence of T3 and 8 of which progressed to T4), including 1 in the stage T1a,2 in the stage T1b, and 10 in the stage T2. Vertical hemilaryngectomy were performed in 4 cases, 3 cases of stage T1a and 1 case of stage T2; 5 cases underwent plasma radiofrequency coblation again, including 3 cases of stage T1b and 2 cases of stage T2,no recurrence was found in all the patients; 1 patient had no obvious recurrence in the larynx but had cervical lymph node metastasis, radical neck dissection was performed; 1 patient with stage T2 recurrence was treated with a tracheotomy to relieve laryngeal obstruction without further treatment;3 cases showed improvement by radiotherapy and chemotherapy treatment after recurrence; 9 death cases,5 patients died after radiotherapy and chemotherapy, and 4 patients stopped getting treatment after recurrence.Conclusion: Low-temperature radiofrequency coblation surgery for patients with early-stage laryngeal cancer has great advantages in the preservation of laryngeal function and reduction of surgical trauma after surgery compared with traditional surgical method, and can obtain satisfactory results, but the selection of surgical indications for some patients with clinical stage T2 is still need to be carefully considered..

Electronic structure of SrSn2As2 near the topological critical point.

Topological materials with exotic quantum properties are promising candidates for quantum spin electronics. Different classes of topological materials, including Weyl semimetal, topological superconductor, topological insulator and Axion insulator, etc., can be connected to each other via quantum phase transition. For example, it is believed that a trivial band insulator can be twisted into topological phase by increasing spin-orbital coupling or changing the parameters of crystal lattice. With the results of LDA calculation and measurement by angle-resolved photoemission spectroscopy (ARPES), we demonstrate in this work that the electronic structure of SrSn2As2 single crystal has the texture of band inversion near the critical point. The results indicate the possibility of realizing topological quantum phase transition in SrSn2As2 single crystal and obtaining different exotic quantum states.

p53 protein aggregation promotes platinum resistance in ovarian cancer.

High-grade serous ovarian carcinoma (HGSOC), the most lethal gynecological cancer, often leads to chemoresistant diseases. The p53 protein is a key transcriptional factor regulating cellular homeostasis. A majority of HGSOCs have inactive p53 because of genetic mutations. However, genetic mutation is not the only cause of p53 inactivation. The aggregation of p53 protein has been discovered in different types of cancers and may be responsible for impairing the normal transcriptional activation and pro-apoptotic functions of p53. We demonstrated that in a unique population of HGSOC cancer cells with cancer stem cell properties, p53 protein aggregation is associated with p53 inactivation and platinum resistance. When these cancer stem cells differentiated into their chemosensitive progeny, they lost tumor-initiating capacity and p53 aggregates. In addition to the association of p53 aggregation and chemoresistance in HGSOC cells, we further demonstrated that the overexpression of a p53-positive regulator, p14ARF, inhibited MDM2-mediated p53 degradation and led to the imbalance of p53 turnover that promoted the formation of p53 aggregates. With in vitro and in vivo models, we demonstrated that the inhibition of p14ARF could suppress p53 aggregation and sensitize cancer cells to platinum treatment. Moreover, by two-dimensional gel electrophoresis and mass spectrometry we discovered that the aggregated p53 may function uniquely by interacting with proteins that are critical for cancer cell survival and tumor progression. Our findings help us understand the poor chemoresponse of a subset of HGSOC patients and suggest p53 aggregation as a new marker for chemoresistance. Our findings also suggest that inhibiting p53 aggregation can reactivate p53 pro-apoptotic function. Therefore, p53 aggregation is a potential therapeutic target for reversing chemoresistance. This is paramount for improving ovarian cancer patients' responses to chemotherapy, and thus increasing their survival rate.

Droplet optofluidic imaging for λ-bacteriophage detection via co-culture with host cell Escherichia coli.

Bacteriophages are considered as attractive indicators for determining drinking water quality since its concentration is strongly correlated with virus concentrations in water samples. Previously, bacteriophage detection was based on a plague assay that required a complicated labelling technique and a time-consuming culture assay. Here, for the first time, a label-free bacteriophage detection is reported by using droplet optofluidic imaging, which uses host-cell-containing microdroplets as reaction carriers for bacteriophage infection due to a higher contact ratio. The optofluidic imaging is based on the effective refractive index changes in the microdroplet correlated with the growth rate of the infected host cells, which is highly sensitive, i.e. can detect one E. coli cell. The droplet optofluidic system is not only used in drinking water quality monitoring, but also has high potential applications for pathogenic bacteria detection in clinical diagnosis and food industry.

Bisoprolol improved endothelial function and myocardium survival of hypertension with stable angina: a randomized double-blinded trial.

AIM: This study was designed to determine the effect of bisoprolol on endothelial function of brachial artery and the myocardium survival in hypertensive patients with stable angina. PATIENTS AND METHODS: 222 subjects with hypertension who had received coronary angiography examination were involved in the study, 162 in bisoprolol therapy group (96 men, 59%) and 60 in non-bisoprolol group (39 men, 65%). In accordance with results of angiography (coronary stenosis ≥ 50%), the patients in bisoprolol group were divided into three sub-groups: (1) single-vessel coronary disease group (n=42); (2) double-vessel coronary disease group (n=44); (3) multi-vessel coronary disease group (n=39) and hypertension-only group (n=37). All the subjects were treated with conventional drugs plus bisoprolol and followed up for 12 months. Parameters of clinical features, echocardiography, radionuclide ventriculographic and laboratory findings were measured and analyzed. RESULTS: After 12 months bisoprolol treatment, the flow-mediated vasodilatation (FMD) and 99Tcm-sestamibi (99Tcm-MIBI) uptake fraction which reflects the survival of myocardium were improved markedly in bisoprolol group (all p < 0.05). Interestingly, a more significant improvement in FMD and 99Tcm-MIBI uptake fraction were observed in severe coronary disease sub-groups (double-vessel group and multi-vessel group) when compared with single-vessel sub-group (p < 0.05). CONCLUSIONS: Hypertensive subjects with stable angina might get benefit from the treatment of bisoprolol in improving endothelial function and the survival of myocardium.

Reversal of physiological stress-induced resistance to topoisomerase II inhibitors using an inducible phosphorylation site-deficient mutant of I kappa B alpha.

Physiological stress conditions associated with the tumor microenvironment play a role in resistance to anticancer therapy. In this study, treatment of EMT6 mouse mammary tumor cells with hypoxia or the chemical stress agents brefeldin A (BFA) or okadaic acid (OA) causes the development of resistance to the topoisomerase II inhibitor etoposide. The mechanism of physiological stress-induced drug resistance may involve the activation of stress-responsive proteins and transcription factors. Our previous work shows that treatment with BFA or OA causes activation of the nuclear transcription factor NF-kappa B. Pretreatment with the proteasome inhibitor carbobenzyoxyl-leucinyl-leucinyl-leucinal inhibits stress-induced NF-kappa B activation and reverses BFA-induced drug resistance. To test whether NF-kappa B specifically mediates stress-induced drug resistance, an inducible phosphorylation site-deficient mutant of I kappa B alpha (I kappa B alpha M, S32/36A) was introduced into EMT6 cells. In this study, we show that I kappa B alpha M expression inhibits stress-induced NF-kappa B activation and prevents BFA-, hypoxia-, and OA-induced resistance to etoposide. These results indicate that NF-kappa B activation mediates both chemical and physiological drug resistance to etoposide. Furthermore, they imply that coadministration of agents that inhibit NF-kappa B may enhance the efficacy of topoisomerase II inhibitors in clinical cancer chemotherapy.

The pharmacological phenotype of combined multidrug-resistance mdr1a/1b- and mrp1-deficient mice.

Two major classes of plasma membrane proteins that actively extrude a wide range of structurally diverse hydrophobic amphipathic antineoplastic agents from cells, with different mechanisms of action, lead to multidrug resistance. To study the importance of these ATP-binding cassette transporters to the toxicity of cancer chemotherapy agents, we have used mice genetically deficient in both the mdr1a and mdr1b genes [mdr1a/1b(-/-) mice], the mrp1 gene [mrp1(-/-) mice], and the combined genes mdr1a/1b and mrp1 [mdr1a/1b(-/-), mrp1(-/-) mice] and embryonic fibroblasts derived from wild-type mice and from the three gene knockout animals. The consequences of export pump deficiencies were evaluated primarily using vincristine and etoposide. Mice deficient in the three genes, mdr1a/1b and mrp1, exhibited a 128-fold increase in toxicity to vincristine and a 3-5-fold increase in toxicity to etoposide; increased toxicity to embryonic fibroblast cells from triple knockout mice also occurred with vincristine and etoposide. Vincristine, which normally does not express toxicity to the bone marrow and to the gastrointestinal mucosa when used at therapeutic doses, caused extensive damage to these tissues in mdr1a/1b(-/-), mrp1(-/-) mice. The findings indicate that the P-glycoprotein and mrpl are compensatory transporters for vincristine and etoposide in the bone marrow and the gastrointestinal mucosa and emphasize the potential for increased toxicities by the combined inhibition of these efflux pumps.

Triplet-state energies and substituent effects of excited aroyl compounds in the gas phase.

Triplet-state energy values obtained from the gas phase are still scarce. In this study, the triplet-state energies of 58 aroyl compounds, introduced as gas chromatographic peaks at atmospheric pressure and typically 473 K, have been determined from the 0-0 bands of their n --> pi* type phosphorescence spectra in excited nitrogen. Correlations of those gas-phase triplet-state energies with Hammett constants could be observed for substituted acetophenones, benzaldehydes and benzophenones.

Quenching and enhancement of aroyl luminescence in excited nitrogen.

This study investigates both decreases and increases of aromatic carbonyl phosphorescence in excited nitrogen, i.e., in a gas-chromatographic device called the aroyl luminescence detector (ALD). The ALD responds, with nigh specificity, to subpicogram amounts of strongly phosphorescing aroyls. Aroyl response may, however, be quenched by coeluting peaks or gaseous impurities. This deleterious effect has been investigated with O2, H2, CH4, and C3H8 as model quenchers. Aroyl phosphorescence is more severely quenched than the nitrogen background, i.e., the so-called second-positive system, N2 (C 3 pi u)-->N2 (B 3 pi g). Oxygen, while being the strongest among the tested quenchers of aroyl phosphorescence, is the weakest quencher of nitrogen emission. The efficiency of various quenchers is similar for aroyl compounds of similar structure. It differs, however--though not by more than a factor of 2--among aroyls of different chemical types. In contrast to these intensity-reducing effects, aroyl phosphorescence is significantly enhanced by the addition of argon to (the carrier and excitation gas) nitrogen. It is proposed that the reaction sequence Ar*(3P0,2) + N2-->N2(C)*-->N2(B)* + hv-->N2(A)* + hv results in an increased yield of the metastable N2(A 3 sigma u+) state (this state being considered responsible for the n-->pi* excitation of aroyl compounds via an efficient triplet-triplet energy-transfer process).

Prostaglandin A1 inhibits stress-induced NF-kappaB activation and reverses resistance to topoisomerase II inhibitors.

Stress conditions associated with solid tumors lead to the formation of heterogeneous tumor cell subpopulations and insensitivity to cancer chemotherapeutics. In this report, we show that EMT6 mouse mammary tumor cells treated with the chemical stress, brefeldin A (BFA), or the physiological stress, hypoxia, develop resistance to the topoisomerase II (topoII) inhibitors teniposide and etoposide. BFA and hypoxia treatment did not alter intracellular drug concentrations, topoll protein levels, or inhibit topoII activity. BFA and hypoxia did cause the activation of the nuclear transcription factor NF-kappaB. We demonstrate that pretreatment with the synthetic cyclopentenone prostaglandin A1 (PGA1) inhibits stress-induced NF-kappaB activation and reverses BFA- and hypoxia-induced resistance. The reversal of BFA-induced resistance can occur when PGA1 is administered either before or several hours after the induction of stress. Taken together, these data support the involvement of NF-kappaB in stress-induced drug resistance, show that pharmacologic inhibitors of NF-kappaB can disrupt the biological consequences of stress, and imply that inhibitors of NF-kappaB may be useful agents to enhance the clinical efficacy of topoII-directed chemotherapeutics.

Gas chromatographic determination of primary alcohols as their formylbenzoic esters by gas-phase luminescence detection.

A new and convenient method is described for the derivatization of primary alcohols with p-formylbenzoyl chloride, and the sensitive photometric detection of the resulting formylbenzoic ester derivatives based on their gas-phase luminescence in excited nitrogen. The coupling reaction proceeds rapidly and quantitatively, and the formylbenzoic esters show good GC properties. The minimum detectable amounts of the derivatized alcohols, at a signal three-times the peak-to-peak noise, lie between 10 and 100 pg per injection, and their linear ranges cover approximately three-orders of magnitude.

Prevention of brefeldin A-induced resistance to teniposide by the proteasome inhibitor MG-132: involvement of NF-kappaB activation in drug resistance.

Brefeldin A, an agent that disrupts protein transport from the endoplasmic reticulum to the Golgi, induces the expression of GRP78 and the activation of nuclear factor (NF)-kappaB in cells. Treatment of cells with brefeldin A causes the development of resistance to topoisomerase II-directed agents, such as etoposide and doxorubicin. In this study, we show that treatment of EMT6 mouse mammary tumor cells with brefeldin A strongly induces GRP78 mRNA (8.5-fold) and resistance to teniposide (VM26). Treatment with okadaic acid causes a minor increase in GRP78 mRNA (2.1-fold) yet still induces resistance to VM26 as effectively as brefeldin A. In contrast, cells treated with castanospermine show a moderate increase in GRP78 mRNA (3.9-fold) but no resistance to VM26. These data imply that GRP78 induction does not mediate the development of drug resistance. An alternative mechanism of drug resistance may involve activation of the transcription factor, NF-kappaB, and we show that both brefeldin A and okadaic acid activate NF-kappaB in EMT6 cells. Furthermore, we demonstrate that treatment with the proteasome inhibitor MG-132 blocks the activation of NF-kappaB and prevents the development of resistance to VM26 induced by brefeldin A. Collectively, these results suggest that the resistance to VM26 in EMT6 cells treated with brefeldin A is mediated by the activation of NF-kappaB rather than the induction of GRP78. Our results also suggest that inhibition of NF-kappaB activation in tumor cells may increase the efficacy of topoisomerase II-directed agents in chemotherapy.

Hormonal regulation of glutamine metabolism by OK cells.

The precise mechanism(s) of action of PTH, insulin or glucagon in the regulation of renal glutamine and ammonia metabolism is unknown. Our aim was to delineate the effects and the site(s) of action of these hormones on renal glutamine metabolism. Experiments were carried out using OK cells as a model system. Cell cultures were incubated for three hours in a bicarbonate buffer of pH 7.4 supplemented with either 1 mM [2-15N] or [5-15N] glutamine and 10(-7) M PTH, insulin or glucagon. Comparative studies were performed at pH 6.8, 7.4 or 7.6 without hormone. PTH and acute acidosis significantly stimulated glutamine metabolism via both the phosphate-dependent glutaminase (PDG) and glutamate dehydrogenase (GLDH) pathways. The opposite was observed at pH 7.6. Insulin augmented flux via PDG with little effect on the GLDH pathway. Glucagon had insignificant effects on either PDG or GLDH pathways. Intracellular [15N] glutamate formed from [2-15N] glutamine was removed partially by transamination to alanine, aspartate and serine and partially by translocation to an extracellular compartment. Acidosis, PTH and insulin enhanced the formation of [15N] alanine with little effect on [15N] aspartate. PTH, insulin and glucagon significantly stimulated the production of [15N]serine, whereas acidosis had little effect. The translocation of intracellular glutamate was significantly increased by acidosis, PTH and insulin and decreased by acute alkalosis. The data indicate that: (a) PTH mimicks the effect of acute acidosis on renal glutamine metabolism, that is, augmented glutamine metabolism through both PDG and GLDH pathways and stimulated the output of intracellular glutamate. This effect might be mediated via decreased activity of the Na(+)-H+ exchanger associated with cellular acidification and/or through a second messenger; (b) insulin, but not glucagon, increased glutamine uptake and metabolism, and simultaneously enhanced output of intracellular glutamate sufficiently to stimulate the PDG pathway; and (c) overall, glucagon had little effect on glutamine metabolism by OK cells compared with either PTH or insulin.

Interrelationships of leucine and glutamate metabolism in cultured astrocytes.

The aim was to study the extent to which leucine furnishes alpha-NH2 groups for glutamate synthesis via branched-chain amino acid aminotransferase. The transfer of N from leucine to glutamate was determined by incubating astrocytes in a medium containing [15N]leucine and 15 unlabeled amino acids; isotopic abundance was measured with gas chromatography-mass spectrometry. The ratio of labeling in both [15N]glutamate/[15N]leucine and [2-15N]glutamine/[15N]leucine suggested that at least one-fifth of all glutamate N had been derived from leucine nitrogen. At the same time, enrichment in [15N]leucine declined, reflecting dilution of the 15N label by the unlabeled amino acids that were in the medium. Isotopic abundance in [15N]isoleucine increased very quickly, suggesting the rapidity of transamination between these amino acids. The appearance of 15N in valine was more gradual. Measurement of branched-chain amino acid transaminase showed that the reaction from leucine to glutamate was approximately six times more active than from glutamate to leucine (8.72 vs. 1.46 nmol/min/mg of protein). However, when the medium was supplemented with alpha-ketoisocaproate (1 mM), the ketoacid of leucine, the reaction readily ran in the "reverse" direction and intraastrocytic [glutamate] was reduced by approximately 50% in only 5 min. Extracellular concentrations of alpha-ketoisocaproate as low as 0.05 mM significantly lowered intracellular [glutamate]. The relative efficiency of branched-chain amino acid transamination was studied by incubating astrocytes with 15 unlabeled amino acids (0.1 mM each) and [15N]glutamate. After 45 min, the most highly labeled amino acid was [15N]alanine, which was closely followed by [15N]leucine and [15N]isoleucine. Relatively little 15N was detected in any other amino acids, except for [15N]serine. The transamination of leucine was approximately 17 times greater than the rate of [1-14C]leucine oxidation. These data indicate that leucine is a major source of glutamate nitrogen. Conversely, reamination of alpha-ketoisocaproate, the ketoacid of leucine, affords a mechanism for the temporary "buffering" of intracellular glutamate.