Biomedical subjects

J Wen

Publications and source records attributed to J Wen.

At least 19 recordsLinked to original sources

PubMed2006

High performance liquid chromatographic determination of a new antifungal compound, ADKZ in rat plasma.

A high performance liquid chromatography (HPLC) method was developed and validated for the determination of ADKZ (1-(1H-1,2,4-triazole)-2-(2,4-diflurophenyl) -3-[N-methyl-N-(4-iodo-benzyl)amino]-2-propanol) in rat plasma. The compound was extracted from plasma samples by liquid-liquid extraction, and an isomeric compound of ADKZ (1-(1H-1,2,4-triazole)-2-(2,4-diflurophenyl)-3-[N-methyl-N -(3-iodo-benzyl)amino]-2-propanol) was used as the internal standard (IS), which were analyzed on a reversed-phase C18 column (5 microm, 200 mm x 4.6 mm i.d.). The extracted plasma samples were eluted with acetonitrile-0.018 M triethylamine solution adjusted to pH 3.2 with phosphoric acid (35:65, v/v). The effluent was monitored by a UV detector at 230 nm. The retention time of ADKZ was 7.1 min and IS 8.2 min. The calibration curves were linear in the concentration range of 0.02-2.00 microg/ml with the correlation coefficients greater than 0.999. The quantification limit of ADKZ in rat plasma was 0.02 microg/ml. Intra- and inter-day precision ranged from 2.6 to 7.9% and 3.1 to 9.6%, respectively. The extraction recovery from plasma was no less than 80%. No endogenous interferences were observed with either ADKZ or IS. The method has been successfully used to support the pre-clinical pharmacokinetic studies of ADKZ in rats.

Acetonitriles↗

PubMed2006

High prevalence of albuminuria in population-based patients diagnosed with type 2 diabetes in the Shanghai downtown.

OBJECTIVE: The prevalence of albuminuria and the risk factors associated with albuminuria were evaluated among the Chinese patients diagnosed with type 2 diabetes aged over 30 in the Shanghai downtown. We also evaluated the variability of urinary albumin-to-creatinine ratio (ACR) among the three measurements and the relationship between diabetic retinopathy (DR) and albuminuria. METHODS: The 1039 Chinese patients diagnosed with type 2 diabetes aged over 30 were investigated by randomized cluster sampling in the Shanghai downtown and 1018 patients were analyzed in this study. Body mass measurements including height, weight, waist circumference and hip circumference, resting blood pressure, fasting blood measures, urinary ACR and the digitally stored fundus images were investigated. The prevalence of albuminuria was calculated and the risk factors associated with albuminuria were evaluated by stepwise logistic regression. The concordance of urinary ACR was evaluated by observed agreement. The relationship between albuminuria and DR was also evaluated. RESULTS: (1) The mean age of all patients was 66.10+/-11.54 years and the duration of diabetes was 7.89+/-7.16 years. (2) The prevalence of albuminuria was 49.6% among the Chinese patients diagnosed with type 2 diabetes aged over 30 in the Shanghai downtown, 41.4% with microalbuminuria and 8.2% with macroalbuminuria. (3) Microalbuminuria was significantly associated with systolic blood pressure, gender and waist circumference. Macroalbuminuria was significantly associated with systolic blood pressure and duration of diabetes. (4) Observed agreement among the three urinary ACR measurement for albuminuria staging was 73.3% (first versus second), 64.5% (first versus third) and 77.5% (second versus third). Observed agreement in the albuminuria staging between the single urinary ACR measurement and all three urinary ACR measurements was 85.8% (first versus all three), 87.6% (second versus all three) and 81.9% (third versus all three). (5) The percentage of DR in the macroalbuminuric group (59.2%) was significantly higher than that in the normalbuminuria group (16.1%) and microalbuminuria group (24.6%). (6) The macroalbuminuric patients with DR had significantly increased fasting blood glucose and HbA1c compared with the macroalbuminuric patients without DR. CONCLUSION: The prevalence of microalbuminuria observed in the Chinese patients diagnosed with type 2 diabetes aged over 30 in the Shanghai downtown reached up to 41.4% though the observations in our study might be representative of the diabetic patients of the Shanghai downtown. We agreed that at least two of the three urinary collections were done in a 3- to 6-month period because of the day-to-day variability in albumin excretion. The percentage of DR among the patients with macroalbuminuria was 59.2%, and the macroalbuminuric patients with the significantly high plasma glucose and DR were prone to diagnose DN.

Adult↗

PubMed2006

Evidence of angiogenesis and microvascular regression in autosomal-dominant polycystic kidney disease kidneys: a corrosion cast study.

Autosomal-dominant polycystic kidney disease (ADPKD) accounts for about 10% of all cases of chronic renal failure requiring dialysis. The disease is characterized by proliferation of renal epithelial cells and formation of cysts that expand over years and replace the normal parenchyma of the kidney. As the cysts grow, the volume of the kidney can increase by more than 10-fold, implying that remodeling and expansion of the vasculature must occur to provide oxygenation and nutrition to the cyst cells. Our previous studies support the notion that there is angiogenesis in ADPKD. We report here results from resin casting of ADPKD kidneys vasculature. In this study, the corrosion-casting method was used in conjunction with scanning electron microscopy to study the vascular architecture and the evidence for angiogenesis in ADPKD kidneys. We found a well-defined vascular network around the cysts forming a 'vascular capsule' somewhat similar to that described in avascular leiomyomata. We also found that the normal vascular architecture is lost and replaced by an assortment of capillaries of larger size than those in the normal kidney, mixed with flattened and spiral arterioles, damaged glomeruli, and atresic venules, indicative of regression of the microvasculature. In the same areas, there was capillary sprouting, considered the hallmark of angiogenesis. The present study documents regression changes of the vasculature and confirms the existence of angiogenesis in ADPKD kidneys, and suggests the use of inhibitors of angiogenesis as a possible avenue for the treatment of the disease.

Adult↗

PubMed2006

Refractoriness to short day lengths augments tonic and gonadotrophin-releasing hormone-stimulated lutenising hormone secretion.

Siberian hamsters (Phodopus sungorus) undergo reproductive involution following exposure to short winter day lengths. Following approximately 20 weeks of exposure to short day (SD) lengths, hamsters become refractory to the inhibitory effects of SD, and reproductive competence is restored in anticipation of spring. The extent to which changes in gonadal steroid-dependent and -independent regulation of gonadotrophin secretion participate in this vernal reactivation of the gonads is not known. This experiment tested whether tonic and gonadotrophin-releasing hormone (GnRH)-stimulated regulation of lutenising hormone (LH) secretion differs between photoresponsive and photorefractory Siberian hamsters. Male hamsters born into long day (LD) lengths were castrated or subjected to a sham-castration surgery at 17 days of age, implanted s.c. with blank or testosterone-filled capsules, and housed in LD or SD thereafter. Baseline LH and LH responses to GnRH (200 ng/kg, s.c) were measured at 14 (photoresponsive) and 40 (photorefractory) weeks of age. Despite lower circulating testosterone concentrations in gonadally regressed SD hamsters on week 14, tonic LH concentrations were comparable among all groups of gonad-intact hamsters on weeks 14 and 40; however, week 14 SD hamsters exhibited significantly higher GnRH-stimulated LH responses. Tonic LH concentrations were indistinguishable among all groups of castrated hamsters bearing empty implants on week 14, but prolonged exposure to LD led to a decrease in resting LH, whereas prolonged exposure to SD resulted in an increase in LH. In castrated hamsters bearing testosterone implants, baseline LH concentrations were comparable in all groups, but GnRH treatment resulted in significantly higher LH concentrations in photorefractory (week 40, SD) hamsters relative to all other groups. The data suggest that the development of photorefractoriness in Siberian hamsters is characterised by enhanced gonadal hormone-independent stimulation of LH secretion, and diminished sensitivity to inhibitory negative-feedback effects of testosterone on LH secretion. Decreases in responsiveness of gonadotrophin secretion to gonadal hormone negative feedback may contribute to the process of photorefractoriness and assist in maintaining the growth of reproductive organs during the process of gonadal recrudescence.

Adaptation, Physiological↗

PubMed2006

Rifampicin plus pyrazinamide versus isoniazid for treating latent tuberculosis infection: a meta-analysis.

SETTING: Six trials from Haiti, Mexico, the U.S.A., Brazil, Spain, Zambia and Hong Kong. OBJECTIVE: To evaluate the efficacy and safety of rifampicin plus pyrazinamide (RZ) vs. isoniazid (INH) for the prevention of tuberculosis (TB) among persons with or without human immunodeficiency virus (HIV) infection. DESIGN: Meta-analysis of randomised controlled trials (RCTs) and quasi-RCTs that compared RZ for 2-3 months with INH for 6-12 months. Endpoints were development of active TB, severe adverse effects and death. Treatment effects were summarised as risk difference (RD) with 95% confidence intervals (CI). RESULTS: Three trials conducted in HIV-infected patients and three trials conducted in non-HIV-infected persons were identified. The rates of TB in the RZ group were similar to those in the INH group, whether the subjects were HIV-infected or not (HIV-infected patients: pooled RD = 0%, 95% CI -1-2, P = 0.89; non-HIV-infected persons: pooled RD = 0%, 95% CI -2-1, P = 0.55). There was no difference in mortality between the two treatment groups (HIV-infected patients: pooled RD = -1%, 95% CI -4-2, P = 0.53; non-HIV-infected persons: pooled RD = 0%, 95% CI -1-1, P = 1.00). However, both subgroup analyses showed that a higher incidence of all severe adverse events was associated with 2RZ than INH among non-HIV-infected persons (RD = 29%, 95% CI 13-46, P = 0.0005 vs. RD = 7%, 95% CI 4-10, P < 0.0001). CONCLUSION: RZ is equivalent to INH in terms of efficacy and mortality in the treatment of latent tuberculosis infection. However, this regimen increases the risk of severe adverse effects compared with INH in non-HIV-infected persons.

Antibiotics, Antitubercular↗

PubMed2005

Chiral liquid chromatography resolution and stereoselective pharmacokinetic study of tetrahydropalmatine enantiomers in dogs.

A selective chiral high performance liquid chromatographic (HPLC) method coupled with achiral column was developed and validated to separate and quantify tetrahydropalmatine (THP) enantiomers in dog plasma. Chromatography was accomplished by two steps: (1) racemic THP was separated from biological matrix and collected on a Kromasil C18 column (150 mmx4.6 mm, 5 microm) with the mobile phase acetonitrile-0.1% phosphoric acid solution, adjusted with triethylamine to pH 6.15 (47:53); (2) enantiomeric separation was performed on a Chiralcel OJ-H column (250 mmx4.6 mm, 5 microm) with the mobile phase anhydrous ethanol. The detection wavelength was set at 230 nm. (+)-THP and (-)-THP were separated with a resolution factor (Rs) of at least 1.6 and a separation factor (alpha) greater than 1.29. Linear calibration curves were obtained over the range of 0.025-4 microg/ml in plasma for each of (+)-THP and (-)-THP (R2>0.999) with a limit of detection (LOD) of 0.005 microg/ml and the recovery was greater than 88% for each enantiomer. The relative standard deviation (R.S.D.) and relative error values were less than 10% at upper and lower concentrations. The method was used to determine the pharmacokinetics of THP enantiomers after oral administration of racemic THP. The results presented herein showed the stereoselective disposition kinetics of THP in dogs and were a further contribution to the understanding of the kinetic behavior of THP analogues.

Animals↗

PubMed2005

A T-786C polymorphism in 5'-flanking region of the endothelial nitric oxide synthase gene and endothelium-dependent arterial dilation in Type 2 diabetes.

OBJECTIVE: Several studies have shown that the T-786C polymorphism in 5'-flanking region of the endothelial nitric oxide synthase (eNOS) gene is associated with coronary artery disease in non-diabetic population. In the present study, we attempted to assess whether the T-786C polymorphism of eNOS gene is associated with endothelial dysfunction Type 2 diabetes. RESEARCH DESIGN AND METHODS: A total of 162 Type 2 diabetic men were studied. PCR/allele-specific probes were used to analyse the T-786C polymorphism of eNOS gene, and high resolution ultrasound was used to measure brachial artery diameter at rest, after reactive hyperaemia and after sublingual glyceryltrinitrate. RESULTS: The flow-mediated arterial dilation among subjects with T/C or C/C was 3.73+/-0.50%, which was significantly lower than that in subjects with T/T (4.15+/-0.49%) (P=0.000). On multiple linear regression analysis, the presence of C allele, mean blood pressure, low-density lipoprotein (LDL) and serum lipoprotein (a) [Lp(a)] were independent determinants for reduced endothelium-dependent arterial dilation (R2=0.175, P=0.0021). The flow-mediated arterial dilation in smokers with T/C or C/C was significantly lower than that in smokers with T/T (P<0.001), but not in non-smokers. In addition, the presence of C allele, LDL and Lp(a) were independent determinants for reduced endothelium-dependent arterial dilation (R2=0.258, P=0.0017) in smokers, but not in non-smokers. CONCLUSION: The C allele of T-786C polymorphism of eNOS gene is a genetic risk factor for endothelial dysfunction in Type 2 diabetic patients, especially among smokers.

Adult↗

PubMed2003

Associations of lead biomarkers with renal function in Korean lead workers.

AIMS: To compare associations of lead biomarkers with renal function in current and former lead workers. METHODS: Cross sectional analysis of first year results from a longitudinal study of 803 lead workers and 135 controls in South Korea. Clinical renal function was assessed by blood urea nitrogen (BUN), serum creatinine, and measured and calculated creatinine clearance. Urinary N-acetyl-beta-D-glucosaminidase (NAG) and retinol-binding protein were also measured. RESULTS: Mean (SD) tibia lead, blood lead, and DMSA chelatable lead levels in lead workers were 37.2 (40.4) micro g/g bone mineral, 32.0 (15.0) micro g/dl, and 767.8 (862.1) micro g/g creatinine, respectively. Higher lead measures were associated with worse renal function in 16/42 models. When influential outliers were removed, higher lead measures remained associated with worse renal function in nine models. An additional five associations were in the opposite direction. Effect modification by age was observed. In 3/16 models, associations between higher lead measures and worse clinical renal function in participants in the oldest age tertile were significantly different from associations in those in the youngest age tertile which were in the opposite direction. Mean urinary cadmium (CdU) was 1.1 micro g/g creatinine (n = 191). Higher CdU levels were associated with higher NAG. CONCLUSIONS: These data suggest that lead has an adverse effect on renal function in the moderate dose range, particularly in older workers. Associations between higher lead measures and lower BUN and serum creatinine and higher creatinine clearances may represent lead induced hyperfiltration. Environmental cadmium may also have an adverse renal impact, at least on NAG.

Adolescent↗

PubMed2003

Does solvent influence the ground-state tautomeric population of hypericin?

Nuclear magnetic resonance measurements indicate that hypericin exists in the same "normal" tautomeric form irrespective of whether the solvent is dimethyl sulfoxide or tetrahydrofuran. This result is discussed in the context of previous experimental and theoretical work. It is concluded that solvent perturbations cannot induce tautomerization in hypericin.

Anthracenes↗

PubMed2001

Determination of carbohydrate contents from excess light scattering.

In the light scattering technique, glycosylation gives rise to excess light scattering for glycoproteins. Assuming additivity of refractive index and using an appropriate refractive index increment for carbohydrate, one can determine the degree of glycosylation from the excess light scattering. Here we have used size-exclusion chromatography in combination with online light scattering, UV absorbance, and refractive index. The results show that the technique accurately determines the carbohydrate content of recombinant stem cell factor.

Animals↗

PubMed2001

8-oxo-dGTPase, which prevents oxidative stress-induced DNA damage, increases in the mitochondria from failing hearts.

BACKGROUND: Reactive oxygen species (ROS) can cause an oxidative modification of nucleotides, such as 8-oxo-7,8-dihydrodeoxyguanosine triphosphate (8-oxo-dGTP), which can lead to defects in DNA replication. The misincorporation of 8-oxo-dGTP into DNA is prevented by 8-oxo-dGTPase, which hydrolyzes 8-oxo-dGTP into 8-oxo-dGMP. The changes in this defensive system have not yet been examined in failing hearts, in which the generation of ROS increases. METHODS AND RESULTS: Myocardial infarction (MI) was created in mice by ligating the left coronary artery. Four weeks later, the left ventricle was dilated and contractility was diminished on echocardiography. The generation of ROS, as measured by electron spin resonance spectroscopy with 4-hydroxy-2,2,6,6-tetramethyl-piperidine-N-oxyl, increased in the noninfarcted left ventricle from MI mice. The formation of thiobarbituric acid-reactive substances also increased in the mitochondria from MI mice. 8-Oxo-dGTPase was detected in the mitochondrial fractions isolated from MI mice using a Western blot analysis with an antibody to its human homologue (hMTH1). Immunohistochemistry showed positive staining for hMTH1 was localized in the cardiac myocytes. CONCLUSIONS: The level of 8-oxo-dGTPase increased in the mitochondria isolated from post-MI hearts as oxidative stress increased, thus suggesting that a preventive mechanism is activated against ROS-induced DNA damage. As a result, 8-oxo-dGTPase is considered a useful marker of mitochondrial oxidative stress in heart failure.

Animals↗

PubMed2001

B7H costimulates clonal expansion of, and cognate destruction of tumor cells by, CD8(+) T lymphocytes in vivo.

B7H/B7RP (hereby called B7H) is a new member of the B7 family of costimulatory molecules and interacts with inducible costimulatory molecule (ICOS). Its function for CD8 T cells has not been reported. We report here that expression of B7H on the tumor cells reduced tumorigenicity and induced immunity to subsequent challenge with parental tumor cells. The immune protection correlates with an enhanced cytotoxic T lymphocyte (CTL) response against P1A, the major tumor antigen expressed in the J558 tumor. To understand the mechanism of immune protection, we adoptively transferred transgenic T cells specific for tumor antigen P1A into mice that bore P1A-expressing tumors. We found that while the transgenic T cells divided faster in mice bearing the B7H(+) tumors, optimal B7H-induced clonal expansion of P1CTL required costimulation by B7-1 and B7-2 on the endogenous host antigen-presenting cells (APCs). Interestingly, when B7H(+) and B7H(-) tumors were coinjected, P1CTL selectively eliminated the B7H(+) tumor cells. Moreover, B7H expressed on the tumor cells made them highly susceptible to destruction by CTL in vivo, even if the CTL was administrated into mice with large tumor burdens. Tumors that recurred in the P1CTL-treated mice lost transfected B7H and/or H-2L(d), the class I molecule that presents the P1A peptide. Taken together, our results reveal that B7H costimulates clonal expansion of, and cognate destruction by CD8(+) T lymphocytes in vivo.

Animals↗

PubMed2001

On the site and mode of antigen presentation for the initiation of clonal expansion of CD8 T cells specific for a natural tumor antigen.

Because of the low frequency of antigen-specific T cells, early events in the activation of tumor-specific T cells in vivo have not been well characterized. There is still no direct documentation on where the clonal expansion begins and how tumor antigens are presented to the host CD8 T cells to initiate it. Here we used transgenic T cells specific for a natural tumor antigen P1A to evaluate the kinetics, location, and modes of antigen presentation for initiating CTL response in vivo. Our results demonstrate that the initial activation of P1A-specific T cells takes place in the lymphoid organs. The activated T cells then migrate into tumors, where they undergo accelerated division and acquire distinct activation markers. The site of initiation cannot be altered by either local expression of costimulatory molecules or by intratumor injection of naïve T cells. Moreover, using genetic models that allow only one mode of antigen presentation, we show here that both cross-presentation of P1A by the host antigen-presenting cells, and direct antigen presentation and costimulation by the tumor cells are sufficient to initiate rapid T cell-clonal expansion in the lymphoid organ. These results provide direct evidence for two fundamental assumptions on the mechanisms of T-cell activation in vivo.

Animals↗

PubMed2001

Enhanced generation of reactive oxygen species in the limb skeletal muscles from a murine infarct model of heart failure.

BACKGROUND: The generation of reactive oxygen species (ROS) is enhanced in the failing myocardium. We hypothesized that ROS were also increased in the limb skeletal muscles in heart failure. Methods and Results-- Myocardial infarction (MI) was created in mice by ligating the left coronary artery. After 4 weeks, the left ventricle was dilated and contractility was diminished by echocardiography. Left ventricular end-diastolic pressure was elevated after MI in association with an increase in lung weight/body weight and the presence of pleural effusion. The generation of ROS in the limb muscles, including the soleus and gastrocnemius muscles, which were excised after MI, was measured by electron spin resonance spectroscopy with 4-hydroxy-2,2,6,6-tetramethyl-piperidine-N-oxyl (hydroxy-TEMPO). Overall, generation was increased, but it was attenuated in the presence of dimethylthiourea or 4,5-dihydroxy-1,2-benzenedisulfonic disodium salt in the reaction mixture, indicating increased generation of hydroxyl radicals originating from superoxide anion. Thiobarbituric acid-reactive substance formation was also increased in muscles after MI. Mitochondrial complex I and III activities were both decreased after MI, which may have caused the functional uncoupling of the respiratory chain and ROS production. Antioxidant enzyme activities, including superoxide dismutase, catalase, and glutathione peroxidase, were comparable between groups. CONCLUSIONS: Skeletal muscle in post-MI heart failure expressed an increased amount of ROS in association with ROS-mediated lipid peroxidation. This supports the hypothesis that oxidative stress may cause (at least in part) skeletal muscle dysfunction in heart failure.

Animals↗

PubMed2001

The E2F6 transcription factor is a component of the mammalian Bmi1-containing polycomb complex.

The E2F transcription factors play a key role in the regulation of cellular proliferation and terminal differentiation. E2F6 is the most recently identified and the least well understood member of the E2F family. It is only distantly related to the other E2Fs and lacks the sequences responsible for both transactivation and binding to the retinoblastoma protein. Consistent with this finding, E2F6 can behave as a dominant negative inhibitor of the other E2F family members. In this study, we continue to investigate the possible role(s) of E2F6 in vivo. We report the isolation of RYBP, a recently identified member of the mammalian polycomb complex, as an E2F6-interacting protein. Mapping studies indicate that RYBP binds within the known "repression domain" of E2F6. Moreover, we demonstrate that endogenous E2F6 and polycomb group proteins, including RYBP, Ring1, MEL-18, mph1, and the oncoprotein Bmi1, associate with one another. These findings suggest that the biological properties of E2F6 are mediated through its ability to recruit the polycomb transcriptional repressor complex.

Animals↗

PubMed2001

NASA/American Cancer Society High-Resolution Flow Cytometry Project - III. Multiparametric analysis of DNA content and electronic nuclear volume in human solid tumors.

BACKGROUND: The NASA/American Cancer Society (ACS) flow cytometer can simultaneously measure electronic nuclear volume (ENV) and DNA content of nuclei. The preceding articles in this volume ("NASA/American Cancer Society High-Resolution Flow Cytometer Project-I") described the schematics, performance, and procedures used for the preparation of nuclei for analysis on this unit. In the present article, we describe the analysis of selected human tumors using the ratio of ENV/DNA content (nuclear packing efficiency [NPE]). METHODS: Tumor specimens (frozen) were minced with scalpels and stained with 1-10 microg/ml of 4',6-diamidino-2-phenylindole (DAPI) dihydrochloride at pH 6.0-7.2. Trout erythrocytes were used as internal standards. Data on ENV and DNA content were collected in list mode files. Propidium iodide-stained nuclei, analyzed on a Coulter XL cytometer, were used for comparison. RESULTS: Simultaneous measurement of ENV and DNA makes it possible to discriminate between hypodiploid or hyperdiploid tumor cells, as well as to differentiate between near-diploid aneuploid and diploid cells on the basis of their increased ENV. The NPE ratio is a valuable parameter for the detection of small quantities of tumor cells, separating overlapping diploid and aneuploid populations for cell cycle analysis and characterizing the level of differentiation in some tumors. CONCLUSION: NPE analysis provides unique measuring capabilities for the study of human solid tumors by flow cytometry.

American Cancer Society↗

PubMed2001

NASA/American Cancer Society High-Resolution Flow Cytometry Project - II. Effect of pH and DAPI concentration on dual parametric analysis of DNA/DAPI fluorescence and electronic nuclear volume.

BACKGROUND: In the present paper, we describe the effect of 4', 6-diamidino-2-phenylindole (DAPI) dihydrochloride concentration and pH on the resolution of DNA distribution histograms generated by dual-parametric simultaneous analysis of DNA content and electronic nuclear volume (ENV). METHODS: Nuclei from tissue culture cell lines and frozen human solid tumors were isolated in nuclear isolation media containing different concentrations of DAPI, at various pH levels, and analyzed on a NASA/American Cancer Society (ACS) flow cytometer. Samples stained with propidium iodide/hypotonic citrate and analyzed in a Coulter XL flow cytometer were used for comparison. RESULTS: Nuclei stained with DAPI concentration of 1-3 microg/ml, pH 6.0, gave the best resolution for the detection of the near-diploid and near-tetraploid populations. Simultaneous use of ENV and DAPI/DNA fluorescence under these conditions identified subpopulations that otherwise could not be detected by DNA analysis alone. CONCLUSIONS: Staining at 1-3 microg/ml DAPI, pH 6.0, was optimal for the detection of aneuploid populations, especially the near-diploid and/or near-tetraploid populations in human tumors.

American Cancer Society↗

PubMed2001

Transfer of cobalamin from intrinsic factor to transcobalamin II.

The process is obscure by which cobalamin (Cbl) in the endocytosed intrinsic factor (IF)-cobalamin (Cbl) complex is released and transferred to transcobalamin II (TCII) within the enterocyte. Using recombinant IF and TCII, binding of Cbl to IF at pH 5.0 was 70% of binding at pH 7.0, whereas for TCII alone, the value was only 12%. TCII binding activity was lost rapidly at lower pH, but this was not due to protease action. TCII incubated at pH 5.0 with cathepsin L was degraded and could not subsequently bind Cbl. Thus, transfer from IF to TCII is unlikely to occur within an acid compartment. Only 13-15% of bound Cbl was released at pH 5.0 and pH 6.0 from either rat IF, human IF, or human TCII. The K(a) of human or rat IF at pH 7.5 was 2.2 nM; for TCII, the value was 0.34 nM. At pH 7.5, Cbl transfers from IF to TCII, but only to a limited extent (21%), as detected by nondenaturing electrophoresis. Transfer of Cbl from IF to TCII could not be demonstrated at pH values of 5.0 or 6.0. Thus, luminal transfer of Cbl between IF and TCII is likely to be limited, but is possible. The most likely mechanism for intracellular transfer of Cbl from IF to TCII involves initial lysosomal proteolysis of IF, with subsequent Cbl binding to TCII in a more neutral cellular compartment.

Journal Article↗