Application value of transcranial contrast-enhanced ultrasonography in evaluating middle cerebral artery stenosis.

OBJECTIVE: To detect the display rate and flow velocity of intracranial circle of Willis (anterior, middle, and posterior cerebral arteries) with transcranial contrast-enhanced transcranial color-coded sonography (CE-TCCS), using digital subtraction angiography (DSA) as the golden diagnostic standard. PATIENTS AND METHODS: We collected data from 104 patients with suspected stroke treated in our hospital between December 2019 and October 2021. The detection rate of the intracranial circle of Willis, anterior cerebral artery (ACA), middle cerebral artery (MCA), and posterior cerebral artery (PCA) were analyzed based on routine TCCS and CE-TCCS data. Based on digital subtraction angiography (DSA) data, the degree of MCA stenosis was divided into mild stenosis (<50%), moderate stenosis (50-69%), severe stenosis (70-99%), and bilateral middle cerebral artery CE-TCCS examinations were performed. We evaluated MCA color blood flow on CE-TCCS, and recorded the peak systolic velocity (PSV), end-diastolic velocity (EDV), and mean flow velocity (MFV). RESULTS: The display rates of ACA, MCA, and PCA were significantly improved on the CE-TCCS, and the PSV, EDV and MFV of the MCA stenosis group were higher than those of the normal group. The flow velocity of each stenosis subgroup was increased compared to the normal group. The optimal cutoff values of normal and stenosis under the receiver operating characteristic (ROC) curve were PSV = 168.5 cm/s, EDV = 61.5 cm/s, and MFV = 110.5 cm/s. The optimal cutoff values for mild and moderate stenosis and for moderate and severe stenosis were PSV = 201.5 cm/s and 249.5 m/s, EDV = 95.2 cm/s and 141.5 cm/s, and MFV = 137.6 cm/s and 160.5 cm/s, respectively. PSV and MFV had the most significant sensitivity, specificity, and accuracy. CONCLUSIONS: Transcranial contrast-enhanced ultrasonography can improve the display rate of intracranial blood vessels and can accurately diagnose MCA stenosis.

[Genetic and clinical analysis of X-linked hypophosphatemic rickets].

Objective: To investigate the clinical and genetic features, and treatment of X-linked hypophosphatemic rickets (XLH). Methods: In this retrospective study, we reviewed the medical records of 25 pediatric patients with XLH who were admitted to Department of Endocrinology Genetics and Metabolism,Beijing Children's Hospital from January 2010 to January 2020. The clinical characteristics, PHEX gene variants, as well as clinical outcome of the patients were summarized. To analyze the correlation between genotype and phenotype, the patients were divided into different subgroups according to the location of the variants, including N-terminal-located vs. C-terminal-located variant, and Zn-binding domain exon 17 or 19 variant vs. non-exon 17 or 19 variant. The age at onset, height standard deviation score (HtSDS), intercondylar or intermalleolar distance, fasting serum phosphorus, and HtSDS and intercondylar or intermalleolar distance at the final follow-up were compared by rank sum test or t text. Results: Among the 25 children with XLH, 8 were boys and 17 were girls. The median age of onset was 1.2 (1.0, 1.8) years, and the median age of diagnosis was 2.5 (1.5, 4.3) years. The main clinical manifestations were abnormal gait and lower limb deformity. The HtSDS was -2.0(-3.2, -0.8), and the intercondylar or intermalleolar distance was 4.5 (3.0, 6.0) cm. The fasting serum phosphorus level was 0.8 (0.7, 0.9) mmol/L, while the serum alkaline phosphatase level was (721±41) U/L and the serum calcium level was (2.5±0.1) mmol/L. Three patients (12%) had parathyroid hormone levels above the upper limit of the normal range. Twenty-five patients (100%) showed radiographic changes of active rickets. Nephrocalcinosis was found in 2 cases (9%). Twenty-four different PHEX variations were detected in 25 patients, among whom 11 (44%) had not been reported previously. No hot spot variation was found. No statistical differences (all P>0.05) were identified in clinical features and outcomes either in comparing patients with N-terminal (21 cases) and C-terminal (4 cases) variants, or in comparing patients with variant located in exon 17 or 19 (4 cases) or not (21 cases). Twenty-four cases (96%) were treated regularly with phosphate supplements and active vitamin D. After 2.7 (1.6, 5.0) years of follow-up, clinical symptoms were relieved in 96% (24/25) of the patients. The HtSDS after treatment had no significant difference compared to that before treatment (-2.0(-3.2, -0.8) vs.-2.0(-2.8, -1.1),Z =-0.156, P>0.05), while the intercondylar or intermalleolar distance after treatment was significantly reduced compared to that before treatment (4.5(3.0, 6.0) vs. 1.5(0, 3.3) cm, Z =-3.043, P<0.05). Bone X-rays were reexamined in 17 cases after treatment, and radiographic signs of rickets were improved. Eighteen cases had secondary hyperparathyroidism and 7 cases had nephrocalcinosis. Conclusions: The main clinical manifestations of XLH are abnormal gait, lower limb deformity and short stature. A high proportion of novel variations of PHEX gene but no hot spot variation neither genotype-phenotype correlation are found. Regular treatment with phosphate supplements and active vitamin D can significantly improve the symptoms except for the height. However, the rate of adverse events including secondary hyperparathyroidism and nephrocalcinosis seems to be high.

[Diagnosis and follow-up of 2 cases of pediatric nephrogenic syndrome of inappropriate antidiuresis resulting from activating mutation in AVPR2 and literature review].

Objective: To analyze the clinical and genetic features, as well as the treatment outcomes of two boys with nephrogenic syndrome of inappropriate antidiuresis (NSIAD) caused by gain-of-function mutations in the V2 vasopressin receptor gene (AVPR2). Methods: The clinical manifestations, genetic testing, therapeutic interventions and the outcomes of two boys with NSIAD hospitalized in the Department of Endocrinology, Beijing Children's Hospital in April 2019 were reported. A literature search with "Nephrogenic syndrome of inappropriate antidiuresis" and "AVPR2 gene" as keywords was conducted at the China national knowledge infrastructure (CNKI), the Wanfang Data Knowledge Service Platform, PubMed and Springer Link up to May 2020. Relevant published articles were reviewed. Results: The two cases presented with chronic and severe hyponatremia with hypo-osmolality, inappropriately elevated urinary osmolality and urinary sodium levels. The onset age was 5.25-years and 2 months respectively. AVPR2 sequencing revealed a previously described hemizygous activating mutation (c.409C>T, p.R137C) in both of boys, each inherited the variant from their mother. Patient 1 limited fluid intake by himself in his daily life, intravenous and oral sodium supplementations showed no significant increase of serum sodium level. Oral furosemide increased the serum sodium level and maintained it within normal range. The serum sodium and potassium levels were in the normal range during the 1-year follow-up period with oral furosemide. The serum sodium level of Patient 2 increased with restricting fluid intake and with salt supplementation. However, after he experienced respiratory infection, the plasma sodium level decreased. Subsequently, oral anti-infection medicine and furosemide were applied. The serum sodium level increased two days later and remained at a normal range afterwards. The boy was 1 year old with normal growth. He stopped taking furosemide after 4 months while taking 1 gram of salt per day, the blood sodium level maintained at normal range. Literature search identified no reports in Chinese journals, whereas 50 publications were found in English journals. A total of 30 NSIAD probands were reported and 16 of those (53%) had childhood onset, most presented with seizures. The majority had a hotspot change at the nucleotide position of 409 in AVPR2. Nine cases had an amino acid change as R137C and five cases as R137L. Fluid restriction and oral urea intake were main treatment options, no report so far was found with oral furosemide treatment. Conclusions: NSIAD presented with hyponatremia without any other specific presentations. Genetic testing for variants in AVPR2 is helpful for early diagnosis and timely treatment. The first two cases of oral furosemide treatment were reported by the article which helped to maintain a normal serum sodium level after limiting fluid intake and supplementing sodium which showed limited effect.

[A rare case of cystadenoma in the small intestine].

In recent years, there have been more and more reports about cystadenoma. Cystadenoma can occur in many parts of the body, and cystadenoma in different parts may show different clinical symptoms, however, some patients with cystadenoma have no symptoms. The vast majority of cystadenomas are benign lesions, but a small number of cystadenomas can be malignant. For example, a small number of ovarian cystadenomas and pancreatic cystadenomas may be malignant. This study reported a patient with small intestinal cystadenoma diagnosed by pathology. The patient's physical examination revealed a lesion in the left upper abdomen. He had only abdominal distension and no other discomfort. His laboratory examination results were basically normal, i.e. blood routine, urine routine, stool routine, liver function, kidney function, myocardial enzyme, tumor marker, etc. The patient underwent sectional small intestine resection and the pathological sample was analyzed. The histological findings of the resected intestinal sample were consistent with cystadenoma. Computed tomography scan of the abdomen was performed 4 months after the surgery. No recurrence of the tumor was found. The patient recovered in good condition. By consulting the literature, I found very few reports of small intestinal cystadenoma before, it was very rare. This article described the clinical manifestation, diagnosis and differential diagnosis, treatment and prognosis of a case of small intestinal cystadenoma, it suggested that cystadenoma can occur in the small intestine, other than the ovary, pancreas, liver, lung, thyroid, prostate, seminal vesicle, skin, etc. The cystadenoma in small intestine is easy to be mistaken with other tumors, such as small intestine stromal tumor, small intestine adenocarcinoma, small intestine lipoma, small intestine hemangiomas, etc., and it is difficult to fully confirm through imaging examinations, such as computed tomography and magnetic resonance imaging. Laparotomy and histopathological examination are necessary before definitive diagnosis. This disease can be treated by small bowel resection at the affected region and good prognosis can be achieved.

[Effect of human Wharton's Jelly or adipose derived mesenchymal stem cells culture supernatant on endothelial cells angiogenesis].

Objective: To compare the curative effect of mesenchymal stem cells derived from human Wharton's Jelly(WJ-MSC) or adipose(AD-MSC) culture supernatant on endothelial cells angiogenesis. Methods: WJ-MSC and AD-MSC were isolated, identified, and the culture supernatant of stem cells was collected.The WJ-MSC or AD-MSC supernatant co-cultured with the endothelial cells. The expression levels of pro-angiogenic and anti-angiogenic genes of endothelial cells were assessed using qRT-PCR analysis, and the effects of stem cell culture supernatant on angiogenesis were evaluated by performing a tube formation assay in vitro. Results: After adding WJ-MSC and AD-MSC culture supernatant, the expression levels of pro-angiogenic genes in endothelial cells were upregulated, and the expression levels of anti-angiogenic genes were downregulated significantly in both experimental groups compared to the control group (P<0.01), and tube formation of endothelial cells was also significantly increased in both experimental groups as determined by the increase of the tube length ((43.2±9.2) mm vs (94.3±13.2)mm, (86.1±7.2)mm, P<0.01). Conclusion: The results showed that AD-MSC culture supernatant can promote endothelial cells angiogenesis and its curative effect is similar to that of WJ-MSC.

Effects of compound porcine cerebroside and ganglioside on neurotoxicity caused by oxaliplatin chemotherapy: preliminary results.

OBJECTIVE: Oxaliplatin has shown good anti-tumour activity in the treatment of tumours involving the digestive system. However, its application is limited because of severe neurotoxicity in some patients. The purpose of this study was to evaluate whether compound porcine cerebroside and ganglioside (CPCG) can reduce or prevent oxaliplatin-induced neurotoxicity. PATIENTS AND METHODS: Patients with digestive system tumour who received oxaliplatin-based chemotherapy were retrospectively divided into experimental and control groups according to the receipt of CPCG during chemotherapy. Adverse events at the end of each chemotherapy cycle were recorded. We compared the incidence of neurotoxicity between the two groups and graded the neurotoxicity symptoms using the Common Terminology Criteria for Adverse Events v5.0. RESULTS: The study included 115 patients (experimental group, 57; control group, 58). The number of chemotherapy cycles (6.65 vs. 6.41, p=0.540) and oxaliplatin dose (775.92 mg/m2 vs. 724.20 mg/m2, p=0.250) were comparable between the two groups. All patients developed grade 1 to 3 neurotoxicity; grade 4-5 neurotoxicity was not observed. The incidence of neurotoxicity and the probability of advanced neurotoxicity were significantly lower in the experimental group than in the control group (p<0.05). After a 6 to 18 months follow-up, the two groups showed no significant differences in the chemotherapy response and recurrence rate (p=0.846). CONCLUSIONS: CPCG reduces oxaliplatin-induced neurotoxicity without reducing the efficacy of oxaliplatin-based regimens; thus, it can be used for preventing oxaliplatin-induced neurotoxicity in patients with cancer.

Controlled reaction for improved CH3NH3PbI3 transition in perovskite solar cells.

Hybrid halide perovskites represent one of the most promising solutions toward the fabrication of all solid nanostructured solar cells, with improved efficiency and long-term stability. This article aims at investigating the properties of CH3NH3PbI3 with controlled loading time in CH3NH3I solution via a two-step sequential deposition and correlating them with their photovoltaic performances. It is found that the optimum PCE of the loading time in the CH3NH3I solution is possible only at a relatively short time (10 min). Prolonging the loading time will degrade the perovskite film, and deteriorate the device performance by introducing a large amount of excessive defects and recombination. However, even if the material band gap remains substantially unchanged, a suitable loading time can dramatically improve the charge transport within the perovskite layer, exhibiting the out-standing performances of meso-superstructured solar cells.

The delayed genotoxic effect of N-nitroso N-propoxur insecticide in mammalian cells.

The N-nitroso derivative of an extensively used insecticide, propoxur, consistently induced dose-responsive chromosome aberrations and sister-chromatid exchanges (SCEs) in Chinese hamster ovary (CHO-W8) cells. Further investigations indicated that post-treatment incubation with a regular 1.5-cell-cycle period did not offer an unbiased estimation of the genotoxicity of N-nitroso carbamate insecticides. The scale of chromosome aberration induction increased with extension of the post-treatment incubation period. Comparable phenomena were not found in CHO-AGT cells proficient for O(6)-methylguanine-DNA-methyltransferase. In CHO-W8 cells, pulsed-treatment of the insecticide in the 1st replication cycle showed higher SCE induction than in the 2nd cycle. Similar phenomenon was also found in SCE induced by N-nitroso derivatives from other carbamate insecticides including aldicarb, carbofuran and methomyl. Treated cells did not show significantly perturbed cell cycle progression until 12 h after treatment removal. Based on the above observations, the O(6)-methylguanine-DNA adduct is suggested to be the major lesion caused by the delayed genotoxic effect of N-methyl carbamate insecticides as described in this report.

Reduction of dietary magnesium by only 50% in the rat disrupts bone and mineral metabolism.

INTRODUCTION: The objective of this study was to determine the effect of a moderate reduction of dietary magnesium [50% of nutrient requirement (50% NR)] on bone and mineral metabolism in the rat, and to explore possible mechanisms for the resultant reduced bone mass. METHODS: Female rats were 6 weeks of age at the start of study. Serum magnesium (Mg), calcium (Ca), parathyroid hormone (PTH), 1,25(OH)(2)-vitamin D, alkaline phosphatase, osteocalcin, and pyridinoline were measured during the study at 3- and 6-month time points in control (dietary Mg of 100% NR) and Mg-deficient animals (dietary Mg at 50% NR). Femurs and tibias were also collected for mineral content analyses, micro-computerized tomography, histomorphometry, and immunohistochemical localization of substance P, TNFalpha, and IL-1beta at 3 and 6 months. RESULTS: Although no significant change in serum Mg was observed, Mg deficiency developed, as assessed by the reduction in bone Mg content at the 3- and 6-month time points (0.69+/-0.05 and 0.62+/-0.04% ash, respectively, in the Mg depletion group compared to 0.74+/-0.04 and 0.67+/-0.04% ash, respectively, in the control group; p=0.0009). Hypercalcemia did not develop. Although serum Ca level remained in the normal range, it fell significantly with Mg depletion at 3 and 6 months (10.4+/-0.3 and 9.6+/-0.3 mg/dl, respectively, compared to 10.5+/-0.4 and 10.1+/-0.6 mg/dl, respectively, in the control group; p=0.0076). The fall in serum Ca in the Mg-depleted animals was associated with a fall in serum PTH concentration between 3 and 6 months (603+/-286 and 505+/-302 pg/ml, respectively, although it was still higher than the control). The serum 1,25(OH)(2)-vitamin D level was significantly lower in the Mg depletion group at 6 months (10.6+/-7.1 pg/ml) than in the control (23.5+/- 12.7 pg/ml) (p<0.01 by the t-test). In Mg-deficient animals, no difference was noted in markers of bone turnover. Trabecular bone mineral content gain was less over time in the distal femur with Mg deficiency at 3 and 6 months (0.028+/-0.005 and 0.038+/-0.007 g, respectively, compared to 0.027+/-0.004 and 0.048+/-0.006 g, respectively, in the control group; p<0.005). Histomorphometry at these time points demonstrated decreased trabecular bone volume (15.76+/-1.93 and 14.19+/-1.85%, respectively, compared to 19.24+/-3.10 and 17.30+/-2.59%, respectively, in the control group; p=0.001). Osteoclast number was also significantly increased with Mg depletion (9.07+/-1.21 and 13.84+/-2.06, respectively, compared to 7.02+/-1.89 and 10.47+/-1.33, respectively, in the control group; p=0.0003). Relative to the control, immunohistochemical staining intensity of the neurotransmitter substance P and of the cytokines TNFalpha and IL-1beta was increased in cells of the bone microenvironment in the Mg depletion group, suggesting that inflammatory cytokines may contribute to bone loss. CONCLUSION: These data demonstrate that Mg intake of 50% NR in the rat causes a reduced bone mineral content and reduced volume of the distal femur. These changes may be related to altered PTH and 1,25(OH)(2)-vitamin D formation or action as well as to an increase release of substance P and the inflammatory cytokines TNFalpha and IL-1beta.

Alterations in growth plate and articular cartilage morphology are associated with reduced SOX9 localization in the magnesium-deficient rat.

Insufficient dietary magnesium (Mg) intake has been associated with low bone mass in humans,and recent basic science studies have indicated that this bone loss may be secondary to increased release of substance P and TNFc Much less is known about the effects of low Mg intake on cartilage. We have evaluated growth plate and articular cartilage in rats following a 6 month dietary Mg restriction. Histomorphometry demonstrated significantly decreased distal femur articular cartilage chondrocyte density and decreased tibial growth plate width in experimental animals compared to controls. Growth plates of Mg-restricted animals showed reduced chondrocyte column formation. Extracellular matrix of both articular cartilage and growth plates in experimental animals contained reduced amounts of proteoglycans. Immunolocalization of Sox9 was decreased in both articular and growth plate cartilage in experimental animals compared to controls, suggesting that reduced Mg intake causes cartilage changes that may be secondary to reduced levels of the SOX9 transcription factor.

Magnesium deficiency: effect on bone and mineral metabolism in the mouse.

Insufficient dietary magnesium (Mg) intake has been associated in humans with low bone mass. Mg deficiency in the rat has suggested bone loss is due to increased bone resorption and/or inadequate bone formation during remodeling. The purpose of this study was to assess the effect of a low Mg diet on bone and mineral metabolism in the young and mature BALB/c mouse and explore the hypothesis that inflammatory cytokines may contribute to Mg deficiency-induced osteoporosis. Using an artificial diet, we induced targeted Mg depletion (0.002% Mg) with all other nutrients maintained at the normal level. In all Mg-depleted mice, hypomagnesemia developed and skeletal Mg content fell significantly. The serum Ca in Mg-deficient mice was higher than in control mice; however, serum PTH levels were not significantly different. Osteoprotegerin (OPG) in dosages that inhibit osteoclastic bone resorption did not prevent hypercalcemia in Mg-deficient animals. No significant difference in serum Ca was observed between groups when dietary Ca was reduced by 50%, suggesting that a compensatory increase in intestinal absorption might account for the hypercalcemia. Growth plate width decreased 33% in young Mg-deficient animals and chondrocyte columns decreased in number and length, suggesting that Mg deficiency reduced bone growth. Trabecular bone volume in the metaphysis of the tibia in these animals was decreased and osteoclast number was increased by 135%. Osteoblast number was significantly reduced. Immunohistochemistry revealed that substance P increased 230% and 200% in megakaryocytes and lymphocytes, respectively, after 1 day of Mg depletion. IL-1 increased by 140% in osteoclasts by day 3 and TNF alpha increased in osteoclasts by 120% and 500% in megakaryocytes on day 12. This study demonstrates a profound effect of Mg depletion on bone characterized by impaired bone growth, decreased osteoblast number, increased osteoclast number in young animals, and loss of trabecular bone with stimulation of cytokine activity in bone.

Separation of pure elemental and oxygen influenced signal in ELNES.

The energy loss near edge structure (ELNES) of many elements is strongly influenced by the presence of oxygen or other elements at surfaces, grain boundaries, or in the bulk material. The presented investigation deals mainly with the influence of oxygen at the surface. A method for the separation of both, the pure bulk signal and the oxidized surface signal, was evaluated and tested on Al, Cu, Mg, and Si. A comparison of experimental data with ab initio bandstructure calculations and other proofs of the accuracy of ELNES separation are presented. Influences of error propagations were tested and are exemplarily given for Al and Si.

Modulation of intraocular pressure by unilateral and forced unilateral nostril breathing in young healthy human subjects.

PURPOSE: To determine the effects of unilateral right/left nostril breathing (URNB/ULNB) and forced unilateral right/left nostril breathing (FURNB/FULNB) on intraocular pressure (IOP) and to examine the differences in the IOP during the various phases of nasal cycle. METHODS: Young healthy volunteers of either sex aged between 19-24 years, participated in the sessions using URNB/ULNB (n = 52) and FURNB/FULNB (n = 28). The nostril dominance was calculated from signals recorded on the PowerLab equipment, representing pressure changes at the end of the nostrils during respiration. The IOP was measured with Tono-Pen. The subjects were divided into 4 groups viz. right nostril dominant (RND), left nostril dominant (LND), transitional right nostril dominant (TRND) and transitional left nostril dominant (TLND) groups. The IOP data 'before and after' URNB/ULNB or FURNB/FULNB were compared by using paired t-test. The baseline data of IOP between the groups were analysed by using independent samples t-test. RESULTS: The URNB decreased the IOP in the LND and TLND (p < 0.01) and also in the RND (p < 0.05) groups but not significantly in the TRND group. The ULNB decreased the IOP in the RND group (p < 0.01) only. The FURNB significantly reduced the IOP (p < 0.05) only in the LND and RND groups. The FULNB decreased the IOP but not significantly. The baseline IOP did not differ significantly between the LND, RND, TLND and TRND groups. CONCLUSION: The URNB/FURNB reduced the IOP, while ULNB/FULNB failed to increase the IOP significantly. It is suggested that the lowering of IOP by URNB indicated sympathetic stimulation.

Genotoxic effects of triphenyltin acetate and triphenyltin hydroxide on mammalian cells in vitro and in vivo.

Two organotin pesticides, triphenyltin acetate (TPTA) and triphenyltin hydroxide (TPTH), were evaluated for their ability to induce micronuclei (MN) and sister chromatid exchange (SCE) in vitro using cultured Chinese hamster ovary (CHO) cells and in vivo BALB/c mouse erythrocytes. Both pesticides induced a dose-dependent increase but only TPTH induced a significant increase in MN at the highest dose (150 ng/ml) tested in CHO cells. With adding S9 microsomal fractions, both pesticides induced a meaningful MN induction at 150 ng/ml and a dose-dependent significant increase in SCE. In vivo MN induction in erythrocytes was conducted by treating BALB/c mice orally or intraperitoneally with these pesticides either in a single or triple treatments. Oral gavage (p.o.) of TPTA resulted in a dose-related significant increase of MN induction in peripheral blood and of TPTH induced a significant increase in micronucleated reticulocyte (MNRETs) only in a single treatment. Intraperitoneal administration of TPTA or TPTH, however, resulted in meaningless random increases in MN though these increases might be attributable to toxic effects. The MNRETs levels in the treatment with both pesticides were independent to the sampling time. This study demonstrated that TPTA and TPTH was potential chromosome mutagens.

Preparation for TEM of layered samples with fragile microstructure and weak layer interface.

The objective of this work was to prepare for transmission electron microscopy (TEM) a layered structure of materials with fragile microstructure. The samples consisted of two layers of different materials, silicon nitride and borosilicate glass, loosely bonded together. The low strength of the sample resulted in fragmentation during more conventional preparation. However, it was possible to prepare the fragments by mounting them in a titanium specimen carrier with aluminium strips as support. After grinding and polishing, a technique of low-angle ion milling was used to obtain electron beam transparent areas at the nitride/glass interface.

Altered pHi regulation in 3T3/CFTR clones and their chemotherapeutic drug-selected derivatives.

Recently (L. Y. Wei, M. J. Stutts, M. M. Hoffman, and P. D. Roepe. Biophys. J. 69: 883-895, 1996), 3T3 cells overexpressing the cystic fibrosis transmembrane conductance regulator (CFTR) were found to exhibit chemotherapeutic drug resistance and other traits of multidrug resistant (MDR) cells. In the present work, NIH 3T3/CFTR clones were selected with either doxorubicin or vincristine in incremental fashion to generate series of stable MDR cell lines that exhibit increasing levels of drug resistance. Thus C3D6 (grown in the presence of 600 nM doxorubicin) was selected from C3D4 (grown in the presence of 400 nM doxorubicin), which was selected from C3D1 (grown in the presence of 100 nM doxorubicin), which was in turn selected from the original 3T3/CFTR clone C3 (M. J. Stutts, S. E. Gabriel, J. C. Olsen, J. T. Gatzy, T. L. O'Connell, E. M. Price, and R. C. Boucher. J. Biol. Chem. 268: 20653-20658, 1993), which was not grown in the presence of chemotherapeutic drug. A similar series was generated via selection with vincristine. In both series, as well as series derived from a different CFTR clone, initial low-level drug selection increases CFTR expression without promoting MDR 1 or multidrug resistance-associated protein expression. On continued selection at higher drug concentrations, CFTR mRNA levels decrease while MDR 1 mRNA levels concomitantly increase. At each incremental step of selection, intracellular pH (pHi) increases (e.g., pHi of C3D6 > C3D4 > C3D1 > C3). Cl-/HCO3- exchange activity is significantly reduced in the drug-selected derivatives overexpressing MDR 1 but not the parental CFTR clones. The apparent set point of Na+/H+ exchange activity is significantly lower for the non-drug-selected 3T3/CFTR clones, relative to controls, but it increases on initial selection with chemotherapeutic drug. Overexpression of MDR 1 in the higher-level selectants does not appear to further perturb apparent Na+/H+ exchange. These data further describe how CFTR and MDR proteins may affect pHi regulation.

Ultramicrotomy of powder material for TEM/STEM study.

This paper summarizes methods conventionally used to prepare thin foil samples of powder materials for transmission electron microscopy (TEM) and introduces another variant, ultramicrotomy, for the preparation of TEM samples of industrial dust powder. The choice of ultramicrotoming in the present work was based on two features of this technique: (1) it can produce thin-sectioned specimens with a uniform thickness; (2) it can retain the original elemental distribution in phases of the sample during sectioning. Dust powder preparation and the sectioning procedure are described in this paper. The results of the method are illustrated by examples of TEM/STEM micrographs of industrial dust.

Assessment of the ability of propoxur, methomyl, and aldicarb, three carbamate insecticides, to induce micronuclei in vitro in cultured Chinese hamster ovary cells and in vivo in BALB/c mice.

Three carbamate insecticides (propoxur, methomyl, and aldicarb) were evaluated for their ability to induce micronuclei (MN) in vitro using cultured Chinese hamster ovary (CHO) cells, and in vivo in mouse bone marrow erythrocytes. In vitro, all three insecticides induced a significant increase in micronucleated binucleate cells, which was generally both dose and sample time dependent. The in vivo studies involved treating male BALB/c mice by different routes, either once or on 3 consecutive days, followed by multiple or single sampling. Treatment by intraperitoneal injection or oral gavage induced a significant increase in micronucleated reticulocytes (MNRETs) in peripheral blood. For all three chemicals, the MN response depended on sample time and the number of treatments, while for aldicarb, the response depended also on the route of exposure. These positive results demonstrate that propoxur, methomyl, and aldicarb are capable of inducing structural and/or numerical chromosomal aberrations in mammalian cells either in vitro or in vivo. Furthermore, based on the results obtained, on optimal in vivo MN protocol for carbamate insecticides is a single treatment followed by blood sampling at 24 and 48 hr after treatment.

Altered drug translocation mediated by the MDR protein: direct, indirect, or both?

Overexpression of the MDR protein, or p-glycoprotein (p-GP), in cells leads to decreased initial rates of accumulation and altered intracellular retention of chemotherapeutic drugs and a variety of other compounds. Thus, increased expression of the protein is related to increased drug resistance. Since several homologues of the MDR protein (CRP, ItpGPA, PDR5, sapABCDF) are also involved in conferring drug resistance phenomena in microorganisms, elucidating the function of the MDR protein at a molecular level will have important general applications. Although MDR protein function has been studied for nearly 20 years, interpretation of most data is complicated by the drug-selection conditions used to create model MDR cell lines. Precisely what level of resistance to particular drugs is conferred by a given amount of MDR protein, as well as a variety of other critical issues, are not yet resolved. Data from a number of laboratories has been gathered in support of at least four different models for the MDR protein. One model is that the protein uses the energy released from ATP hydrolysis to directly translocate drugs out of cells in some fashion. Another is that MDR protein overexpression perturbs electrical membrane potential (delta psi) and/or intracellular pH (pHi) and thereby indirectly alters translocation and intracellular retention of hydrophobic drugs that are cationic, weakly basic, and/or that react with intracellular targets in a pHi or delta psi-dependent manner. A third model proposes that the protein alternates between drug pump and Cl- channel (or channel regulator) conformations, implying that both direct and indirect mechanisms of altered drug translocation may be catalyzed by MDR protein. A fourth is that the protein acts as an ATP channel. Our recent work has tested predictions of these models via kinetic analysis of drug transport and single-cell photometry analysis of pHi, delta psi, and volume regulation in novel MDR and CFTR transfectants that have not been exposed to chemotherapeutic drugs prior to analysis. This paper reviews these data and previous work from other laboratories, as well as relevant transport physiology concepts, and summarizes how they either support or contradict the different models for MDR protein function.