The membrane transport system responsible for multidrug resistance is operating in nonresistant cells

Cultured hamster fibroblasts of the DM-15 cell line stained by rhodamine 123 gradually release the dye when placed in dye-free medium. Here we demonstrate that reserpine, verapamil, and trifluoperazine are capable of blocking this release. We also show that reserpine can inhibit the efflux of another dye, phosphine 3R, from DM-15 cells and the release of rhodamine 123 from mouse embryo fibroblasts, four mouse cell lines, and MDCK cells. The three substances that block the release of the dyes are potent inhibitors of the membrane transport system implicated in the phenomenon of multidrug resistance (MDR). By using this system MDR cells can pump many structurally unrelated drugs and dyes, including rhodamine 123 and phosphine 3R, from the cytoplasm to the outer medium. It appears from our results that the membrane transport system responsible for MDR operates slowly in nonresistant cells and can play a role in normal cell physiology.     

Multidrug-resistance phenotype of a subpopulation of T-lymphocytes without drug selection

Multidrug-resistant (MDR) cells demonstrate the increased activity of the membrane transport system performing efflux of diverse lipophylic drugs and fluorescent dyes from the cells. In order to detect MDR cells we have developed a simple test consisting of three steps: staining of the cells with fluorescent dye rhodamine 123, incubation in the dye-free medium and, finally, detection by fluorescence microscopy of the cells that have lost accumulated dye. The experiments with B-lymphoma cell lines with different degrees of MDR have shown that the cell fluorescence after the poststaining incubation is indeed inversely proportional to the degree of resistance. Application of this testing procedure to normal human or mouse leukocytes revealed the presence of the cells rapidly losing the dye in these populations. Cell fractionation experiments have shown that there are T-lymphocytes (most T-killers/suppressors and a part of T-helpers) that demonstrate rapid efflux of rhodamine 123. This characteristic was detected also in T-killer clones and cell line and in some T-lymphomas. The inhibitors of the MDR transport system, reserpine and verapamil, blocked the efflux of the dye from these cells. Rhodamine-losing T-lymphoma contained large amounts of the mRNA coding P-glycoprotein, the MDR efflux pump, and demonstrated increased resistance to rhodamine 123, gramicidin D, colchicine, and vincristine, the drugs belonging to the cross-resistance group for the MDR cells. The role of the increased activity of the MDR membrane transport system in T-lymphocytes is discussed.     

Kinetic analysis of rhodamines efflux mediated by the multidrug resistance protein (MRP1)

Characterization of rhodamine 123 as functional assay for MDR has been primarily focused on P-glycoprotein-mediated MDR. Several studies have suggested that Rh123 is also a substrate for MRP1. However, no quantitative studies of the MRP1-mediated efflux of rhodamines have, up to now, been performed. Measurement of the kinetic characteristics of substrate transport is a powerful approach to enhancing our understanding of their function and mechanism. In the present study, we have used a continuous fluorescence assay with four rhodamine dyes (rhodamine 6G, tetramethylrosamine, tetramethylrhodamine ethyl ester, and tetramethylrhodamine methyl ester) to quantify drug transport by MRP1 in living GLC4/ADR cells. The formation of a substrate concentration gradient was observed. MRP1-mediated transport of rhodamine was glutathione-dependent. The kinetics parameter, k(a) = V(M)/k(m), was very similar for the four rhodamine analogs but approximately 10-fold less than the values of the same parameter determined previously for the MRP1-mediated efflux of anthracycline. The findings presented here are the first to show quantitative information about the kinetics parameters for MRP1-mediated efflux of rhodamine dyes.     

Microplate screening of the differential effects of test agents on Hoechst 33342, rhodamine 123, and rhodamine 6G accumulation in breast cancer cells that overexpress P-glycoprotein

A microplate screening method has been developed to evaluate the effects of test agents on the accumulation of the fluorescent P-glycoprotein (Pgp) substrates Hoechst 33342, rhodamine 123, and rhodamine 6G in multidrug-resistant (MDR) breast cancer cells that overexpress Pgp. All three substrates exhibit substantially higher accumulation in MCF7 non-MDR cells versus NCI/ADR-RES MDR cells, while incubation with 50 microM reserpine significantly reduces or eliminates these differences. Rhodamine 123 shows the lowest substrate accumulation efficiency in non-MDR cells relative to the substrate incubation level. The effects of several chemosensitizing agents and a series of paclitaxel analogs on the accumulation of each fluorescent substrate suggest that there are distinct differences in the substrate interaction profiles exhibited by these different agents. The described methods may be useful in Pgp-related research in the areas of cancer MDR, oral drug absorption, the blood-brain barrier, renal/hepatic transport processes, and drug-drug interactions.     

Evaluation of the Role of P-Glycoprotein in Ivermectin Uptake by Primary Cultures of Bovine Brain Microvessel Endothelial Cells

The P-glycoprotein efflux system located on the apical membrane of brain capillary endothelial cells functions as part of the blood-brain barrier. In this study, primary cultures of bovine brain microvessel endothelial cells (BMECs) were investigated for the presence of a P-glycoprotein system and its contribution in regulating ivermectin distribution across the blood-brain barrier. Results of rhodamine 123 uptake studies with cyclosporin A and verapamil as substrates indicated that a functional efflux system was present on BMECs. Immunoblot analysis with the C219 monoclonal antibody to the product of the multidrug resistant member 1(MDR1) gene also confirmed the expression of MDR1 in the BMECs. Unbound ivermectin was shown to significantly increase the uptake of rhodamine 123 in BMECs, however, the drug only modestly enhanced the transcellular passage of rhodamine. The results of these studies affirmed that unbound ivermectin is an inhibitor of the MDR1 efflux system in BMECs.     

Forest Canopy Gap Distributions in the Southern Peruvian Amazon

Canopy gaps express the time-integrated effects of tree failure and mortality as well as regrowth and succession in tropical forests. Quantifying the size and spatial distribution of canopy gaps is requisite to modeling forest functional processes ranging from carbon fluxes to species interactions and biological diversity. Using high-resolution airborne Light Detection and Ranging (LiDAR), we mapped and analyzed 5,877,937 static canopy gaps throughout 125,581 ha of lowland Amazonian forest in Peru. Our LiDAR sampling covered a wide range of forest physiognomies across contrasting geologic and topographic conditions, and on depositional floodplain and erosional terra firme substrates. We used the scaling exponent of the Zeta distribution (λ) as a metric to quantify and compare the negative relationship between canopy gap frequency and size across sites. Despite variable canopy height and forest type, values of λ were highly conservative (λ _mean  = 1.83, s  = 0.09), and little variation was observed regionally among geologic substrates and forest types, or at the landscape level comparing depositional-floodplain and erosional terra firme landscapes. λ-values less than 2.0 indicate that these forests are subjected to large gaps that reset carbon stocks when they occur. Consistency of λ-values strongly suggests similarity in the mechanisms of canopy failure across a diverse array of lowland forests in southwestern Amazonia.     

MDR1 P-glycoprotein transports endogenous opioid peptides

MDR1 P-glycoprotein is generally regarded as an efflux pump for amphipathic toxic compounds. The question remains, however, whether certain endogenous compounds are also substrates for this transporter. Certain peptides have been shown to interact with MDR1 Pgp as well and we have therefore investigated whether endogenous bioactive peptides are substrates. We demonstrate here that the synthetic μ-opioid peptide DAMGO is a good substrate for MDR1 Pgp. In view of its low interaction with the membrane it is an attractive ligand for measurement of MDR1 Pgp-mediated transport activity in membrane vesicles. Various linear peptides with amidated C-termini were found to inhibit MDR1 Pgp-mediated DAMGO transport. This group includes endogenous opioid peptides such as adrenorphin and endomorphin 1 and 2, as well as the neurokinin, Substance P. The latter bioactive peptides have a relatively high affinity for the transporter. Transport of endomorphin 1 and 2 could be directly demonstrated by the uptake of the radiolabeled opioid peptides in membrane vesicles from MDR1-transfected cells with a K_m of 15 and 12 μM, respectively. This opens the possibility that MDR1 Pgp is involved in the elimination and/or tissue distribution of these bioactive peptides.     

Studies of Human MDR1-MDR2 Chimeras Demonstrate the Functional Exchangeability of a Major Transmembrane Segment of the Multidrug Transporter and Phosphatidylcholine Flippase

P-glycoprotein (P-gp), encoded by the MDR1 gene, is a plasma membrane transporter which effluxes a large number of structurally nonrelated hydrophobic compounds. The molecular basis of the broad substrate recognition of P-gp is not well understood. Despite the 78% amino acid sequence identity of the MDR1 and MDR2 transporter, MDR2, which has been identified as a phosphatidylcholine transporter, does not transport most MDR1 substrates. The structural and functional differences between MDR1 and MDR2 provide an opportunity to identify the residues essential for the broad substrate spectrum of MDR1. Using an approach involving exchanging homologous segments of MDR1 and MDR2 and site-directed mutagenesis, we have demonstrated that MDR1 residues Q330, V331, and L332 in transmembrane domain 6 are sufficient to allow an MDR2 backbone in the N-terminal half of P-gp to transport several MDR1 substrates, including bisantrene, colchicine, vinblastine, and rhodamine-123. These studies help define some residues important for multidrug transport and indicate the close functional relationship between the multidrug transporter (MDR1) and phosphatidylcholine flippase (MDR2).     

Expression and activity of P-glycoprotein, a multidrug efflux pump, in human hematopoietic stem cells.

Hematopoietic stem cells show reduced staining with a mitochondrial fluorescent dye, rhodamine 123 (Rh-123), which was supposed to indicate decreased mitochondrial activity in these cells. Rh123 and several other fluorescent dyes are substrates for transport mediated by P-glycoprotein (P-gp), an efflux pump responsible for multidrug resistance in tumor cells. We have found that staining of human bone marrow cells with fluorescent dyes is potentiated by P-gp inhibitors and inversely correlated with P-gp expression. P-gp is expressed in practically all hematopoietic progenitor cells, including long-term culture-initiating cells. The highest levels of P-gp among the progenitors are associated with cells displaying characteristics of pluripotent stem cells. These results have implications for stem cell purification and bone marrow resistance to cancer chemotherapy.     

Sub-diffraction Limit Localization of Proteins in Volumetric Space Using Bayesian Restoration of Fluorescence Images from Ultrathin Specimens

Photon diffraction limits the resolution of conventional light microscopy at the lateral focal plane to 0.61λ/NA (λ = wavelength of light, NA = numerical aperture of the objective) and at the axial plane to 1.4nλ/NA² (n = refractive index of the imaging medium, 1.51 for oil immersion), which with visible wavelengths and a 1.4NA oil immersion objective is ∼220 nm and ∼600 nm in the lateral plane and axial plane respectively. This volumetric resolution is too large for the proper localization of protein clustering in subcellular structures. Here we combine the newly developed proteomic imaging technique, Array Tomography (AT), with its native 50–100 nm axial resolution achieved by physical sectioning of resin embedded tissue, and a 2D maximum likelihood deconvolution method, based on Bayes'' rule, which significantly improves the resolution of protein puncta in the lateral plane to allow accurate and fast computational segmentation and analysis of labeled proteins. The physical sectioning of AT allows tissue specimens to be imaged at the physical optimum of modern high NA plan-apochormatic objectives. This translates to images that have little out of focus light, minimal aberrations and wave-front distortions. Thus, AT is able to provide images with truly invariant point spread functions (PSF), a property critical for accurate deconvolution. We show that AT with deconvolution increases the volumetric analytical fidelity of protein localization by significantly improving the modulation of high spatial frequencies up to and potentially beyond the spatial frequency cut-off of the objective. Moreover, we are able to achieve this improvement with no noticeable introduction of noise or artifacts and arrive at object segmentation and localization accuracies on par with image volumes captured using commercial implementations of super-resolution microscopes.     

Effects of sample size on estimates of population growth rates calculated with matrix models

Background

Matrix models are widely used to study the dynamics and demography of populations. An important but overlooked issue is how the number of individuals sampled influences estimates of the population growth rate (λ) calculated with matrix models. Even unbiased estimates of vital rates do not ensure unbiased estimates of λ–Jensen''s Inequality implies that even when the estimates of the vital rates are accurate, small sample sizes lead to biased estimates of λ due to increased sampling variance. We investigated if sampling variability and the distribution of sampling effort among size classes lead to biases in estimates of λ.

Methodology/Principal Findings

Using data from a long-term field study of plant demography, we simulated the effects of sampling variance by drawing vital rates and calculating λ for increasingly larger populations drawn from a total population of 3842 plants. We then compared these estimates of λ with those based on the entire population and calculated the resulting bias. Finally, we conducted a review of the literature to determine the sample sizes typically used when parameterizing matrix models used to study plant demography.

Conclusions/Significance

We found significant bias at small sample sizes when survival was low (survival = 0.5), and that sampling with a more-realistic inverse J-shaped population structure exacerbated this bias. However our simulations also demonstrate that these biases rapidly become negligible with increasing sample sizes or as survival increases. For many of the sample sizes used in demographic studies, matrix models are probably robust to the biases resulting from sampling variance of vital rates. However, this conclusion may depend on the structure of populations or the distribution of sampling effort in ways that are unexplored. We suggest more intensive sampling of populations when individual survival is low and greater sampling of stages with high elasticities.     

Localization and activity of multidrug resistance protein 1 in the secretory pathway of Leishmania parasites

Upregulation of the multidrug resistance protein 1 (LeMDR1) in the protozoan parasite, Leishmania enriettii, confers resistance to hydrophobic drugs such as vinblastine, but increases the sensitivity of these parasites to the mitochondrial drug, rhodamine 123. In order to investigate the mechanism of action of LeMDR1, the subcellular localization of green fluorescent protein (GFP)-tagged versions of LeMDR1 and the fate of the traceable-fluorescent LeMDR1 substrate calcein AM were examined in both Leishmania mexicana and L. enriettii LeMDR1 -/- and overexpressing cell lines. The LeMDR1-GFP chimera was localized by fluorescence microscopy to a number of secretory and endocytic compartments, including the Golgi apparatus, endoplasmic reticulum (ER) and a multivesicular tubule (MVT)-lysosome. Pulse-chase labelling experiments with calcein AM suggested that the Golgi and ER pools, but not the MVT-lysosome pool, of LeMDR1 were active in pumping calcein AM out of the cell. Cells labelled with calcein AM under conditions that slow vesicular transport (low temperature and stationary growth) inhibited export and resulted in the accumulation of fluorescent calcein in both the Golgi and the mitochondria. We propose that LeMDR1 substrates are pumped into secretory compartments and exported from the parasite by exocytosis. Accumulation of MDR substrates in the ER can result in alternative transport to the mitochondrion, explaining the reciprocal sensitivity of drug-resistant Leishmania to vinblastine and rhodamine 123.     

Expression of the T cell receptor αβ on a CD123+ BDCA2+ HLA-DR+ subpopulation in head and neck squamous cell carcinoma

Human Plasmacytoid Dendritic Cells (PDCs) infiltrating solid tumor tissues and draining lymph nodes of Head and Neck Squamous Cell Carcinoma (HNSCC) show an impaired immune response. In addition to an attenuated secretion of IFN-α little is known about other HNSCC-induced functional alterations in PDCs. Particular objectives in this project were to gain new insights regarding tumor-induced phenotypical and functional alterations in the PDC population. We showed by FACS analysis and RT-PCR that HNSCC orchestrates an as yet unknown subpopulation exhibiting functional autonomy in-vitro and in-vivo besides bearing phenotypical resemblance to PDCs and T cells. A subset, positive for the PDC markers CD123, BDCA-2, HLA-DR and the T cell receptor αβ (TCR-αβ) was significantly induced subsequent to stimulation with HNSCC in-vitro (p = 0.009) and also present in metastatic lymph nodes in-vivo. This subgroup could be functionally distinguished due to an enhanced production of IL-2 (p = 0.02), IL-6 (p = 0.0007) and TGF-β (not significant). Furthermore, after exposure to HNSCC cells, mRNA levels revealed a D-J-beta rearrangement of the TCR-beta chain besides a strong enhancement of the CD3ε chain in the PDC population. Our data indicate an interface between the PDC and T cell lineage. These findings will improve our understanding of phenotypical and functional intricacies concerning the very heterogeneous PDC population in-vivo.     

Multiple insulin degrading enzyme variants alter in vitro reporter gene expression

The insulin degrading enzyme (IDE) variant, v311 (rs6583817), is associated with increased post-mortem cerebellar IDE mRNA, decreased plasma β-amyloid (Aβ), decreased risk for Alzheimer''s disease (AD) and increased reporter gene expression, suggesting that it is a functional variant driving increased IDE expression. To identify other functional IDE variants, we have tested v685, rs11187061 (associated with decreased cerebellar IDE mRNA) and variants on H6, the haplotype tagged by v311 (v10; rs4646958, v315; rs7895832, v687; rs17107734 and v154; rs4646957), for altered in vitro reporter gene expression. The reporter gene expression levels associated with the second most common haplotype (H2) successfully replicated the post-mortem findings in hepatocytoma (0.89 fold-change, p = 0.04) but not neuroblastoma cells. Successful in vitro replication was achieved for H6 in neuroblastoma cells when the sequence was cloned 5′ to the promoter (1.18 fold-change, p = 0.006) and 3′ to the reporter gene (1.29 fold change, p = 0.003), an effect contributed to by four variants (v10, v315, v154 and v311). Since IDE mediates Aβ degradation, variants that regulate IDE expression could represent good therapeutic targets for AD.     

Diurnal Microstructural Variations in Healthy Adult Brain Revealed by Diffusion Tensor Imaging

Biorhythm is a fundamental property of human physiology. Changes in the extracellular space induced by cell swelling in response to the neural activity enable the in vivo characterization of cerebral microstructure by measuring the water diffusivity using diffusion tensor imaging (DTI). To study the diurnal microstructural alterations of human brain, fifteen right-handed healthy adult subjects were recruited for DTI studies in two repeated sessions (8∶30 AM and 8∶30 PM) within a 24-hour interval. Fractional anisotropy (FA), apparent diffusion coefficient (ADC), axial (λ_//) and radial diffusivity (λ_⊥) were compared pixel by pixel between the sessions for each subject. Significant increased morning measurements in FA, ADC, λ_// and λ_⊥ were seen in a wide range of brain areas involving frontal, parietal, temporal and occipital lobes. Prominent evening dominant λ_⊥ (18.58%) was detected in the right inferior temporal and ventral fusiform gyri. AM-PM variation of λ_⊥ was substantially left side hemisphere dominant (p<0.05), while no hemispheric preference was observed for the same analysis for ADC (p = 0.77), λ_// (p = 0.08) or FA (p = 0.25). The percentage change of ADC, λ_//, λ_⊥, and FA were 1.59%, 2.15%, 1.20% and 2.84%, respectively, for brain areas without diurnal diffusivity contrast. Microstructural variations may function as the substrates of the phasic neural activities in correspondence to the environment adaptation in a light-dark cycle. This research provided a baseline for researches in neuroscience, sleep medicine, psychological and psychiatric disorders, and necessitates that diurnal effect should be taken into account in following up studies using diffusion tensor quantities.     

Type 2 diabetes is associated with reduced ATP-binding cassette transporter A1 gene expression, protein and function

Objective

Increasing plasma glucose levels are associated with increasing risk of vascular disease. We tested the hypothesis that there is a glycaemia-mediated impairment of reverse cholesterol transport (RCT). We studied the influence of plasma glucose on expression and function of a key mediator in RCT, the ATP binding cassette transporter-A1 (ABCA1) and expression of its regulators, liver X receptor-α (LXRα) and peroxisome proliferator-activated receptor–γ (PPARγ).

Methods and Results

Leukocyte ABCA1, LXRα and PPARγ expression was measured by polymerase chain reaction in 63 men with varying degrees of glucose homeostasis. ABCA1 protein concentrations were measured in leukocytes. In a sub-group of 25 men, ABCA1 function was quantified as apolipoprotein-A1-mediated cholesterol efflux from 2–3 week cultured skin fibroblasts. Leukocyte ABCA1 expression correlated negatively with circulating HbA1c and glucose (rho = −0.41, p<0.001; rho = −0.34, p = 0.006 respectively) and was reduced in Type 2 diabetes (T2DM) (p = 0.03). Leukocyte ABCA1 protein was lower in T2DM (p = 0.03) and positively associated with plasma HDL cholesterol (HDL-C) (rho = 0.34, p = 0.02). Apolipoprotein-A1-mediated cholesterol efflux correlated negatively with fasting glucose (rho = −0.50, p = 0.01) and positively with HDL-C (rho = 0.41, p = 0.02). It was reduced in T2DM compared with controls (p = 0.04). These relationships were independent of LXRα and PPARγ expression.

Conclusions

ABCA1 expression and protein concentrations in leukocytes, as well as function in cultured skin fibroblasts, are reduced in T2DM. ABCA1 protein concentration and function are associated with HDL-C levels. These findings indicate a glycaemia- related, persistent disruption of a key component of RCT.     

Sex-dependent influences of obesity on cerebral white matter investigated by diffusion-tensor imaging

Several studies have shown that obesity is associated with changes in human brain function and structure. Since women are more susceptible to obesity than men, it seems plausible that neural correlates may also be different. However, this has not been demonstrated so far. To address this issue, we systematically investigated the brain''s white matter (WM) structure in 23 lean to obese women (mean age 25.5 y, std 5.1 y; mean body mass index (BMI) 29.5 kg/m², std 7.3 kg/m²) and 26 lean to obese men (mean age 27.1 y, std 5.0 y; mean BMI 28.8 kg/m², std 6.8 kg/m²) with diffusion-weighted magnetic resonance imaging (MRI). There was no significant age (p>0.2) or BMI (p>0.7) difference between female and male participants. Using tract-based spatial statistics, we correlated several diffusion parameters including the apparent diffusion coefficient, fractional anisotropy (FA), as well as axial (λ_∥) and radial diffusivity (λ_⊥) with BMI and serum leptin levels. In female and male subjects, the putative axon marker λ_∥ was consistently reduced throughout the corpus callosum, particularly in the splenium (r = −0.62, p<0.005). This suggests that obesity may be associated with axonal degeneration. Only in women, the putative myelin marker λ_⊥ significantly increased with increasing BMI (r = 0.57, p<0.005) and serum leptin levels (r = 0.62, p<0.005) predominantly in the genu of the corpus callosum, suggesting additional myelin degeneration. Comparable structural changes were reported for the aging brain, which may point to accelerated aging of WM structure in obese subjects. In conclusion, we demonstrate structural WM changes related to an elevated body weight, but with differences between men and women. Future studies on obesity-related functional and structural brain changes should therefore account for sex-related differences.     

Retention of vital dyes correlates inversely with the multidrug-resistant phenotype of adriamycin-selected murine fibrosarcoma variants

Retention of the vital dyes rhodamine 123 (R-123) and hydroethidine (HET) correlates inversely with the multidrug resistant phenotypes of the adriamycin (ADM)-selected variants of a uv-induced murine fibrosarcoma cell line (UV-2237M). The differential affinity of these dyes for specific cellular organelles makes them unique compounds for studies of cellular transport. HET enters viable cells freely, is dehydrogenated to ethidium bromide (EtBr), and is subsequently accumulated in the nucleus. Viable cells are impermeable to extracellular EtBr, facilitating kinetic analysis of the efflux of intracellular EtBr. We found that the metabolite EtBr was rapidly cleared by ADM-resistant but not by ADM-sensitive cells. R-123 has a high affinity to mitochondria. Our results show that ADM-sensitive cells retain R-123 whereas the ADM-resistant cells do not. The clearance of both R-123 and EtBr from these cells was inhibited by verapamil. Therefore, R-123 and HET may be considered MDR-associated compounds useful in studying the MDR phenotype of cancer cells. Previously we reported a direct correlation between the level of activity of the calcium- and phospholipid-dependent protein kinase (protein kinases C) and ADM resistance in UV-2237M variant lines. In this report, we demonstrate a direct correlation between cellular calcium and MDR in these cells. Although chelation of extracellular calcium by EDTA did not alter the fluorescence profile of R-123 of the various cell lines, treating the ADM-resistant variants with verapamil restored cellular calcium to the same level as that of the parental cells and, at the same time, retarded the facilitated efflux of R-123 and EtBr and partially reversed cancer cell resistance to ADM.     

Evidence that a cell-type-specific efflux pump regulates cell differentiation in Dictyostelium

We have identified a cellular efflux pump, RhT, with the properties of an MDR transporter-a type of ATP-binding cassette transporter whose substrates include small hydrophobic molecules. RhT transports rhodamine 123 (Rh123) and is inhibited by low temperature, energy poisons, and several MDR transport inhibitors, such as verapamil. All vegetative cells have RhT activity, but during development prestalk cells lose RhT activity while prespore cells retain it. We also identified several RhT inhibitors. The most effective inhibitor is the stalk cell-inducing chlorinated alkyl phenone, DIF-1. The RhT inhibitors disrupted development, to varying degrees, and induced stalk cell formation in submerged culture. The inhibitors displayed the same rank order of pharmacological efficacy for stalk cell induction as they did for Rh123 transport inhibition. We also found that cerulenin, a specific inhibitor of DIF-1 biosynthesis (R. R. Kay, 1998, J. Biol. Chem. 273, 2669-2675), abolished the induction of stalk cells by each of the RhT inhibitors, and this effect could be reversed by DIF-1. Thus, DIF-1 synthesis appears to be required for the induction of stalk cells by the RhT inhibitors. Since DIF-1 is the most potent inhibitor of RhT activity, and thus a likely transport substrate itself, we propose that RhT inhibitors induce stalk cell differentiation by blocking DIF-1 export, causing DIF-1 to build up within cells. Our results provide evidence for a prespore-specific efflux pump that regulates cell fate determination, perhaps by regulating the cellular concentration of DIF-1.     

Renal Water Molecular Diffusion Characteristics in Healthy Native Kidneys: Assessment with Diffusion Tensor MR Imaging

Background

To explore the characteristics of diffusion tensor imaging (DTI) and magnetic resonance (MR) imaging in healthy native kidneys.

Methods

Seventy-three patients without chronic kidney disease underwent DTI-MRI with spin echo-echo planar (SE-EPI) sequences accompanied by an array spatial sensitivity encoding technique (ASSET). Cortical and medullary mean, axial and radial diffusivity (MD, AD and RD), fractional anisotropy (FA) and primary, secondary and tertiary eigenvalues (λ₁, λ₂, λ₃) were analysed in both kidneys and in different genders.

Results

Cortical MD, λ₂, λ₃, and RD values were higher than corresponding medullary values. The cortical FA value was lower than the medullary FA value. Medullary λ₁ and RD values in the left kidney were lower than in the right kidney. Medullary λ₂, and λ₃ values in women were higher than those in men. Medullary FA values in women were lower than those in men. Medullary FA (r = 0.351, P = 0.002) and λ₁ (r = 0.277, P = 0.018) positively correlated with eGFR. Medullary FA (r = −0.25, P = 0.033) negatively correlated with age.

Conclusions

Renal water molecular diffusion differences exist in human kidneys and genders. Age and eGFR correlate with medullary FA and primary eigenvalue.