Comparison of axisymmetric and three-dimensional models for gas uptake in a single bifurcation during steady expiration

Reactive gas uptake is predicted and compared in a single bifurcation at steady expiratory flow in terms of Sherwood number using an axisymmetric single-path model (ASPM) and a three-dimensional computational fluid dynamics model (CFDM). ASPM is validated in a two-generation geometry by comparing the average gas-phase mass transfer coefficients with the experimental values. ASPM predicted mass transfer coefficients within 20% of the experimental values. The flow and concentration variables in the ASPM were solved using Galerkin finite element method and in the CFDM using commercial finite element software FIDAP. The simulations were performed for reactive gas flowing at Reynolds numbers ranging from 60 to 350 in both symmetric bifurcation for three bifurcation angles, 30 deg, 70 deg, and 90 deg, and in an asymmetric bifurcation. The numerical models compared with each other qualitatively but quantitatively they were within 0.4-8% due to nonfully developed flow in the parent branch predicted by the CFDM. The radially averaged concentration variation along the axial location matched qualitatively between the CFDM and ASPM but quantitatively they were within 32% due to differences in the flow field. ASPM predictions compared well with the CFDM predictions for an asymmetric bifurcation. These results validate the simplified ASPM and the complex CFDM. ASPM predicts higher Sherwood number with a flat velocity inlet profile compared to a parabolic inlet velocity profile. Sherwood number increases with the inlet average velocity, wall mass transfer coefficient, and bifurcation angle since the boundary layer grows slower in the parent and daughter branches.     

The penetration of a dinucleoside monophosphate into rabbit blood cells in vitro

Uridylyl 3′-5′[¹⁴C] adenosine and uridylyl 2′-5′[¹⁴C] adenosine penetrate into rabbit blood cell sap at the same rate. No enzymatic or chemical hydrolysis of the dinucleoside monophosphates was observed.     

Hepatocyte uptake of alpha 1-proteinase inhibitor-trypsin complexes in vitro: evidence for a shared uptake mechanism for proteinase complexes of alpha 1-proteinase inhibitor and antithrombin III

In vivo clearance studies have indicated that the clearance of proteinase complexes of the homologous serine proteinase inhibitors alpha 1-proteinase inhibitor and antithrombin III occurs via a specific and saturable pathway located on hepatocytes. In vitro hepatocyte-uptake studies with antithrombin III-proteinase complexes confirmed the hepatocyte uptake and degradation of these complexes, and demonstrated the formation of a disulfide interchange product between the ligand and a cellular protein. We now report the results of in vitro hepatocyte uptake studies with alpha 1-proteinase inhibitor-trypsin complexes. Trypsin complexes of alpha 1-proteinase inhibitor were prepared and purified to homogeneity. Uptake of these complexes by hepatocytes was time and concentration-dependent. Competition experiments with alpha 1-proteinase inhibitor, alpha 1-proteinase inhibitor-trypsin, and antithrombin III-thrombin indicated that the proteinase complexes of these two inhibitors are recognized by the same uptake mechanism, whereas the native inhibitor is not. Uptake studies were performed at 37 degrees C with 125I-alpha 1-proteinase inhibitor-trypsin and analyzed by sodium dodecyl sulfate-gel electrophoresis in conjunction with autoradiography. These studies demonstrated time-dependent uptake and degradation of the ligand to low molecular weight peptides. In addition, there was a time-dependent accumulation of a high molecular weight complex of ligand and a cellular protein. This complex disappeared when gels were performed under reducing conditions. The sole cysteine residue in alpha 1-proteinase inhibitor was reduced and alkylated with iodoacetamide. Trypsin complexes of the modified inhibitor were prepared and purified to homogeneity. Uptake and degradation studies demonstrated no differences in the results obtained with this modified complex as compared to unmodified alpha 1-proteinase inhibitor-trypsin complex. In addition, the high molecular weight disulfide interchange product was still present on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized cells. Clearance and clearance competition studies with alpha 1-proteinase inhibitor-trypsin, alkylated alpha 1-proteinase inhibitor-trypsin, antithrombin III-thrombin, and anti-thrombin III-factor IXa further demonstrated the shared hepatocyte uptake mechanism for all these complexes.     

A novel 11-residual peptaibol-derived carrier peptide for in vitro oligodeoxynucleotide delivery into cell

Using a pore- and channel-forming peptide, TV-XIIa, which is an 11-residual peptaibol isolated from the fungus Trichoderma viride, we developed a vehicle for the cellular delivery of such polar biologically active agents as antisense oligodeoxynucleotides (ODNs). To function as an ODN carrier, basic amino acids, 10-mer of lysine, were conjugated to the C-terminus of TV-XIIa and the designed carrier peptide, Ac-U-N-I-I-U-P-L-L-U-P-I-K-K-K-K-K-K-K-K-K-K-OH (U: alpha-aminoisobutyric acid), was synthesized by the Fmoc-based solid-phase method. The complex between the carrier peptide and ODNs, which was electrostatically formed, was capable of crossing the membranes of NIH3T3 cells and the ODNs were accumulated in the cytoplasm and the nucleus. However, the complex was not taken up by A549 cells. The translocation of the complex occurred at both 4 and 37 degrees C in NIH3T3 cells and did not seem to involve an energy-dependent endocytic process.     

Evidence for a single steroid-binding protein in the rabbit progesterone receptor

The rabbit uterine progesterone receptor copurifies as two molecular weight (Mr) forms of about 105,000 and 78,000. To investigate whether these are different proteins, we have used protease digestion, reversible denaturation, and photoaffinity labeling in studies on the steroid-binding domain of the receptor. Digestion of the Mr 105,000 and 78,000 forms, photoaffinity labeled with [3H]R5020, with Staphylococcus aureus V8 protease revealed identical peptide fragments of Mr 43,000, 39,000, and 27,000-30,000. When receptor in cytosol was denatured, separated by electrophoresis, and then reconstituted, [3H]progesterone bound specifically to a single form at about Mr 105,000. After partial purification, the reversible denaturation procedure revealed both the larger and the smaller progesterone-binding species similar to the photoaffinity-labeled species in this preparation. Receptor in uterine cytosol prepared under mild conditions appeared as a predominant large molecular weight form on photoaffinity labeling with [17 alpha-methyl-3H]R5020, [6,7-3H]R5020, or [3H]RU27987. Further purification of this cytosol showed the generation of a smaller labeled species. These results from three different approaches reinforce the view that the rabbit progesterone receptor contains a single steroid-binding protein.     

Hybrid embryos produced by transferring panda or cat somatic nuclei into rabbit MII oocytes can develop to blastocyst in vitro

The developmental potential of hybrid embryos produced by transferring panda or cat fibroblasts into nucleated rabbit oocytes was assessed. Both the panda-rabbit and the cat-rabbit hybrid embryos were able to form blastocysts in vitro. However, the rates of attaining the two-cell, four-cell, eight-cell, morula, or blastocyst stages for panda-rabbit hybrids were significantly greater than those of cat-rabbit hybrids (P<0.05). Transferring the rabbit fibroblasts into nucleated rabbit oocytes, 31.0% of the blastocyst rate was obtained, which was significantly higher than that of both the panda-rabbit and the cat-rabbit hybrid embryos (P<0.05). Whether or not the second polar body (PB2) was extruded from the one-cell hybrid embryos (both panda-rabbit and cat-rabbit hybrids) significantly affected their developmental capacity. Embryos without an extruded PB2 showed a higher capacity to develop into blastocysts (panda-rabbit: 19.2%; cat-rabbit: 4.3%), while embryos with extruded PB2 could only develop to the morula stage. The hybrid embryos formed pronucleus-like structures (PN) in 2-4 hr after activation, and the number of PN in one-cell embryos varied from one to five. Tracking of the nucleus in the egg after fusion revealed that the somatic nucleus could approach and aggregate with the oocyte nucleus spontaneously. Chromosome analysis of the panda-rabbit blastocysts showed that the karyotype of the hybrid embryos (2n=86) consisted of chromosomes from both the panda (2n=42) and the rabbit (2n=44). The results demonstrate that (1) it is possible to produce genetic hybrid embryos by interspecies nuclear transfer; (2) the developmental potential of the hybrid embryos is highly correlated to the donor nucleus species; and (3) the hybrid genome is able to support the complete preimplantation embryonic development of the hybrids.     

Identification of a third secondary carrier (DcuC) for anaerobic C4-dicarboxylate transport in Escherichia coli: roles of the three Dcu carriers in uptake and exchange.

In Escherichia coli, two carriers (DcuA and DcuB) for the transport of C4 dicarboxylates in anaerobic growth were known. Here a novel gene dcuC was identified encoding a secondary carrier (DcuC) for C4 dicarboxylates which is functional in anaerobic growth. The dcuC gene is located at min 14.1 of the E. coli map in the counterclockwise orientation. The dcuC gene combines two open reading frames found in other strains of E. coli K-12. The gene product (DcuC) is responsible for the transport of C4 dicarboxylates in DcuA-DcuB-deficient cells. The triple mutant (dcuA dcuB dcuC) is completely devoid of C4-dicarboxylate transport (exchange and uptake) during anaerobic growth, and the bacteria are no longer capable of growth by fumarate respiration. DcuC, however, is not required for C4-dicarboxylate uptake in aerobic growth. The dcuC gene encodes a putative protein of 461 amino acid residues with properties typical for secondary procaryotic carriers. DcuC shows sequence similarity to the two major anaerobic C4-dicarboxylate carriers DcuA and DcuB. Mutants producing only DcuA, DcuB, or DcuC were prepared. In the mutants, DcuA, DcuB, and DcuC were each able to operate in the exchange and uptake mode.     

Development of a renal microchip for in vitro distal tubule models

Current developments in tissue engineering and microtechnology fields have allowed the proposal of pertinent tools, microchips, to investigate in vitro toxicity. In the framework of the proposed REACH European directive and the 3R recommendations, the purpose of these microtools is to mimic organs in vitro to refine in vitro culture models and to ultimately reduce animal testing. The microchip consists of functional living cell microchambers interconnected by a microfluidic network that allows continuous cell feeding and waste removal controls by fluid microflow. To validate this approach, Madin Darby Canine Kidney (MDCK) cells were cultivated inside a polydimethylsiloxane microchip. To assess the cell proliferation and feeding, the number of inoculated cells varied from 5 to 10 x 10(5) cells/microchip (corresponding roughly to 2.5 to 5 x 10(5) cells/cm2) and from four flow rates 0, 10, 25, and 50 microL/min were tested. Morphological observations have shown successful cell attachment and proliferation inside the microchips. The best flow rate appears to be 10 microL/min with which the cell population was multiplied by about 2.2 +/- 0.1 after 4 days of culture, including 3 days of perfusion (in comparison to 1.7 +/- 0.2 at 25 microL/min). At 10 microL/min flow rate, maximal cell population reached about 2.1 +/- 0.2 x 10(6) (corresponding to 7 +/- 0.7 x 10(7) cells/cm(3)). The viability, assessed by trypan blue and lactate deshydrogenase measurements, was found to be above 90% in all experiments. At 10 microL/min, glucose monitoring indicated a cell consumption of 16 +/- 2 microg/h/10(6) cells, whereas the glutamine metabolism was demonstrated with the production of NH3 by the cells about 0.8 +/- 0.4 micromol/day/10(6) cells. Augmentation of the flow rate appeared to increase the glucose consumption and the NH3 production by about 1.5- to 2-fold, in agreement with the tendencies reported in the literature. As a basic chronic toxicity assessment in the microchips, 5 mM and 10 mM ammonium chloride loadings, supplemented in the culture media, at 0, 10, and 25 micaroL/min flow rates were performed. At 10 microL/min, a reduction of 35% of the growth ratio with 5 mM and of 50% at 10 mM was found, whereas at 25 microL/min, a reduction of 10% with 5 mM and of 30% at 10 mM was obtained. Ammonium chloride contributed to increase the glucose consumption and to reduce the NH3 production. The microchip advantages, high surface/volume ratio, and dynamic loadings, coupled with the concordance between the present and literature results dealing with ammonia/ammonium effects on MDCK illustrate the potential of our microchip for wider in vitro chronic toxicity investigations.     

A single channel for nitrate uptake, nitrite export and nitrite uptake by Escherichia coli NarU and a role for NirC in nitrite export and uptake

Two related polytopic membrane proteins of the major facilitator family, NarK and NarU, catalyse nitrate uptake, nitrite export and nitrite uptake across the Escherichia coli cytoplasmic membrane by an unknown mechanism. A 12-helix model of NarU was constructed based upon six alkaline phosphatase and beta-galactosidase fusions to NarK and the predicted hydropathy for the NarK family. Fifteen residues conserved in the NarK-NarU protein family were substituted by site-directed mutagenesis, including four residues that are essential for nitrate uptake by Aspergillus nidulans: arginines Arg(87) and Arg(303) in helices 2 and 8, and two glycines in a nitrate signature motif. Despite the wide range of substitutions studied, in no case did mutation result in loss of one biochemical function without simultaneous loss of all other functions. A NarU+ NirC+ strain grew more rapidly and accumulated nitrite more rapidly than the isogenic NarU+ NirC(-) strain. Only the NirC+ strain consumed nitrite rapidly during the later stages of growth. Under conditions in which the rate of nitrite reduction was limited by the rate of nitrite uptake, NirC+ strains reduced nitrite up to 10 times more rapidly than isogenic NarU+ strains, indicating that both nitrite efflux and nitrite uptake are largely dependent on NirC. Isotope tracer experiments with [15N]nitrate and [14N]nitrite revealed that [15N]nitrite accumulated in the extracellular medium even when there was a net rate of nitrite uptake and reduction. We propose that NarU functions as a single channel for nitrate uptake and nitrite expulsion, either as a nitrate-nitrite antiporter, or more likely as a nitrate/H+ or nitrite/H+ channel.     

Folate-targeted PEG as a potential carrier for carboplatin analogs. Synthesis and in vitro studies

Like most low molecular weight drugs, carboplatin has a short blood circulation time, which reduces tumor uptake and intracellular DNA binding. Drugs conjugated to PEG carriers benefit from prolonged blood circulation, but suffer from reduced cell permeability. In this work we attempted to develop long-circulating PEGylated carboplatin analogues with improved cell permeation abilities, by conjugating the platinum moiety to folate-targeted PEG carriers capable of utilizing the folate receptor-mediated endocytosis (FRME). Two bifunctional FA-PEG conjugates, FA-PEG-Pt and FA-PEG-FITC, were prepared, and their cell uptake, DNA binding, and cytotoxicity were studied by fluorescent microscopy, FACS, and platinum analysis. Folate-targeted PEG conjugates enter the cells efficiently by the FRME pathway but form relatively few DNA adducts and have higher IC(50) values than carboplatin and their nontargeted analogues. Nontargeted PEG-Pt conjugates have a lower cellular uptake but produce higher levels of DNA binding and improved cytotoxicity. Carboplatin, used as a control, has the fastest cellular uptake, but after 16 h of postincubation a large percentage of the drug is excreted from the cells. The findings of this study suggest that folate-targeted conjugates such as FA-PEG-Pt, may not be an optimal prodrug for the carboplatin family compounds, because the conjugates or the active moieties are neutralized or blocked during the FRME process and do not manage to effectively reach the nuclear DNA.     

Fertilization in vitro of rabbit eggs with or without follicular cells by epididymal spermatozoa capacitated in a chemically defined medium

A portion of eggs from mature does treated with FSH and HCG to induce superovulation was freed from the follicular cells by treatment with hyaluronidase and repeated pipetting. Denuded and untreated eggs with follicular cells were placed in a defined medium with epididymal spermatozoa which had been exposed to the medium for 15 min and preincubated for 10–10.5 h in a CO₂ incubator. No significant difference was noted in the penetration rate of the eggs with (92%) and without (99%) follicular cells 5–5.5 h after insemination. Although there was no difference in the incidence of polyspermy, many more spermatozoa had penetrated into the perivitelline space of the denuded eggs than into those with follicular cells. Regardless of the presence of follicular cells, most of the penetrated eggs were undergoing cleavage or had developed to the 2- or 4-cell stage 24 h after insemination.     

Insights into a single rod-like helix in activated radixin required for membrane-cytoskeletal cross-linking

The members of the ezrin-radixin-moesin (ERM) family of proteins function as membrane-cytoskeletal cross-linkers in actin-rich cell surface structures. ERM proteins are thereby thought to be essential for cortical cytoskeleton organization, cell motility, adhesion, and proliferation. These modular polypeptides consist of a central helix-rich region, termed the alpha-domain, that connects an N-terminal FERM domain required for membrane binding and a C-terminal region which contains a major actin-binding motif. Conformational regulation of ERM protein function occurs by association of the FERM and C-terminal domains, whereby the membrane- and actin-binding activities are mutually suppressed and the protein is thought to take an inactive "closed" form. Here we report in vitro and in vivo studies of radixin to address the role of the alpha-domain in conformational activation of ERM proteins. Remarkably, an isolated alpha-domain comprised of radixin(311-469) forms a monomeric, stable helical rod that spans 240 A in length from the N-terminus to the C-terminus, most likely stabilized by extensive salt bridge interactions. By fusing green fluorescent protein variants to the FERM and C-terminal domains, we probed in vitroconformational changes impacted by the presence of the alpha-domain using fluorescence resonance energy transfer (FRET). Furthermore, deletion of this unusually long alpha-helical structure (radixin residues 314-411) prevents ERM membrane targeting in vivo.     

Alternate Requirement for Vitamin B12 or Methionine in Mutants of Pseudomonas denitrificans, a Vitamin B12-producing Bacterium

Experiments are described which indicate that Pseudomonas denitrificans, an organism that overproduces vitamin B(12), uses the B(12) pathway exclusively for methionine synthesis.     

Addressing the design of in vitro models for the investigation of microcystins: The essential role of transporter‐mediated uptake

This article discusses the need for transporter‐mediated uptake for investigations addressing the mechanism of action of microcystins [with reference to the previous article in Cell Biology International by de Souza Votto (2007) 31:1359–1366].     

Stimulation of hexose uptake by haemopoietic cell growth factor occurs in WEHI-3B myelomonocytic leukaemia cells: a possible mechanism for loss of growth control

WEHI-3B myelomonocytic leukaemia cells secrete a haemopoietic cell growth factor (HCGF) which facilitates the proliferation and development of multipotential stem cells and committed progenitor cells. Several cloned, nonleukaemic cell lines (FDC-P cells) are absolutely dependent on HCGF and die in the absence of it. In these cell lines, factor dependence is associated with the ability of HCGF to increase glucose uptake, thereby controlling glycolytic flux and intracellular ATP levels. We have now investigated the effects of HCGF on glucose uptake in WEHI-3B cells. At 20 degrees C 2-deoxyglucose uptake could be stimulated by the addition of HCGF to the extracellular medium. L-glucose uptake was markedly lower than 2-deoxyglucose uptake and did not respond to the addition of HCGF. At 37 degrees C no HCGF stimulation of 2-deoxyglucose uptake was found. However, at this temperature HCGF release from WEHI-3B cells was markedly higher than at 20 degrees C. Our experiments indicate that HCGF stimulates the glucose transport system in both WEHI-3 cells and FDC-P cells. The similarities between the WEHI-3B cell and FDC-P2 cell polypeptide phenotype were investigated using two-dimensional isoelectric focussing/poly-acrylamide gel electrophoresis. This revealed a high degree of correlation between the two cell types in their protein constituents, indicating a close relationship between the normal and leukaemic cells. These similarities between WEHI-3B cells and FDC-P2 cells are considered and their relevance to haemopoiesis and leukaemogenesis is discussed.