Relation between the anion exchange protein in kidney medullary collecting duct cells and red cell band 3

Summary A membrane protein that is immunochemically similar to the red cell anion exchange protein, band 3, has been identified on the basolateral face of the outer medullary collecting duct (MCD) cells in rabbit kidney. In freshly prepared separated rabbit MCD cells, M.L. Zeidel, P. Silva and J.L. Seifter (J. Clin. Invest. 77:1682–1688, 1986) found that Cl^–/HCO ₃ ^- exchange was inhibited by the stilbene anion exchange inhibitor, DIDS (4,4-diisothiocyano-2,2-disulfonic stilbene), with aK ₁ similar to that for the red cell. We have measured the binding affinities of a fluorescent stilbene inhibitor, DBDS (4,4-dibenzamido-2,2-disulfonic stilbene), to MCD cells in 28.5 mM citrate and have characterized both a high-affinity site (K ₁ ^s =93±24 mM) and a lower affinity site (K ₂ ^s =430±260 nM), which are closely similar to values for the red cell of 110±51 nM for the high-affinity site and 980±200 nM for the lower affinity site (A.S. Verkman, J.A. Dix & A.K. Solomon,J. Gen. Physiol. 81:421–449, 1983). When Cl^– replaces citrate in the buffer, the two sites collapse into a single one withK ₁ ^s =1500±400 nM, similar to the singleK ₁ ^s =1200±200 nM in the red cell (J.A. Dix, A.S. Verkman & A.K. Solomon,J. Membrane Biol. 89:211–223, 1986). The kinetics of DBDS binding to MCD cells at 0.25 M^–1 are characterized by a fast process, =0.14±0.03 sec, similar to =0.12±0.03 sec in the red cell. These similarities show that the physical chemical characteristics of stilbene inhibitor binding to MCD cell band 3 closely resemble those for red cell band 3, which suggests that the molecular structure is highly conserved.     

A dynamic metabolite valve for the control of central carbon metabolism

Successful redirection of endogenous resources into heterologous pathways is a central tenet in the creation of efficient microbial cell factories. This redirection, however, may come at a price of poor biomass accumulation, reduced cofactor regeneration and low recombinant enzyme expression. In this study, we propose a metabolite valve to mitigate these issues by dynamically tuning endogenous processes to balance the demands of cell health and pathway efficiency. A control node of glucose utilization, glucokinase (Glk), was exogenously manipulated through either engineered antisense RNA or an inverting gene circuit. Using these techniques, we were able to directly control glycolytic flux, reducing the specific growth rate of engineered Escherichia coli by up to 50% without altering final biomass accumulation. This modulation was accompanied by successful redirection of glucose into a model pathway leading to an increase in the pathway yield and reduced carbon waste to acetate. This work represents one of the first examples of the dynamic redirection of glucose away from central carbon metabolism and enables the creation of novel, efficient intracellular pathways with glucose used directly as a substrate.     

Function of tubulin binding proteins in vivo

Overexpression of the beta-tubulin binding protein Rbl2p/cofactor A is lethal in yeast cells expressing a mutant alpha-tubulin, tub1-724, that produces unstable heterodimer. Here we use RBL2 overexpression to identify mutations in other genes that affect formation or stability of heterodimer. This approach identifies four genes-CIN1, CIN2, CIN4, and PAC2-as affecting heterodimer formation in vivo. The vertebrate homologues of two of these gene products-Cin1p/cofactor D and Pac2p/cofactor E-can catalyze exchange of tubulin polypeptides into preexisting heterodimer in vitro. Previous work suggests that both Cin2p or Cin4p act in concert with Cin1p in yeast, but no role for vertebrate homologues of either has been reported in the in vitro reaction. Results presented here demonstrate that these proteins can promote heterodimer formation in vivo. RBL2 overexpression in cin1 and pac2 mutant cells causes microtubule disassembly and enhanced formation of Rbl2p-beta-tubulin complex, as it does in the alpha-tubulin mutant that produces weakened heterodimer. Significantly, excess Cin1p/cofactor D suppresses the conditional phenotypes of that mutant alpha-tubulin. Although none of the four genes is essential for viability under normal conditions, they become essential under conditions where the levels of dissociated tubulin polypeptides increase. Therefore, these proteins may provide a salvage pathway for dissociated tubulin heterodimers and so rescue cells from the deleterious effects of free beta-tubulin.     

The isolation of peptides by high-performance liquid chromatography using predicted elution positions

This paper describes the derivation and use of predictive retention coefficients for the reversed-phase high-performance liquid chromatography of peptides. The use of predicted elution positions in the isolation of peptides is illustrated by two examples where peptides, whose existence was postulated from cDNA sequence data, have been successfully isolated. The combination of the powerful chromatographic technology and the ability to predict the elution positions of peptides based on their composition provides a very potent method for the isolation of peptides from biological tissues.     

Chemical composition and in vitro ruminal fermentation of common tree forages in the semi-arid rangelands of Swaziland

Browse plants play an important role in providing feed for livestock in semi-arid rangelands of Africa. Chemical composition and in vitro ruminal fermentation of leaves collected from Acacia burkei, Acacia tortilis, Acacia nilotica, Dichrostachys cinerea and Ehretia obtusifolia in communal grazing lands in the lowveld of Swaziland is presented. Leaves were collected from trees located on two soil types (i.e., lithosol and vertisol) in the communal land but it had no effect on the chemical composition of tree leaves. The NDFom and ADFom content were highest in D. cinerea and A. burkei and lowest in E. obtusifolia and A. nilotica. Crude protein (CP) contents ranged between 108 g/kg and 122 g/kg DM. D. cinerea had the highest Ca and Mg content, while A. tortilis had the lowest. There were marked variations in K level amongst browse species, with A. tortilis (9.1 g/kg DM) having the highest value. The P, Zn and Fe did not differ between browse species. Soil type and tree species interaction impacted in vitro fermentation parameters. Extent of fermentation, as measured by 48 h cumulative gas production, and organic matter degradability was highest in E. obtusifolia leaves and lowest in D. cinerea leaves within soil type. Fermentation efficiency, as measured by partitioning factors, was highest in A. nilotica leaves. Leaves of E. obtusifolia could be a valuable supplementary feedstuff for ruminant livestock due to its in vitro fermentation characteristics as well as low fibre and moderate CP levels.     

Analysis of Adenosine Immunoreactivity, Uptake, and Release in Purified Cultures of Developing Chick Embryo Retinal Neurons and Photoreceptors

We have investigated the presence of endogenous adenosine and of mechanisms for adenosine uptake and release in chick embryo retinal neurons and photoreceptors grown in purified cultures in the absence of glial cells. Simultaneous autoradiographic and immunocytochemical analysis showed that endogenous adenosine and the uptake mechanism for this nucleoside colocalize in practically all the photoreceptors, but only in approximately 20% of the neurons. Approximately 25% of the neurons showed either immunocytochemical labeling or autoradiographic labeling, while greater than 50% of the neurons were unlabeled with both techniques. [3H]Adenosine uptake was saturable and could be inhibited by nitrobenzylthioinosine and dipyridamole and by pretreatment of the [3H]adenosine with adenosine deaminase. Although these observations indicate that the uptake is specific for adenosine, only 35% of accumulated radioactivity was associated with adenosine, with the remaining 65% representing inosine, hypoxanthine, and nucleotides plus uric acid. Adenosine as well as several of its metabolites were released by the cells under basal as well as K(+)-stimulated conditions. Potassium-enhanced release was blocked by 10 mM CoCl2 or in Ca2(+)-free, Mg2(+)-rich solutions. The results indicate that retinal cells that synthesize, store, and release adenosine differentiate early during embryogenesis and are therefore consistent with a hypothetical role for adenosine in retinal development.     

Bleomycin-iron can degrade DNA in the presence of excess ethylenediaminetetraacetic acid in vitro

The antineoplastic drug bleomycin, when complexed to Fe(II), causes both single- and double-stranded lesions in DNA in vitro. EDTA is commonly used to inhibit the reaction of bleomycin-Fe with DNA, presumably by removing the metal cofactor. In this study, we utilized a simple assay involving the conversion of supercoiled plasmid DNA to the nicked or linear forms to further investigate the ability of bleomycin-Fe to degrade DNA in the presence of EDTA. We found that a 1:1 complex of bleomycin and Fe can degrade plasmid DNA even in the presence of a 10(6) molar excess of EDTA over bleomycin. Furthermore, we found that the half-life for inactivation of bleomycin-Fe by excess EDTA is about 1.5 h. Finally, we demonstrate that excess bleomycin associated with the outer plasma membranes of cells can damage DNA after the cells are lysed in buffers containing EDTA and SDS. These results suggest that EDTA may not be an efficient inhibitor of the reaction of bleomycin-Fe with DNA.     

Identification of nonsulfated cholecystokinin-58 in canine intestinal extracts and its biological properties

Nonsulfated CCK(58) [CCK(58)(ns)] has not been considered to be of biological importance because CCK(58)(ns) binds poorly to the CCK(A) receptor and has only been identified once in intestinal extracts. In this work, a radioimmunoassay specific for the COOH-terminal region of gastrin and CCK (antibody 5135) was used to monitor the purification of CCK molecular forms from canine intestinal extracts. A minor immunoreactive peak was associated with a major absorbance peak during an ion-exchange, HPLC step. Characterization of this minor immunoreactive peak demonstrated that it was CCK(58)(ns). CCK(58)(ns) is 14% as immunoreactive as sulfated CCK(8) [CCK(8)(s)]. Amino acid analysis demonstrated that CCK(58)(ns) was present at 50% the amount of CCK(58)(s). In addition, we found that CCK(58)(ns) does not potently displace an (125)I-labeled CCK(10) analog from the CCK(A) receptor in mouse pancreatic membranes and does not stimulate amylase release from isolated pancreatic acini, or stimulate pancreatic secretion in an anesthetized rat model. By contrast, CCK(58)(ns) does bind to CCK(B) receptors and stimulates gastric acid secretion via this receptor. The presence of CCK(58)(ns) and its ability to selectively stimulate the CCK(B) receptor without stimulation of the CCK(A) receptor suggest that CCK(58)(ns) may have unique physiological properties, especially tissues where the nonsulfated peptide can act as a paracrine or neurocrine agent.