Transcriptomic Analysis of the Dual Response of Rhodococcus aetherivorans BCP1 to Inorganic Arsenic Oxyanions.

Bacterial strains belonging to the genus Rhodococcus are able to degrade various toxic organic compounds and tolerate high concentrations of metal(loid)s. We have previously shown that Rhodococcus aetherivorans BCP1 is resistant to various levels of the two arsenic inorganic species, arsenite [As(III)] and arsenate [As(V)]. However, while arsenite showed toxic effects at concentrations as low as 5 mM, arsenate at 30 mM boosted the growth rate of BCP1 cells and was toxic only at concentrations of >100 mM. Since such behavior could be linked to peculiar aspects of its metabolism, the transcriptomic analysis of BCP1 cells exposed to 5 mM As(III) and 30 mM As(V) was performed in this work. The aim was to clarify the mechanisms underlying the arsenic stress response of the two growth phenotypes in the presence of the two different oxyanions. The results revealed that As(III) induced higher activity of reactive oxygen species (ROS)-scavenging enzymes than As(V) in relation to the expression of enzymes involved in cellular damage recovery and redox buffers/cofactors (ergothioneine, mycofactocin, and mycothiol). Further, As(III) downregulated pathways related to cell division, while both oxyanions downregulated genes involved in glycolysis. Notably, As(V) induced the expression of enzymes participating in the synthesis of metallophores and rearranged the central and energetic metabolism, also inducing alternative pathways for ATP synthesis and glucose consumption. This study, in providing transcriptomic data on R. aetherivorans exposed to arsenic oxyanions, sheds some light on the plasticity of the rhodococcal response to arsenic stress, which may be important for the improvement of biotechnological applications. IMPORTANCE Members of the genus Rhodococcus show high metabolic versatility and the ability to tolerate/resist numerous stress conditions, including toxic metals. R. aetherivorans BCP1 is able to tolerate high concentrations of the two inorganic arsenic oxyanions, arsenite [As(III)] and arsenate [As(V)]. Despite the fact that BCP1 intracellularly converts As(V) into As(III), this strain responds very differently to the presence of these two oxyanions in terms of cell growth and toxic effects. Indeed, while As(III) is highly toxic, exposure to specific concentrations of As(V) seems to boost cell growth. In this work, we investigated the transcriptomic response, ATP synthesis, glucose consumption, and H2O2 degradation in BCP1 cells exposed to As(III) and As(V), inducing two different growth phenotypes. Our results give an overview of the transcriptional rearrangements associated with the dual response of BCP1 to the two oxyanions and provide novel insights into the energetic metabolism of Rhodococcus under arsenic stress.

Examining solvent effects on the ultrafast dynamics of catechol.

We consider the effect of a polar, hydrogen bond accepting, solvent environment on the excited state decay of catechol following excitation to its first excited singlet state (S1). A comparison of Fourier transform infrared spectroscopy and explicit-solvent ab initio frequency prediction suggests that 5 mM catechol in acetonitrile is both nonaggregated and in its "closed" conformation, contrary to what has been previously proposed. Using ultrafast transient absorption spectroscopy, we then demonstrate the effects of aggregation on the photoexcited S1 lifetime: at 5 mM catechol (nonaggregated) in acetonitrile, the S1 lifetime is 713 ps. In contrast at 75 mM catechol in acetonitrile, the S1 lifetime increases to 1700 ps. We attribute this difference to aggregation effects on the excited-state landscape. This work has shown that explicit-solvent methodology is key when calculating the vibrational frequencies of molecules in a strongly interacting solvent. Combining this with highly complementary steady-state and transient absorption spectroscopy enables us to gain key dynamical insights into how a prominent eumelanin building block behaves when in polar, hydrogen bond accepting solvents both as a monomer and as an aggregated species.

Mutation of a type II keratin gene (K6a) in pachyonychia congenita.

Pachyonychia congenita (PC) is a rare autosomal dominant condition characterized by multiple ectodermal abnormalities. Patients with Jadassohn-Lewandowsky Syndrome (MIM #167200; PC-1) have nail defects (onchyogryposis), palmoplantar hyperkeratosis, follicular hyperkeratosis and oral leukokeratosis. Those with the rarer Jackson-Lawler Syndrome (MIM #167210; PC-2) lack oral involvement but have natal teeth and cutaneous cysts. Ultra-structural studies have identified abnormal keratin tonofilaments and linkage to the keratin gene cluster on chromosome 17 has been found in PC families. Keratins are the major structural proteins of the epidermis and associated appendages and the nail, hair follicle, palm, sole and tongue are the main sites of constitutive K6, K16 and K17 expression. Furthermore, mutations in K16 and K17 have recently been identified in some PC patients. Although we did not detect K16 or K17 mutations in PC families from Slovenia, we have found a heterozygous deletion in a K6 isoform (K6a) in the affected members of one family. This 3 bp deletion (AAC) in exon 1 of K6a removes a highly conserved asparagine residue (delta N170) from position 8 of the 1A helical domain (delta N8). This is the first K6a mutation to be described and this heterozygous K6a deletion is sufficient to explain the pathology observed in this PC-1 family.

Analyses of both the alkB gene transcriptional start site and alkB promoter-inducing properties of Rhodococcus sp. strain BCP1 grown on n-alkanes.

Rhodococcus sp. strain BCP1, known for its capacity to grow on short-chain n-alkanes (C(2) to C(7)) and to cometabolize chlorinated solvents, was found to also utilize medium- and long-chain n-alkanes (C(12) to C(24)) as energy and carbon sources. To examine this feature in detail, a chromosomal region which includes the alkB gene cluster encoding a non-heme di-iron monooxygenase (alkB), two rubredoxins, and one rubredoxin reductase was cloned from the BCP1 genome. Furthermore, the activity of the alkB gene promoter (P(alkB)) was examined in the presence of gaseous, liquid, and solid n-alkanes along with intermediates of the putative n-alkane degradation pathway. A recombinant plasmid, pTP(alkB)LacZ, was constructed by inserting the lacZ gene downstream of P(alkB), and it was used to transform Rhodococcus sp. strain BCP1. Measurements of ß-galactosidase activity showed that P(alkB) is induced by C(6) to C(22) n-alkanes. Conversely, C(2) to C(5) and >C(22) n-alkanes and alkenes, such as hexene, were not inducers of alkB expression. The effects on P(alkB) expression induced by alternative carbon sources along with putative products of n-hexane metabolism were also evaluated. This report highlights the great versatility of Rhodococcus sp. strain BCP1 and defines for the first time the alkB gene transcriptional start site and the alkB promoter-inducing capacities for substrates different from n-alkanes in a Rhodococcus strain.

A histidine-kinase cheA gene of Pseudomonas pseudoalcaligens KF707 not only has a key role in chemotaxis but also affects biofilm formation and cell metabolism.

A histidine-kinase cheA gene in Pseudomonas pseudoalcaligenes KF707 plays a central role in the regulation of metabolic responses as well as in chemotaxis. Non-chemotactic mutants harboring insertions into the cheA gene were screened for their ability to form biofilms in the Calgary biofilm device. Notably, ≥95% decrease in the number of cells attached to the polystyrene surface was observed in cheA mutants compared to the KF707 wild-type biofilm phenotype. The ability to form mature biofilms was restored to wild-type levels, providing functional copies of the KF707 cheA gene to the mutants. In addition, phenotype micro-arrays and proteomic analyses revealed that several basic metabolic activities and a few periplasmic binding proteins of cheA mutant cells differed compared to those of wild-type cells. These results are interpreted as evidence of a strong integration between chemotactic and metabolic pathways in the process of biofilm development by P. pseudoalcaligenes KF707.

Calcium release in HSY cells conforms to a steady-state mechanism involving regulation of the inositol 1,4,5-trisphosphate receptor Ca2+ channel by luminal [Ca2+].

In many cell types, low concentrations of inositol 1,4,5-trisphosphate (IP3) release only a portion of the intracellular IP3-sensitive Ca2+ store, a phenomenon known as "quantal" Ca2+ release. It has been suggested that this effect is a result of reduced activity of the IP3-dependent Ca2+ channel with decreasing calcium concentration within the IP3-sensitive store ([Ca2+]s). To test this hypothesis, the properties of IP3-dependent Ca2+ release in single saponin-permeabilized HSY cells were studied by monitoring [Ca2+]s using the Ca(2+)-sensitive fluorescent dye mag-fura-2. In permeabilized cells, blockade of the sarco/ER Ca(2+)-ATPase pump in stores partially depleted by IP3 induced further Ca2+ release via an IP3-dependent route, indicating that Ca2+ entry via the sarco/ER Ca(2+)-ATPase pump had been balanced by Ca2+ loss via the IP3-sensitive channel before pump inhibition. IP3-dependent Mn2+ entry, monitored via quenching of luminal mag-fura-2 fluorescence, was readily apparent in filled stores but undetectable in Ca(2+)-depleted stores, indicating markedly reduced IP3-sensitive channel activity in the latter. Also consistent with reduced responsiveness of Ca(2+)-depleted stores to IP3, the initial rate of refilling of these stores was unaffected by the presence of 0.3 microM IP3, a concentration that was clearly effective in eliciting Ca2+ release from filled stores. Analysis of the rate of Ca2+ release at various IP3 concentrations indicated a significant shift of the IP3 dose response toward higher [IP3] with decreasing [Ca2+]s. We conclude that IP3-dependent Ca2+ release in HSY cells is a steady-state process wherein Ca2+ efflux via the IP3 receptor Ca2+ channel is regulated by [Ca2+]s, apparently via changes in the sensitivity of the channel to IP3.

Monoclonal antibodies as probes of epithelial membrane polarization.

Monoclonal antibodies directed against antigens in the apical plasma membrane of the toad kidney epithelial cell line A6 were produced to probe the phenomena that underlie the genesis and maintenance of epithelial polarity. Two of these antibodies, 17D7 and 18C3, were selected for detailed study here. 17D7 is directed against a 23-kD peptide found on both the apical and basolateral surfaces of the A6 epithelium whereas 18C3 recognizes a lipid localized to the apical membrane only. This novel observation of an apically localized epithelial lipid species indicates the existence of a specific sorting and insertion process for this, and perhaps other, epithelial plasma membrane lipids. The antibody-antigen complexes formed by both these monoclonal antibodies are rapidly internalized by the A6 cells, but only the 18C3-antigen complex is recycled to the plasma membrane. In contrast to the apical localization of the free antigen, however, the 18C3-antigen complex is recycled to both the apical and basolateral surface of the epithelium, which indicates that monoclonal antibody binding interferes in some way with the normal sorting process for this apical lipid antigen.

Anti-adhesion activity of two biosurfactants produced by Bacillus spp. prevents biofilm formation of human bacterial pathogens.

In this work, two biosurfactant-producing strains, Bacillus subtilis and Bacillus licheniformis, have been characterized. Both strains were able to grow at high salinity conditions and produce biosurfactants up to 10% NaCl. Both extracted-enriched biosurfactants showed good surface tension reduction of water, from 72 to 26-30 mN/m, low critical micelle concentration, and high resistance to pH and salinity. The potential of the two lipopeptide biosurfactants at inhibiting biofilm adhesion of pathogenic bacteria was demonstrated by using the MBEC device. The two biosurfactants showed interesting specific anti-adhesion activity being able to inhibit selectively biofilm formation of two pathogenic strains. In particular, Escherichia coli CFT073 and Staphylococcus aureus ATCC 29213 biofilm formation was decreased of 97% and 90%, respectively. The V9T14 biosurfactant active on the Gram-negative strain was ineffective against the Gram-positive and the opposite for the V19T21. This activity was observed either by coating the polystyrene surface or by adding the biosurfactant to the inoculum. Two fractions from each purified biosurfactant, obtained by flash chromatography, fractions (I) and (II), showed that fraction (II), belonging to fengycin-like family, was responsible for the anti-adhesion activity against biofilm of both strains.

Towards standardized criteria for diagnosing chronic intervillositis of unknown etiology: A systematic review.

Chronic intervillositis of unknown etiology (CIUE) is a poorly understood, relatively rare condition characterized histologically by the intervillous infiltration of mononuclear cells in the placenta. Clinically, CIUE is associated with poor pregnancy outcome (e.g., impaired fetal growth, preterm birth, fetal death) and high risk of recurrence in subsequent pregnancies. Because CIUE is not defined consistently, it is essential to clearly define this condition. We therefore review the published definitions of CIUE. In addition, we provide an overview of the reviewed histopathological and maternal characteristics, obstetric features, and pregnancy outcomes. Medical publication databases were searched for articles published through February 2017. Eighteen studies were included in our systematic review. The sole inclusion criterion used in all studies was the presence of intervillous infiltrates. Overall, CIUE was characterized by adverse pregnancy outcome. Miscarriage occurred in 24% of cases, with approximately half of these miscarriages defined as late. Impaired growth was commonly observed, 32.4% of pregnancies reached term, and the live birth rate was 54.9%. The high recurrence rate (25.1%) of the intervillous infiltrates in subsequent pregnancies underscores the clinical relevance of CIUE, the need for increased awareness among pathologists and clinicians, and the need for further research. Criteria for the diagnosis of CIUE are proposed and a Delphi study could be used to resolve any controversy regarding these criteria. Future studies should be designed to characterize the full clinical spectrum of CIUE.

Beta-adrenergic upregulation of the Na(+)-K(+)-2Cl- cotransporter in rat parotid acinar cells.

We used the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6')-carboxyfluorescein to monitor the recovery of the intracellular pH (pHi) of rat parotid acini from an NH4(+)-induced alkaline load. This recovery was markedly inhibited by the loop diuretic bumetanide and by Cl- removal, indicating that it is largely due to NH4+ entry via the basolateral Na(+)-K(+)-2Cl- cotransporter. The rate of recovery of pHi was enhanced threefold by pretreatment (37.5 s) with isoproterenol (K1/2 = 21.5 nM) or norepinephrine (in the presence of phentolamine), and blocked by the beta 1-specific antagonist atenolol, indicating an upregulation of cotransport activity by beta 1-adrenergic stimulation. The effect of isoproterenol was prevented by protein kinase inhibitors and mimicked by cAMP analogues, and by maneuvers known to increase cytosolic cAMP levels in these cells, consistent with the involvement of protein kinase A. Physiologically, such an upregulation of the acinar Na(+)-K(+)-2Cl- cotransporter would lead to an increase in acinar chloride uptake across the basolateral membrane, and consequently, an increase in overall chloride and fluid secretion. Prevention of this upregulation by beta-blockers and possibly by other commonly used clinical agents may account for the dry mouth and dry eyes experienced by some patients taking these medications.

99mTc-pertechnetate uptake in parotid acinar cells by the Na+/K+/Cl- co-transport system.

99mTc-Pertechnetate (99mTcO4-) has widespread clinical use in the diagnosis and evaluation of dysfunctions in many different tissues. However, despite the broad clinical application of this radionuclide, very little is known about the mechanism by which 99mTcO4- enters a cell. We report evidence here that 99mTcO4- shares the Na+/K+/Cl- co-transport system localized to the basolateral membrane of rat parotid acinar cells. 99mTcO4- uptake by these cells was quite rapid (t1/2 approximately 30 s), was completely inhibited by the loop diuretics furosemide and bumetanide, and was markedly dependent on the presence of Na+, K+, and Cl- in the extracellular medium. Relative to uptake measured in the presence of physiological extracellular salt concentrations (Hanks' salts), 99mTcO4- uptake was inhibited 80% by sodium replacement and 50% by potassium replacement. When Cl- was replaced with the physiologically inert anion gluconate a threefold stimulation in 99mTcO4- uptake resulted. These observations provide strong evidence that 99mTcO4- can substitute for Cl- as a substrate for the Na+/K+/Cl- co-transporter and indicate that 99mTcO4- uptake by salivary glands (e.g., as seen with salivary scintiscans), and possibly by a variety of other tissues, reflects the functional activity of this co-transport mechanism.

Circumferential deformation and shear stress induce differential responses in saphenous vein endothelium exposed to arterial flow.

Adaptation of saphenous vein to the hemodynamic stresses of the arterial circulation is critical to the maturation of vein bypass grafts. We have investigated early adaptive responses of venous endothelium by placing excised human saphenous vein in a bypass circuit with either venous or arterial flow conditions, using external stenting to resolve the effects of longitudinal (shear) from circumferential stress. Endothelial protein concentrations were assessed by immunostaining area (ratio of protein/CD31) and Western blotting of endothelial cell lysates (staining ratio protein/vWf). In both unstented and stented veins nitric oxide synthase increased after 90 min of arterial flow: twofold increase of immunostaining area (P = 0.001), four- to fivefold increase by Western blotting (P = 0.02), and increased A23187mediated maximum endothelium-dependent relaxation of vein rings (P = 0.01). In unstented veins, ICAM-1 concentration was increased after 45 min of arterial flow: twofold increase by immunostaining (P = 0.001) and Western blotting (P = 0.038), with maximum fibrinogen-mediated endothelium-dependent relaxation increasing from 55.9+/-4.9 to 97+/-2.1% (P = 0.01). In contrast, in unstented veins there was a threefold decrease of VCAM-1 and no change in P-selectin after arterial flow for 45 and 90 min, respectively. However, no changes in ICAM-1 and VCAM-1 were observed in stented veins. The flow-induced alterations in nitric oxide synthase, ICAM-1, and VCAM-1 were abolished when 3 mM tetraethylammonium ion (K+ channel blocker) was included in the vein perfusate. The very rapid changes in ICAM-1 and VCAM-1 expression are a response to circumferential stress, whereas the slower upregulation of nitric oxide synthase is a response to longitudinal (shear) stress. Similar changes could influence the adhesiveness of endothelium in newly implanted saphenous vein bypass grafts.

Increased substance P immunoreactivity and edema formation following reversible ischemic stroke.

Previous results from our laboratory have shown that neurogenic inflammation is associated with edema formation after traumatic brain injury (TBI). This neurogenic inflammation was characterized by increased substance P (SP) immunoreactivity and could be attenuated with administration of SP antagonists with a resultant decrease in edema formation. Few studies have examined whether neurogenic inflammation, as identified by increased SP immunoreactivity, occurs after stroke and its potential role in edema formation. The present study examines SP immunoreactivity and edema formation following stroke. Experimental stroke was induced in halothane anaesthetized male Sprague-Dawley rats using a reversible thread model of middle cerebral artery occlusion. Increased SP immunoreactivity at 24 hours relative to the non-infarcted hemisphere was observed in perivascular, neuronal, and glial tissue, and within the penumbra of the infarcted hemisphere. It was not as apparent in the infarct core. This increased SP immunoreactivity was associated with edema formation. We conclude that neurogenic inflammation, as reflected by increased SP immunoreactivity, occurs following experimental stroke, and that this may be associated with edema formation. As such, inhibition of neurogenic inflammation may represent a novel therapeutic target for the treatment of edema following reversible, ischemic stroke.

Adaptation of an enzyme to regulatory function: structure of Bacillus subtilis PyrR, a pyr RNA-binding attenuation protein and uracil phosphoribosyltransferase.

BACKGROUND: The expression of pyrimidine nucleotide biosynthetic (pyr) genes in Bacillus subtilis is regulated by transcriptional attenuation. The PyrR attenuation protein binds to specific sites in pyr mRNA, allowing the formation of downstream terminator structures. UMP and 5-phosphoribosyl-1-pyrophosphate (PRPP), a nucleotide metabolite, are co-regulators with PyrR. The smallest RNA shown to bind tightly to PyrR is a 28-30 nucleotide stem-loop that contains a purine-rich bulge and a putative-GNRA tetraloop. PyrR is also a uracil phosphoribosyltransferase (UPRTase), although the relationship between enzymatic activity and RNA recognition is unclear, and the UPRTase activity of PyrR is not physiologically significant in B. subtilis. Elucidating the role of PyrR structural motifs in UMP-dependent RNA binding is an important step towards understanding the mechanism of pyr transcriptional attenuation. RESULTS: The 1.6 A crystal structure of B. subtilis PyrR has been determined by multiwavelength anomalous diffraction, using a Sm co-crystal. As expected, the structure of PyrR is homologous to those proteins of the large type I PRTase structural family; it is most similar to hypoxanthine-guanine-xanthine PRTase (HGXPRTase). The PyrR dimer differs from other PRTase dimers, suggesting it may have evolved specifically for RNA binding. A large, basic, surface at the dimer interface is an obvious RNA-binding site and uracil specificity is probably provided by hydrogen bonds from mainchain and sidechain atoms in the hood subdomain. These models of RNA and UMP binding are consistent with biological data. CONCLUSIONS: The B. subtilis protein PyrR has adapted the substrate- and product-binding capacities of a PRTase, probably an HGXPRTase, producing a new regulatory function in which the substrate and product are co-regulators of transcription termination. The structure is consistent with the idea that PyrR regulatory function is independent of catalytic activity, which is likely to be extremely low under physiological conditions.

Regulation of the Bacillus subtilis pyrimidine biosynthetic operon by transcriptional attenuation: control of gene expression by an mRNA-binding protein.

The pyrimidine nucleotide biosynthetic (pyr) operon of Bacillus subtilis is regulated by a transcriptional attenuation mechanism in which termination of transcription at points upstream of the genes being regulated is promoted by the binding of a regulatory protein, PyrR, to specific sequences in the pyr mRNA. Binding of PyrR to pyr mRNA is stimulated by uridine nucleotides and causes changes in the mRNA secondary structure. This model is supported by extensive molecular genetic analysis. PyrR, which is encoded by the first gene of the pyr operon, is also a uracil phosphoribosyltransferase, although it has little amino acid sequence resemblance to other bacterial uracil phosphoribosyltransferases. Purified B. subtilis pyrR promotes attenuation of pyr transcription in vitro and binds specifically to pyr RNA sequences. The crystallographic structure of PyrR demonstrates the similarity of its tertiary structure to other phosphoribosyltransferases and suggests the surface to which RNA binds. PyrR is widely distributed among eubacteria and appears to regulate pyr genes not only by the attenuation mechanism found in B. subtilis, but also by a coupled transcription-translation attenuation mechanism and by acting as a translational repressor. PyrR illustrates the concept that transcriptional attenuation is a much more widespread and mechanistically versatile mechanism for the regulation of gene expression in bacteria than is generally recognized.

Inflammation in acute CNS injury: a focus on the role of substance P.

Recently, a number of reports have shown that neurogenic inflammation may play a role in the secondary injury response following acute injury to the CNS, including traumatic brain injury (TBI) and stroke. In particular substance P (SP) release appears to be critically involved. Specifically, the expression of the neuropeptide SP is increased in acute CNS injury, with the magnitude of SP release being related to both the frequency and magnitude of the insult. SP release is associated with an increase in blood-brain barrier permeability and the development of vasogenic oedema as well as neuronal injury and worse functional outcome. Moreover, inhibiting the actions of SP through use of a NK1 receptor antagonist is highly beneficial in both focal and diffuse models of TBI, as well as in ischaemic stroke, with a therapeutic window of up to 12 h. We propose that NK1 receptor antagonists represent a novel therapeutic option for treatment of neurogenic inflammation following acute CNS injury.

Stability and Species Specificity of Renal VEGF-A Splicing Patterns in Kidney Disease.

Vascular endothelial growth factor A (VEGF-A) is essential for maintaining the glomerular filtration barrier. Absolute renal levels of VEGF-A change in patients with diabetic nephropathy and inflammatory kidney diseases, but whether changes in the renal splicing patterns of VEGF-A play a role remains unclear. In this study, we investigated mRNA splicing patterns of pro-angiogenic isoforms of VEGF-A in glomeruli and whole kidney samples from human patients with kidney disease and from mouse models of kidney disease. Kidney biopsies were obtained from patients with acute rejection following kidney transplantation, patients with diabetic nephropathy, and control subjects. In addition, kidney samples were obtained from mice with lupus nephritis, mice with diabetes mellitus, and control mice. The relative expression of each VEGF-A splice variant was measured using RT-PCR followed by quantitative fragment analysis. The pattern of renal VEGF-A splice variants was unchanged in diabetic nephropathy and lupus nephritis and was stable throughout disease progression in acute transplant rejection and diabetic nephropathy; these results suggest renal VEGF-A splicing stability during kidney disease. The splicing patterns were species-specific; in the control human kidney samples, VEGF-A 121 was the dominant isoform, whereas VEGF-A 164 was the dominant isoform measured in the mouse kidney samples.

General rate equations and rejection criteria for the rapid equilibrium carrier model of cotransport.

It is demonstrated that under fixed activator conditions the general flux equation for the rapid equilibrium carrier model of cotransport can be written entirely in terms of five independent kinetic constants. Thus the kinetic parameters from any experiment carried out under the same activator conditions must necessarily be expressible in terms of these five constants. These predicted relationships between experimental kinetic parameters provide rejection criteria for the model, a number of which are derived here. Generalization of the treatment to the case where a competitive substrate is present on both sides of the membrane is also given.