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C P Smith

Publications and source records attributed to C P Smith.

At least 19 recordsLinked to original sources
PubMed2005

UT-B is expressed in bovine rumen: potential role in ruminal urea transport.

The UT-A (SLC14a2) and UT-B (SLC14a1) genes encode a family of specialized urea transporter proteins that regulate urea movement across plasma membranes. In this report, we describe the structure of the bovine UT-B (bUT-B) gene and characterize UT-B expression in bovine rumen. Northern analysis using a full-length bUT-B probe detected a 3.7-kb UT-B signal in rumen. RT-PCR of bovine mRNA revealed the presence of two UT-B splice variants, bUT-B1 and bUT-B2, with bUT-B2 the predominant variant in rumen. Immunoblotting studies of bovine rumen tissue, using an antibody targeted to the NH2-terminus of mouse UT-B, confirmed the presence of 43- to 54-kDa UT-B proteins. Immunolocalization studies showed that UT-B was mainly located on cell plasma membranes in epithelial layers of the bovine rumen. Ussing chamber measurements of ruminal transepithelial transport of (14)C-labeled urea indicated that urea flux was characteristically inhibited by phloretin. We conclude that bUT-B is expressed in the bovine rumen and may function to transport urea into the rumen as part of the ruminant urea nitrogen salvaging process.

Amino Acid Sequence↗
PubMed2005

Altered expression of iron transport proteins in streptozotocin-induced diabetic rat kidney.

Diabetes mellitus is associated with altered iron homeostasis in both human and animal diabetic models. Iron is a metal oxidant capable of generating reactive oxygen species (ROS) and has been postulated to contribute to diabetic nephropathy. Two proteins involved in iron metabolism that are expressed in the kidney are the divalent metal transporter, DMT1 (Slc11a2), and the Transferrin Receptor (TfR). Thus, we investigated whether renal DMT1 or TfR expression is altered in diabetes, as this could potentially affect ROS generation and contribute to diabetic nephropathy. Rats were rendered diabetic with streptozotocin (STZ-diabetes) and renal DMT1 and TfR expression studied using semi-quantitative immunoblotting and immunofluorescence. In STZ-diabetic Sprague-Dawley rats, renal DMT1 expression was significantly reduced and TfR expression increased after 2 weeks. DMT1 downregulation was observed in both proximal tubules and collecting ducts. Renal DMT1 expression was also decreased in Wistar rats following 12 weeks of STZ-diabetes, an effect that was fully corrected by insulin-replacement but not by cotreatment with the aldose reductase inhibitor, sorbinil. Increased renal TfR expression was also observed in STZ-diabetic Wistar rats together with elevated cellular iron accumulation. Together these data demonstrate renal DMT1 downregulation and TfR upregulation in STZ-diabetes. Whilst the consequence of altered DMT1 expression on renal iron handling and oxidant damage remains to be determined, the attenuation of the putative lysosomal iron exit pathway in proximal tubules could potentially explain lysosomal iron accumulation reported in human diabetes and STZ-diabetic animals.

Animals↗
PubMed2004

An experimental evaluation of a loop versus a reference design for two-channel microarrays.

MOTIVATION: Despite theoretical arguments that so-called 'loop designs' for two-channel DNA microarray experiments are more efficient, biologists continue to use 'reference designs'. We describe two sets of microarray experiments with RNA from two different biological systems (TPA-stimulated mammalian cells and Streptomyces coelicolor). In each case, both a loop and a reference design were used with the same RNA preparations with the aim of studying their relative efficiency. RESULTS: The results of these experiments show that (1) the loop design attains a much higher precision than the reference design, (2) multiplicative spot effects are a large source of variability, and if they are not accounted for in the mathematical model, for example, by taking log-ratios or including spot effects, then the model will perform poorly. The first result is reinforced by a simulation study. Practical recommendations are given on how simple loop designs can be extended to more realistic experimental designs and how standard statistical methods allow the experimentalist to use and interpret the results from loop designs in practice. AVAILABILITY: The data and R code are available at http://exgen.ma.umist.ac.uk CONTACT: veronica.vinciotti@brunel.ac.uk.

Algorithms↗
PubMed2003

Education and training in the paediatric senior house officer grade: analysis of RCPCH hospital/child health visits reports, 1997-2001.

AIMS: To review the process and outcome of education and training visits to paediatric departments by the RCPCH. METHODS: Retrospective audit of visits reports (1997-2001) against the RCPCH criteria for general professional training. Hospital and/or community child health departments who were responsible for training paediatric senior house officers were visited to assess whether RCPCH criteria of education were being met. Follow up visits were undertaken where limited education and training approval was given. Reports were received from 214 of 242 (88%) hospital and/or community based departments in England, Wales, and Northern Ireland. RESULTS: Satisfactory achievement of the 12 training criteria by departments varied widely: 39-95% (median 66%) achieved. Follow up visits reported significant improvements in most departments. Criteria which departments struggled to achieve reasonable standards were: (1) ensuring SHOs were performing educationally appropriate duties (39% achieved); and (2) satisfactory outpatient experience (41% achieved). Twenty four per cent of hospital based departments did not have a paediatrician with 12 months or more experience of paediatrics resident on call. CONCLUSIONS: The visiting process highlighted areas of good practice, encouraged change to meet the criteria, and recommended increased resources and staffing where necessary to improve training and hence the service. The need for continuing approval for education and training in these departments encouraged significant efforts on the part of trainers and managers to meet the requirements, and consequently the quality of service to children has been enhanced.

Educational Measurement↗
PubMed2002

Botulinum toxin urethral sphincter injection resolves urinary retention after pubovaginal sling operation.

The management of prolonged urinary retention following pubovaginal sling surgery typically involves transvaginal urethrolysis for anatomical urethral obstruction. Brubaker recently reported on urethral sphincter abnormalities as a cause of postoperative urinary retention following either Burch suspension or a pubovaginal sling procedure. We report a case of functional urethral obstruction and detrusor acontractility following pubovaginal sling surgery that was successfully treated by botulinum A toxin urethral sphincter injection.

Aged↗
PubMed2002

Neurovesical dysfunction in postural tachycardia syndrome (POTS).

This is the first report of neurovesical dysfunction in a woman with postural tachycardia syndrome (POTS). The patient had both symptoms and urodynamic findings diagnostic of detrusor hyperreflexia. Management consisted of anticholinergic medication and timed voiding. Lower urinary tract dysfunction may be underrecognized in POTS.

Adult↗
PubMed2002

Botulinum toxin urethral sphincter injection resolves urinary retention after pubovaginal sling operation.

The management of prolonged urinary retention following pubovaginal sling surgery typically involves transvaginal urethrolysis for anatomical urethral obstruction. Brubaker [1] recently reported on urethral sphincter abnormalities as a cause of postoperative urinary retention following either Burch suspension or pubovaginal sling procedure. We report a case of functional urethral obstruction and detrusor acontractility following pubovaginal sling surgery that was successfully treated by botulinum A toxin urethral sphincter injection.

Aged↗
PubMed2002

Expression of a sodium bicarbonate cotransporter in human parotid salivary glands.

The human parotid gland secretes much of the bicarbonate that enters the mouth. Prompted by studies of animal models, this study sought evidence for the expression of a functional Na(+)-HCO(3)(-) cotransporter (NBC) in human parotid acinar cells. Microfluorometric measurements of intracellular pH in isolated acini showed that the recovery from an acid load was achieved in part by HCO(3)(-) uptake via a Na(+)-dependent, DIDS-sensitive mechanism. By reverse transcriptase-polymerase chain reaction, a full-length NBC1 clone was obtained showing more than 99% homology with the human pancreatic isoform hpNBC1. Expressed in Xenopus oocytes, the electrogenicity of the transporter was detected as an inwardly directed, Na(+)- and HCO(3)(-)-dependent flux of negative charge. Immunohistochemistry using antibodies raised to NBC1 showed strong staining of the basolateral membrane of the acinar cells. Therefore, it was concluded that a functional electrogenic Na(+)-HCO(3)(-) cotransporter is expressed in the human parotid gland, and that it contributes to pH regulation in the acinar cells and could play a significant part in salivary secretion.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2002

Coordinated expression of UT-A and UT-B urea transporters in rat testis.

The blood-seminiferous tubule barrier is responsible for maintaining the unique microenvironment conducive to spermatogenesis. A key feature of the blood-testis barrier is selective permeability to solutes and water transport, conferred by the Sertoli cells of the seminiferous tubules (SMTs). Movement of fluid into the lumen of the seminiferous tubule is crucial to spermatogenesis. By Northern analysis, we have shown that 4.0-, 3.3-, 2.8-, and ~1.7-kb UT-A mRNA transcripts and a 3.8-kb UT-B mRNA transcript are detected within rat testis. Western analysis revealed the expression of both characterized and novel UT-A and UT-B proteins within the testis. Immunolocalization studies determined that UT-A and UT-B protein expression are coordinated with the developmental stage of the SMT. UT-A proteins were detected in Sertoli cell nuclei at all stages of tubule development and in residual bodies of stage VIII tubules. UT-B protein was expressed on Sertoli cell membranes of stage II-III tubules. Using in vitro perfusion, we determined that a phloretin-inhibitable urea pathway exists across the SMTs of rat testis and conclude that UT-B is likely to participate in this pathway.

Animals↗
PubMed2002

Characterization of mouse urea transporters UT-A1 and UT-A2.

Specialized transporter proteins that are the products of two closely related genes, UT-A (Slc14a2) and UT-B (Slc14a1), modulate the movement of urea across cell membranes. The purpose of this study was to characterize the mouse variants of two major products of the UT-A gene, UT-A1 and UT-A2. Screening a mouse kidney inner medulla cDNA library yielded 4,047- and 2,876-bp cDNAs, the mouse homologues of UT-A1 and UT-A2. Northern blot analysis showed high levels of UT-A mRNAs in kidney medulla. UT-A transcripts were also present in testes, heart, brain, and liver. Immunoblots with an antiserum raised to the 19 COOH-terminal amino acids of rat UT-A1 (L194) identified immunoreactive proteins in kidney, testes, heart, brain, and liver and showed a complex pattern of differential expression. Relative to other tissues, kidney and brain had the highest levels of UT-A protein expression. In kidney sections, immunostaining with L194 revealed immunoreactive proteins in type 1 (short) and type 3 (long) thin descending limbs of the loop of Henle and in the middle and terminal inner medullary collecting ducts. Expression in Xenopus laevis oocytes showed that, characteristic of UT-A family members, the cDNAs encoded phloretin-inhibitable urea transporters. Acute application of PKA agonists (cAMP/forskolin/IBMX) caused a significant increase in UT-A1- and UT-A3-, but not UT-A2-mediated, urea transport.

Amino Acid Sequence↗
PubMed2002

Structure and characterization of the mouse UT-A gene (Slc14a2).

The movement of urea across plasma membranes is modulated by facilitated urea transporter proteins. These proteins are the products of two closely related genes, termed UT-A (Slc14a2) and UT-B (Slc14a1). By genomic library screening and P1 artificial chromosome "shotgun" sequencing, we have determined the structure of the mouse UT-A gene. The gene is >300 kb in length, contains 24 exons, and has 2 distinct promoters. Flanking the 5'-region of the gene is the UT-Aalpha promoter that regulates transcription of UT-A1 and UT-A3. The second promoter, termed UT-Abeta, is present in intron 13 and regulates transcription of UT-A2. cAMP agonists (100 microM dibutryl cAMP, 25 microM forskolin, 0.5 mM IBMX) increased the activity of a 2.2-kb UT-Aalpha promoter construct 6.2-fold [from 0.026 +/- 0.003 to 0.160 +/- 0.004, relative light units (RLU)/microg protein] and a 2.4-kb UT-Abeta promoter construct 9.5-fold (from 0.020 +/- 0.002 to 0.190 +/- 0.043 RLU/microg protein) above that in untreated controls. Interestingly, only the UT-Abeta promoter contained consensus sequences for CREs and deletion of these elements abolished cAMP sensitivity. Increasing the tonicity of culture medium from 300 to 600 mosmol/kg H(2)O with NaCl caused a significant increase (from 0.060 +/- 0.004 to 0.095 +/- 0.010 RLU/microg protein) in UT-Aalpha promoter activity but had no effect on the UT-Abeta promoter. A tonicity-responsive enhancer was identified in UT-Aalpha and is suggested to be responsible for mediating this effect. Levels of UT-A2 and UT-A3 mRNA were increased in thirsted mice compared with control animals, indicating that the activities of both promoters are likely to be elevated during prolonged antidiuresis.

Animals↗
PubMed2001

ARDS after pulmonary contusion: accurate measurement of contusion volume identifies high-risk patients.

BACKGROUND: The pathophysiology of pulmonary contusion (PC) is poorly understood, and only minimal advances have been made in management of this entity over the past 20 years. Improvement in understanding of PC has been hindered by the fact that there has been no accurate way to quantitate the amount of pulmonary injury. With this project, we examine a method of accurately measuring degree of PC by quantifying contusion volume relative to pulmonary function and outcome. METHODS: Patients with PC from isolated chest trauma who had admission chest computed tomographic scan were identified from the registry of a Level I trauma center over a 1.5-year period. Subsequently, prospective data on all patients admitted to the intensive care unit with PC during a 5-month period were collected and added to the retrospective database. Using computer-generated three-dimensional reconstruction from admission chest computed tomographic scan, contusion volume was measured and expressed as a percentage of total lung volume. Admission pulmonary function variables (Pao2/FiO2, static compliance), injury descriptors (chest Abbreviated Injury Score, Injury Severity Score, injury distribution), and indicators of degree of shock (admission systolic blood pressure, admission base deficit) were documented. Outcomes included maximum positive end-expiratory pressure, ventilator days, pneumonia, and acute respiratory distress syndrome (ARDS). RESULTS: Forty-nine patients with PC (35 bilateral) were identified. The average severity of contusion was 18% (range, 5-55%). Patients were classified using contusion volume as severe PC (> or =20%, n = 17) and moderate PC (< 20%, n = 32). Injury Severity Score was similar in the severe and moderate groups (23.3 vs. 26.5, p = 0.33), as were admission Glasgow Coma Scale score (12 vs. 13, p = 0.30), admission blood pressure (131 vs. 129 mm Hg, p = 0.90), and admission Pao2/Fio2 (197 vs. 255, p = 0.14). However, there was a much higher rate of ARDS in the severe group as compared with the moderate group (82% vs. 22%, p < 0.001). There was a trend toward higher pneumonia rate in the severe group, with 50% of patients in the severe group developing pneumonia as compared with 28% in the moderate group (p = 0.20). CONCLUSION: Extent of contusion volumes measured using three-dimensional reconstruction allows identification of patients at high risk of pulmonary dysfunction as characterized by development of ARDS. This method of measurement may provide a useful tool for the further study of PC as well as for the identification of patients at high risk of complications at whom future advances in therapy may be directed.

Abbreviated Injury Scale↗
PubMed2001

Cellular localization of divalent metal transporter DMT-1 in rat kidney.

We have demonstrated that the kidney plays an important role in iron balance and that metabolically significant reabsorption of this ion occurs in the loop of Henle and the collecting ducts [Wareing M, Ferguson CJ, Green R, Riccardi D, and Smith CP. J Physiol (Lond) 524: 581-586, 2000]. To test the possibility that the divalent metal transporter DMT1 (Gunshin H, Mackenzie B, Berger UV, Gunshin Y, Romero MF, Boron WF, Nussberger S, Gollan JL, and Hediger MA. Nature 388: 482-488, 1997) could represent the apical route for iron entry in the kidney, we raised and affinity-purified an anti-DMT-1 polyclonal antibody and determined DMT-1 distribution in rat kidney by Western analysis, immunofluorescence, and confocal microscopy. The strongest DMT1-specific (i.e., peptide-protectable) immunoreactivity was found in the collecting ducts, in both principal and intercalated cells. Thick ascending limbs of Henle's loop and, more intensely, distal convoluted tubules exhibited apical immunostaining. Considerable intracellular DMT-1 immunoreactivity was seen throughout the nephron, particularly in S3 segments. The described distribution of DMT-1 protein is in agreement with our previous identification of nephron sites of iron reabsorption, suggesting that DMT-1 provides the molecular mechanism for apical iron entry in the distal nephron but not in the proximal tubule. Basolateral iron exit may be facilitated by a different system.

Animals↗
PubMed2001

Physiological and molecular characterization of urea transport by the gills of the Lake Magadi tilapia (Alcolapia grahami).

The Lake Magadi tilapia (Alcolapia grahami) is an unusual fish, excreting all its nitrogenous waste as urea because of its highly alkaline and buffered aquatic habitat. Here, using both physiological and molecular studies, we describe the mechanism of branchial urea excretion in this species. In vivo, repeated short-interval sampling revealed that urea excretion is continuous. The computed urea permeability of A. grahami gill is 4.74x10(-)(5)+/-0.38x10(-)(5 )cm s(-)(1) (mean +/- s.e.m., N=11), some 10 times higher than passive permeability through a lipid bilayer and some five times higher than that of even the most urea-permeable teleosts studied to date (e.g. the gulf toadfish). Transport of urea was bidirectional, as demonstrated by experiments in which external [urea] was elevated. Furthermore, urea transport was inhibited by classic inhibitors of mammalian and piscine urea transporters in the order thiourea>N-methylurea>acetamide. A 1700 base pair cDNA for a putative Magadi tilapia urea transporter (mtUT) was cloned, sequenced and found to display high homology with urea transporters from mammals, amphibians and other fishes. When cRNA transcribed from mtUT cDNA was injected into Xenopus laevis oocytes, phloretin-inhibitable urea uptake was enhanced 3.4-fold relative to water-injected controls. Northern analysis of gill, red blood cells, liver, muscle and brain using a portion of mtUT as a probe revealed that gill is the only tissue in which mtUT RNA is expressed. Magadi tilapia gill pavement cells exhibited a trafficking of dense-cored vesicles between the well-developed Golgi cisternae and the apical membrane. The absence of this trafficking and the poor development of the Golgi system in a non-ureotelic relative (Oreochromis niloticus) suggest that vesicle trafficking could be related to urea excretion in Alcolapia grahami. Taken together, the above findings suggest that the gills of this alkaline-lake-adapted species excrete urea constitutively via the specific facilitated urea transporter mtUT.

Amino Acid Sequence↗
PubMed2001

Oral gabapentin (neurontin) treatment of refractory genitourinary tract pain.

PURPOSE: Refractory genitourinary pain is a common but difficult condition to treat. Examples of chronic genitourinary pain include orchalgia, interstitial cystitis, pain after bladder suspension surgery, nonbacterial prostatitis, and genital pain related to lumbosacral neuropathy. We report our experience with oral gabapentin treatment for this condition. Gabapentin is an anticonvulsant with unclear but therapeutic effects on neurologic pain. MATERIALS AND METHODS: Twenty-one patients referred with refractory genitourinary pain were treated with oral gabapentin. There were 9 men and 12 women. In the male patients, the location of pain was testicle (4), bladder (2), penis (1), or prostate (2). In female patients, the pain was located in the urethra (4), bladder (6), vulva (1), or vagina (1). The dose of gabapentin was titrated from 300 up to 2,100 mg/day. Subjective pain severity and 10-cm visual pain scale was used before and 6 months after therapy. RESULTS: The mean dose of gabapentin was 1,200 mg/day (range 300-2,100 mg). Ten of 21 patients reported subjective improvement of their pain. The remaining patients did not perceive any improvement. Gabapentin was well tolerated; only 4 patients dropped out due to side effects. The most common adverse effects were dizziness and drowsiness. Five of 8 patients with interstitial cystitis reported improvement. CONCLUSIONS: Although only 10 of 21 patients improved with gabapentin, this cohort included only patients with refractory genitourinary pain that failed a wide range of prior treatments. Gabapentin belongs in the armaterium of the urologist who treats genitourinary pain.

Acetates↗
PubMed2000

In vivo characterization of renal iron transport in the anaesthetized rat.

1. In vivo microinjections of 55FeCl3 were made to assess renal iron (Fe2+/3+) transport in the anaesthetized rat. 2. Following microinjection into proximal convoluted tubules (PCTs), 18.5 +/- 2.9 % (mean +/- s.e.m., n = 11) of the 55Fe was recovered in the urine. This recovery was not dependent on the injection site indicating that iron is not reabsorbed across the surface convolutions of the proximal tubule. 3. Following microinjection into distal convoluted tubules (DCTs) 46.1 +/- 6.1 % (n = 8) of the injected 55Fe was recovered. Taken together the recovery data from the PCT and DCT microinjection studies indicate that the transport of iron occurs in the loop of Henle (LH) and collecting duct system. 4. In vivo luminal microperfusion was used to examine iron transport by the LH in more detail. In tubules perfused with 7 micromol l-1 55FeCl3, 52.7 +/- 8. 3 % (n = 8) of the perfused 55Fe was recovered in the collected fluid, indicating significant iron reabsorption in the LH. Addition of copper (Cu2+ as 7 micromol l-1 CuSO4), manganese (Mn2+ as 7 micromol l-1 MnSO4) or zinc (Zn2+ as 7 micromol l-1 ZnSO4) to the perfusate did not affect reabsorption of water, Na+ or K+, but increased recovery of 55Fe to 83.5 +/- 6.8 % (n = 8, P < 0.04), 75.8 +/- 5.9 (n = 6, not significant, n.s.) and 67.9 +/- 3.8; (n = 9, n.s. ), respectively. 5. Thus, iron transport in the LH can be reduced by the addition of copper or manganese to the luminal perfusate suggesting that these ions may compete with iron for a common transport pathway. However, this pathway may not be shared by zinc.

Anesthesia↗
PubMed2000

The HspR regulon of Streptomyces coelicolor: a role for the DnaK chaperone as a transcriptional co-repressordagger.

The dnaK operon of Streptomyces coelicolor encodes the DnaK chaperone machine and HspR, the transcriptional repressor of the operon; HspR confers repression by binding to several inverted repeat sequences in the promoter region, dnaKp. Here, we demonstrate that HspR specifically requires the presence of DnaK protein to retard a dnaKp fragment in gel-shift assays. This requirement is independent of the co-chaperones, DnaJ and GrpE, and it is ATP independent. Furthermore the retarded protein-DNA complex can be 'supershifted' by anti-DnaK monoclonal antibody, demonstrating that DnaK forms an integral component of the complex. It was shown in DNase I footprinting experiments that refolding and specific binding of HspR to its DNA target does not require DnaK. We conclude that the formation of the stable DnaK-HspR-DNA ternary complex does not depend on the chaperoning activity of DnaK. In affinity chromatography experiments using whole-cell extracts, DnaK was shown to co-purify with HspR, providing additional evidence that the two proteins interact in vivo; it was not possible to purify HspR away from DnaK in any experiments unless a powerful denaturant was used. The level of heat shock induction of chromosomal DnaK could be partially suppressed by expressing dnaK extrachromosomally from a heterologous promoter. In addition, it is shown that DnaK confers enhanced HspR-mediated repression of transcription in vitro. Taken together, these results suggest that DnaK functions as a transcriptional co-repressor by binding to HspR at its operator sites. In this model, the DnaK-HspR system would represent a novel example of feedback regulation of gene expression by a molecular chaperone, in which DnaK directly activates a repressor, rather than inactivates an activator (as is the case in the DnaK-sigma32 and Hsp70-HSF systems of other organisms).

Bacterial Proteins↗