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Non-technical summary Due to the conservation of developmental pathways and genetic material over the course of evolution, non-mammalian ‘model organisms’ such as the zebrafish embryo are emerging as valuable tools to explore causes and potential treatments for human diseases. Ion channels are proteins that form pores and help to establish and control electrical gradients by allowing the flow of ions across biological membranes. A diverse range of key physiological mechanisms in every organ in the body depends on the activity of ion channels. In this paper, we show that a potassium-selective channel that underlies salt reabsorption and potassium excretion in the human kidney is also expressed in zebrafish in cells that are important regulators of salt balance. Disruption of the channel's expression in zebrafish leads to effects on the activity of the heart, consistent with a role for this channel in the control of potassium balance in the embryo.

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common inherited renal diseases. It is associated with the progressive development of renal tubular cysts, which may subsequently lead to renal failure. Studies into the genetic basis of ADPKD have identified two genes, PKD1 and PKD2, that are mutated in ADPKD patients. The PKD1 and PKD2 genes encode for two different proteins, TRPP1 and TRPP2. Previous studies have demonstrated the presence of both TRPP1 and TRPP2 in the renal collecting duct cell line M8. The aim of the following study was to investigate the functional properties of cation currents in these cells and to examine the effect of overexpression of TRPP1 using a transgenic cell model (M7). In M8 cells, initial whole cell currents were low. However, over time there was activation of a flow-sensitive current, which was inhibited by gadolinium (IGd). The IGd was more selective for cations over anions, but did not discriminate between monovalent cations and was Ca2+ permeable. Activation of IGd was dependent on the presence of Ca2+ and also required dephosphorylation. The protein phosphatase 2A inhibitor okadaic acid prevented activation of IGd, suggesting that protein phosphatase 2A plays an important role in channel activation. The properties and magnitude of IGd were unaffected in M7 cells, suggesting that overexpression of TRPP1 was without effect. IGd was selectively inhibited by an antibody raised against the C-terminus of TRPP2. However, its selectivity profile was different to TRPP2, suggesting that it is attributable to a TRPP2-like channel or a TRPP2-containing heteromeric channel. In conclusion, these data describe the functional identification of a novel dephosphorylation- and flow-activated TRPP2-related channel in mouse collecting duct cells.

Facilitative urea transporters in the mammalian kidney play a vital role in the urinary concentrating mechanism. The urea transporters located in the renal inner medullary collecting duct, namely UT-A1 and UT-A3, are acutely regulated by the antidiuretic hormone vasopressin. In this study, we investigated the vasopressin regulation of the basolateral urea transporter UT-A3 using an MDCK-mUT-A3 cell line. Within 10 min, vasopressin stimulates urea flux through UT-A3 transporters already present at the plasma membrane, via a PKA-dependent process. Within 1 h, vasopressin significantly increases UT-A3 localization at the basolateral membrane, causing a further increase in urea transport. While the basic trafficking of UT-A3 to basolateral membranes involves both protein kinase C and calmodulin, its regulation by vasopressin specifically occurs through a casein kinase II-dependent pathway. In conclusion, this study details the effects of vasopressin on UT-A3 urea transporter function and hence its role in regulating urea permeability within the renal inner medullary collecting duct.

TWIK-1, a member of the two-pore domain K(+) channel family, is expressed in brain, kidney, and lung. The aim of this study was to examine the effect of loss of TWIK-1 on the renal cortical collecting duct. Ducts were isolated from wild-type and TWIK-1 knockout mice by enzyme digestion and whole-cell clamp obtained via the basolateral membrane. Current- and voltage-clamp approaches were used to examine K(+) conductances. No difference was observed between intercalated cells from wild-type or knockout ducts. In contrast, knockout principal cells were hyperpolarized compared to wild-type cells and had a reduced membrane conductance. This was a consequence of a fall in a barium-insensitive, quinidine-sensitive conductance (G (Quin)). G (Quin) demonstrated outward rectification and had a relatively low K(+) to Na(+) selectivity ratio. Loss of G (Quin) would be expected to lead to the hyperpolarization observed in knockout ducts by increasing fractional K(+) conductance and Na(+) uptake by the cell. Consistent with this hypothesis, knockout ducts had an increased diameter in comparison to wild-type ducts. These data suggest that G (Quin) contributes to the resting membrane potential in the cortical collecting duct and that a fall in G (Quin) could be an adaptive response in TWIK-1 knockout ducts.

The amphibian urea transporter (fUT) shares many properties with the mammalian urea transporters (UT) derived from UT-A and UT-B genes. The transport of urea by fUT is inhibited by the mercurial agent p-chloromercuribenzenesulfonic acid (pCMBS). We found that in oocytes expressing cRNA encoding fUT, a 5-min preincubation in 0.5 mM mercury chloride (HgCl2) also significantly reduced urea uptake. The transport of urea by fUT was rendered mercury (Hg2+) insensitive by mutating either of the residues C185 or H187, both of which lie within the M-I region (close to the hypothetical UT pore). In oocytes expressing a mixture of the C185 and H187 mutants, Hg2+sensitivity was reestablished. The transport of urea by the mouse UTs mUT-A2 and mUT-A3 was not sensitive to Hg2+. Introducing cysteine residues analogous to that mutated in fUT into mUT-A2 or mUT-A3 did not induce Hg2+sensitivity. Additionally, introducing the double cysteine, histidine mutations into mUT-A2 or mUT-A3 still did not induce Hg2+sensitivity, indicating that a region outside of the M-I region also contributes to the Hg2+-induced block of fUT. Using a series of chimeras formed between UT-A3 and fUT, we found that as well as C185 and H187, residues within the COOH terminal of fUT determine Hg2+sensitivity, and we propose that differences in the folding of this region between fUT and mUT-A2/mUT-A3 allow access of Hg2+to the fUT channel pore.

K+ channels play an important role in renal collecting duct cell function. The current study examined barium (Ba2+)-sensitive whole-cell K+ currents (IKBa) in mouse isolated collecting duct principal cells. IKBa demonstrated strong inward rectification and was inhibited by Ba2+ in a dose- and voltage-dependent fashion, with the K d decreasing with hyperpolarization. The electrical distance of block by Ba2+ was around 8.5%. As expected for voltage-dependent inhibition, the association constant increased with hyperpolarization, suggesting that the rate of Ba2+ entry was increased at negative potentials. The dissociation constant also increased with hyperpolarization, consistent with the movement of Ba2+ ions into the intracellular compartment at negative potentials. These properties are not consistent with ROMK but are consistent with the properties of Kir2.3. Kir2.3 is thought to be the dominant basolateral K+ channel in principal cells. This study provides functional evidence for the expression of Kir2.3 in mouse cortical collecting ducts and confirms the expression of Kir2.3 in this segment of the renal tubule using reverse-transcriptase polymerase chain reaction. The conductance described here is the first report of a macroscopic K+ conductance in mouse principal cells that shares the biophysical profile of Kir2.3. The properties and dominant nature of the conductance suggest that it plays an important role in K+ handling in the principal cells of the cortical collecting duct.

The early distal tubule (EDT) of the frog nephron, similar to the thick ascending limb in mammals, mediates the transepithelial absorption of NaCl. The continued absorption of NaCl in the face of varying Na+load is maintained by coordination of the activity of ion-transporting proteins in the apical and basolateral membranes, so-called pump-leak coupling. Previous studies identified intracellular Ca2+, originating from an intracellular Ca2+store, as playing a key role in pump-leak coupling in the EDT (Cooper GJ, Fowler MR, and Hunter M. Pflügers Arch 442: 243–247, 2001). The purpose of the experiments described in this paper was to identify the intracellular Ca2+storage pools in the renal diluting segment. Store Ca2+movements were monitored by the fluorescence of mag-fura 2 in permeabilized segments of frog EDTs. The presence of both ATP and Ca2+was required to maintain store Ca2+content. Removal of either of these substrates resulted in a passive leak of Ca2+from the stores. The uptake of Ca2+into the store was sensitive to the SERCA inhibitor 2,5-di( tert-butyl) hydroquinone, whereas Ca2+release from the store was stimulated by IP3but not cADPR. Store Ca2+was insensitive to the mitochondrial ATP synthase inhibitor oligomycin, and, under conditions that energized Δψm, the complex 1 inhibitor rotenone and the protonophore FCCP. Ionomycin was able to mobilize store Ca2+following exposure to IP3. These results suggest that the endoplasmic reticulum is a dominant Ca2+store in the frog EDT. A second pool, sensitive to ionomycin but not IP3, may overlap with the IP3-sensitve pool. The data also rule out any contribution by mitochondria to EDT Ca2+cycling.

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.

Adrenomedullin is a 52-amino acid peptide first described in a human phaeochromocytoma but since been found to be present in many tissues, including the vascular system and bone. Because of its structural similarity to amylin and calcitonin gene-related peptide, both of which have actions on bone cells, we have previously assessed the effects of adrenomedullin on the skeleton, and found that it increases osteoblast proliferation in vitro and bone formation following local injection in vivo. The present study carries this work forward by assessing the effects on bone of the systemic administration of a fragment of this peptide lacking the structural requirements for vasodilator activity. Two groups of 20 adult male mice received 20 injections of human adrenomedullin(27-52) 8.1 microg or vehicle over a 4-week period and bone histomorphometry and strength were assessed. In the tibia, adrenomedullin(27-52) produced increases in the indices of osteoblast activity, osteoid perimeter and osteoblast perimeter (P

The activities of transport mechanisms in epithelial cells are generally coordinated in order to minimise disturbances in cellular ion content and volume. Furosemide, a potent inhibitor of transport in the renal diluting segment, up-regulates apical K+ channel activity following the release of calcium from intracellular stores. The signal pathway between furosemide application and this calcium release is not known. Single early distal tubule segments from frog kidney were permeabilised with saponin in order to monitor calcium levels within cytoplasmic stores using the calcium-sensitive dye, mag-fura. The uptake (or release) of calcium to (or from) stores was initiated by adding agents to the bath solution, which is in direct contact with the intracellular organelles. ATP promoted calcium uptake into stores, whereas ATP removal led to a slower, spontaneous calcium release. Following loading, calcium stores could be rapidly depleted by inositol 1,4,5-trisphosphate (IP3), but not ryanodine. Calcium release was evident upon lowering the "intracellular" chloride concentration from 12 to 4 mM, equivalent to the fall in chloride induced by furosemide in intact cells. These results suggest that intracellular chloride may function as a second messenger, mediating cross-talk between the apical membrane and intracellular calcium stores.

Background Despite the predominance of superficial injuries after explosive blast exposure, major morbidity or mortality among immediate survivors is caused by delayed perforation of intestinal mural contusions. Previous studies have suggested that small bowel and colonic contusions larger than 10 mm in diameter are at high risk. This experimental study aimed to identify contusions at high risk of late perforation. Methods Histological features of injury were classified in 188 blast-induced intestinal contusions in 16 anaesthetized Large White pigs. Results Some 16 per cent of small bowel and 12 per cent of colonic contusions were at high risk of late perforation. Small bowel contusions larger than 15 mm in diameter had a worse histological grading than those smaller than 15 mm (X 2 = 0.09, 2 d.f., P = 0.01). Contusions that extended over more than half the bowel circumference (X 2 =14.79, 2 d.f., P = 0.0006) and those affecting the mesenteric border (X 2 = 7.5, 2 d.f., P= 0.024) were more severe injuries. Colonic contusions larger than 20 mm in diameter had a worse histological grading than smaller ones (X 2 =14.95, 2 d.f., P = 0.0006). Confluent, rather than diffuse, colonic contusions were more severe injuries (X 2 = 6.37, 2 d.f., P = 0.04). Conclusion Once identified at laparotomy, the number of small bowel contusions requiring excision may he reduced from 86 to 60 per cent; similarly, excision of colonic contusions can be reduced from 73 to 27 per cent if small bowel contusions smaller than 15 mm in diameter and colonic contusions of less than 20 mm are managed conservatively.

This paper considers the wounding effects of small fragments in modern warfare. Small fragment wounds may be expected to predominate on a future conventional battlefield; however, studies and models of "military" wounds have tended to focus on bullets as the wounding projectile. This paper discusses briefly the types of fragment projectile expected from modern munitions. It goes on to define a model for such projectiles, and describes the interaction with soft tissue simulants. The extent of penetration, temporary cavitation, and contamination by foreign material are all considered. This work with simulants is validated by experimental shots against animal tissue. A wound model in an experimental animal is described. This model was used to investigate the hematologic, biochemical, and histologic effects of a small fragment wound. The effects on skin and skeletal muscle are described. By sampling at various times (up to 1 week) after wounding, the natural progress of these wounds has been ascertained. The results from 28 experimental animals, with untreated fragment wounds, are reported. The most important findings are that the skin damage is very localized and that the muscle damage is limited, with little necrotic tissue in the track. Furthermore, the extent of the muscle damage, peripheral to the wound track, improves with time, healing within a few days, provided the wound remains free from infection. There was no clinical or microbiologic evidence of infection in those animals followed for up to 3 days. However, of eight animals followed to 1 week, three developed infected wounds. This work has implications for the management of soft tissue wounds caused by fragmentation munitions. The conventional military approach has been to treat penetrating war wounds by exploration, debridement, excision of dead tissue, and delayed primary closure; conservative treatment has largely been regarded as inappropriate. The work presented here shows that the potential culture medium within the wound is small and can be removed by the normal bodily responses. There is no need for surgery, provided that infection can be prevented. It may be inferred that if bacterial colonization can be prevented in the early stages by the timely use of antibiotics, surgery may be unnecessary. Further studies are planned to investigate this possibility.

In war, uncomplicated penetrating injuries to limbs require evacuation to a surgical facility. A delay is inevitable between injury and definitive surgical treatment. This paper describes an experimental model that has been developed to assess the efficacy of antibiotics in such war wounds ; the aim is to develop a treatment protocol to prevent the development of infection before casualties reach a surgical facility. The model is described in detail. Its use to determine efficacy of an antibiotic regimen, and the consequences of delay in the initial administration of antibiotie, are summarized. Streptococcus Lance field Group L (Strep L) is a pig pathogen analogous to the Streptococcus pyogenes Lancefield Group A in man. The latter is perceived as a major threat to the casualty with a neglected penetrating wound. Strep L caused reproducible infection in a low-energy-transfer fragment wound to a pig thigh. When the wound was observed over a period of 7 days, the only other organisms that appeared consistently were pig pathogenic and nonpathogenic staphylococcal species. Despite the site of the wound, fecal organisms were not observed to colonize the wound significantly, neither were they a cause of early wound infection. The treatment regimen studied was benzylpenicillin, 1 megaunit (6 hourly), given intramuscularly. When commenced 1 hour after wounding, this prevented wound infection for 3 days, when Strep L was deliberately inoculated at the time of wounding. If the start of antibiotics was delayed until 6 hours after wounding, antibiotic regimen was less effective ; the wound track remained infected at 3 days. In a further series of experiments, Staphylococcus hyicus, a potential pig pathogen (which is penicillin resistant through β-lactamase production), was introduced in addition to Strep L. The antibiotic regimen remained effective when the Strep L plus 10 3 S. hyicus were inoculated. When 10 4 S. hyicus were introduced, treatment was less effective, but still of some benefit. Inoculation of >10 5 S. hyicus rendered treatment commencing at 1 hour ineffective. If the start of treatment was delayed to 6 hours after injury, this treatment regimen was adversely affected by the introduction of only 10 3 S. hyicus. In conclusion, an intramuscular benzylpenicillin regimen, commenced 1 hour after wounding, can prevent infection in penetrating soft tissue missile wounds for up to 3 days. A delay of 6 hours renders treatment ineffective.

The mechanism of traumatic amputation of limbs by explosion is presented. A survey of blast casualties from Northern Ireland revealed that amputations through joints were very uncommon--the principal site was through the shaft of the long bones. Computer modelling of a bone exposed to blast forces reinforced the hypothesis developed from the casualty survey, that the primary mechanism of the bone injury was the direct coupling of the blast wave into the tissues. The fracture occurs from the resulting axial stresses in the bone, prior to limb flailing from the gas flow over the limb. The gas flow completes the amputation. Field trials employing a goat hind limb model have confirmed the hypothesis. Having identified the mechanism, concepts to develop protective clothing may now be proposed.

This article examines stability properties of some linear iterative schemes that have been proposed for the solution of the nonlinear algebraic equations arising in the use of implicit Runge-Kutta methods to solve a differential systemx′ =f(x). Each iteration step requires the solution of a set of linear equations, with constant matrixI −hλJ, whereJ is the Jacobian off evaluated at some fixed point. It is shown that the stability properties of a Runge-Kutta method can be preserved only if λ is an eigenvalue of the coefficient matrixA. SupposeA has minimal polynomial (x − λ)mp(x),p(λ) ≠ 0. Then stability can be preserved only if the order of the method is at mostm + 2 (at mostm + 1 except for one case).

Although the outcome of patients transplanted after univentricular cardiac support is similar to that of conventional patients, death on the device remains a substantial problem. The wearable Novacor left ventricular assist system (LVAS) may offer advantages over console-based systems by improving rehabilitation before transplantation. For these advantages to be realised, however, a smooth perioperative course is necessary. We describe our operative technique, based on minimising blood loss and preserving right ventricular function, and the results in the first three patients to have the wearable Novacor LVAS implanted in this institution.

1. Intracellular pH (pHi) regulation was investigated in frog early distal tubule. Single tubules were dissected and perfused, such that the compositions of apical and basolateral solutions could be varied independently. pHi was measured using the fluorescent probe 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). 2. Brief exposure to NH4+ on the basolateral aspect of the tubules elicited an intracellular acidification, followed by an active recovery. The recovery was inhibited by amiloride and its analogue 5-(N-ethyl-N-isopropyl) amiloride (EIPA) when added to the basolateral, but not the apical, solution. Omission of Na+ from the basolateral solution alone completely inhibited pHi recovery. Thus the Na(+)-H+ exchangers appear to be located on the basolateral membrane. 3. Neither amiloride nor EIPA had any effect on pHi under control conditions, suggesting that the activity of the Na(+)-H+ exchangers at the resting pHi is low. However, removal of basolateral Na+ caused an acidification that was blocked by amiloride, indicating that the Na(+)-H+ exchangers can be activated from the resting state. 4. Intrinsic buffering power (beta i) was determined by stepwise removal of ammonium from the cells in Na(+)-free conditions, to prevent pH regulation, and in the presence of Ba2+ and furosemide (frusemide), to inhibit ammonium transport. beta i was a function of pHi, increasing as pHi decreased. 5. Proton efflux was calculated during the recovery from an acid load in tubules from normal and K(+)-loaded frogs and in tubules which had been incubated for 30 min with aldosterone. Potassium loading produces a chronic increase in plasma aldosterone. Both acute and chronic aldosterone treatment caused an intracellular alkalinization. This was due to an alkaline shift in the set-point of the basolateral Na(+)-H+ exchanger, with no change in the density and/or turnover rate.

After vascular injury, intraluminal arterial shunting may be employed if definitive surgery must be delayed. This paper describes use of a heparin-bonded polyurethane shunt to restore distal circulation after ligation of the femoral artery in sheep. In studies to determine tissue viability for 12 h after insertion into the femoral artery, five of seven shunts remained patent. In another series of experiments to study tissue viability after limb ischaemia, the femoral artery was ligated and a tight Esmarch bandage applied to the limb for 6 h before shunt insertion. The shunt remained patent for 12 h in five of six cases. Nearly all shunt failures occurred shortly after placement and were attributable to intimal damage arising from difficulties during insertion.

The nonlinear equations, arising in the implementation of implicit Runge-Kutta methods, may be solved by a modified Newton iteration, but alternative iteration schemes have been suggested to reduce the linear algebra costs. A linear iteration scheme is examined in this article. When applied to an s -stage Runge-Kutta method, each step of the iteration requires s function evaluations and the solution of s sets of linear equations. For the scalar differential equation x ′ = qx , the convergence rate of the scheme depends on the spectral radius π[ M ( z )] of the iteration matrix M , a function of z = hq where h is the steplength. A lower bound for π[ M ( z )] is established and new schemes are obtained for the two-stage Gauss method by minimizing the supremum of this lower bound over regions of the complex plane. In one scheme the supremum on the negative real axis is minimized. The iteration scheme is generalized in order to obtain improved convergence rates. When applied to an s -stage Runge-Kutta method, each step of this new scheme still requires just s function evaluations. However r sets of linear equations, r > s , have to be solved in each step. Some results are obtained for the Gauss methods and some numerical experiments reported.

This chapter outlines the approach to mental health care developed and currently being implemented by Pathways New Zealand for reducing disease risk factors in patients treated for mental health problems. Pathways New Zealand was formed in 1989 following the closure of the major mental service facility for the Waikato-Hauraki Region of New Zealand, Tokonui Hospital. Since that time Pathways has grown to a national level service offering services to its clients ranging from 24-hour supported accommodation, through healthy lifestyles programs, to outcomes based services including patient access to and involvement in the management of their medical and personal history data (ICAN). Gavin Cooper, Pathways Housing Management Coordinator for the Waikato-Hauraki Region, in conjunction with the Waikato Institute of Technology (WINTEC) has developed a holistic system for the treatment of environmentally induced mental illness that includes chemical treatment, exercise programs, self-help training and community support. The results of a two year program of research into the impact of this program are reported on in this chapter, and its suitability for wider adoption discussed. These comments are partly based on research statistics provided by the Centre for Sports Exercise Science (WINTEC) and Mike Dove, Team Leader Residential, Pathways.

Two slightly different test problems have been used to examine nonlinear stability behaviour of numerical methods for solving systems of ordinary differential equations. These test problems, for BN-stability and for monotonicity, both lead to the concept of algebraic stability. In this paper, it is shown that these two problems may be combined to yield weaker stability conditions. Nonautonomous systems give a more restrictive stability condition than autonomous systems. Irreducible methods with some zero quadrature weights can be stable in this weak sense

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common inherited renal diseases. It is associated with the progressive development of renal tubular cysts, which may subsequently lead to renal failure. Studies into the genetic basis of ADPKD have identified two genes, PKD1 and PKD2, that are mutated in ADPKD patients. The PKD1 and PKD2 genes encode for two different proteins, TRPP1 and TRPP2. Previous studies have demonstrated the presence of both TRPP1 and TRPP2 in the renal collecting duct cell line M8. The aim of the following study was to investigate the functional properties of cation currents in these cells and to examine the effect of overexpression of TRPP1 using a transgenic cell model (M7). In M8 cells, initial whole cell currents were low. However, over time there was activation of a flow-sensitive current, which was inhibited by gadolinium (I(Gd)). The I(Gd) was more selective for cations over anions, but did not discriminate between monovalent cations and was Ca2+ permeable. Activation of I(Gd) was dependent on the presence of Ca2+ and also required dephosphorylation. The protein phosphatase 2A inhibitor okadaic acid prevented activation of I(Gd), suggesting that protein phosphatase 2A plays an important role in channel activation. The properties and magnitude of I(Gd) were unaffected in M7 cells, suggesting that overexpression of TRPP1 was without effect. I(Gd) was selectively inhibited by an antibody raised against the C-terminus of TRPP2. However, its selectivity profile was different to TRPP2, suggesting that it is attributable to a TRPP2-like channel or a TRPP2-containing heteromeric channel. In conclusion, these data describe the functional identification of a novel dephosphorylation- and flow-activated TRPP2-related channel in mouse collecting duct cells.

The prevalence of anti-hepatitis C virus (HCV) enzyme immunoassay (EIA)- positive in 167,511 Australian volunteer blood donors from Adelaide, Melbourne, Perth, and Sydney was 0.78%. One thousand two-hundred and eighteen EIA-positive serum samples were assessed by supplemental tests including a blocking EIA and two peptide EIAs corresponding to major epitopes of the HCV C-100–3 antigen. Seven hundred and eighteen samples (59%) were negative by supplemental testing; no evidence of reactivity with other HCV gene products or HCV RNA detected by cDNA polymerase chain reaction was found in selected samples from this group. In contrast, of 43 samples randomly selected from 400 samples (32.8%) positive by supplemental testing, 88% were reactive with HCV 33-C or core antigens and 52% contained HCV RNA, suggesting contact with HCV and infectivity of most donors in this group. Most samples equivocal by supplemental testing reacted only with C-100 and not with other HCV antigens when tested by dot immunoblot assay. Only 21% had detectable HCV RNA. The battery of assays used in this study indicated that approximately 32% of HCV EIA repeatably reactive serum samples were serologically related to HCV, corresponding to a 0.25% prevalence of potentially infectious donors.

A modified Newton method is often used to solve the algebraic equations that arise in the application of implicit Runge-Kutta methods. When the Runge-Kutta method has a coefficient matrix A with a single point spectrum (with eigenvalue λ \lambda ), the efficiency of the modified Newton method is much improved by using a similarity transformation of A. Each iteration involves vector transformations. In this article an alternative iteration scheme is obtained which does not require vector transformations and which is simpler in other respects also. Both schemes converge in a finite number of iterations when applied to linear systems of differential equations, but the new scheme uses the nilpotency of A − λ I A - \lambda I to achieve this. Numerical results confirm the predicted convergence for nonlinear systems and indicate that the scheme may be a useful alternative to the modified Newton method for low-dimensional systems. The scheme seems to become less effective as the dimension increases. However, it has clear advantages for parallel computation, making it competitive for high-dimensional systems.

The computational work required to implement implicit Runge-Kutta methods is often dominated by the cost of solving large sets of nonlinear equations. As an alternative to modified Newton methods, iteration schemes, which sacrifice superlinear convergence for reduced linear algebra costs, have been proposed. A new scheme of this type is considered here. This scheme avoids expensive vector transformations, is computationally more efficient, and gives improved performance.

Injuries to the intestine and mesentery are often found in patients undergoing laparotomy for blunt abdominal trauma. Although treatment of perforations is relatively straightforward, the same is not true for contusions. Few guidelines exist at present to aid the surgeon in deciding which injuries require resection in order to avoid the complications of delayed perforation and late stricture formation. The natural history of these non-perforating intestinal and mesenteric injuries has been examined in an experimental model to identify possible criteria on which future management can be based. In the immediate postinjury period peristalsis and local mesenteric pulsation were absent in the majority of injuries which went on to full recovery and these observations are thus of little predictive value in predicting outcome. The initial size of contusion (length of contusion along longitudinal axis of bowel) relative to bowel wall circumference (BWC) was related to complications as follows: contusion < BWC (n = 47) - one complication; contusion > BWC (n = 8) - three complications (P = 0·02). Similarly, six mesenteric injuries which produced an initial ischaemia (assessed by fluorescein) less than twice the BWC did not result in any complications, compared with four complications which occurred in ten cases when the initial ischaemia was greater than twice the BWC. These results go some way towards providing a better understanding of these injuries and in turn may help the emergency surgeon in deciding which injuries require resection.

It is apparent from review of published papers and books that misunderstanding and confusion exists in the minds of many authors describing the interaction of penetrating missiles with tissues. These misapprehensions may influence the management of wounds by suggesting didactic approaches based upon a preconceived notion of the nature and severity of the wound for different types of projectiles. This review considers the biophysics of penetrating missile wounds, highlights some of the more common misconceptions and seeks to reconcile the conflicting and confusing management doctrines that are promulgated in the literature - differences that arise not only from two scenarios, peace and war, but also from misapprehensions of the wounding process. Wounds of war and of peacetime differ both in the nature of the wound and in the propensity for wound infection. Additionally, the limitations imposed by war dictate the type of management that may be practised and result in procedures that would be considered inappropriate by some in civilian clinical practice. Many of the procedures described in civilian peacetime settings, such as reliance on antibiotics alone for the control of infection in penetrating wounds, or minimal excision and debridement, can yield good results but would herald disaster if transposed to a war setting.

We report here the application of a previously described method to directly determine the CO2 permeability (P(CO2)) of the cell membranes of normal human red blood cells (RBCs) vs. those deficient in aquaporin 1 (AQP1), as well as AQP1-expressing Xenopus laevis oocytes. This method measures the exchange of (18)O between CO2, HCO3(-), and H2O in cell suspensions. In addition, we measure the alkaline surface pH (pH(S)) transients caused by the dominant effect of entry of CO2 vs. HCO3(-) into oocytes exposed to step increases in [CO2]. We report that 1) AQP1 constitutes the major pathway for molecular CO2 in human RBCs; lack of AQP1 reduces P(CO2) from the normal value of 0.15 +/- 0.08 (SD; n=85) cm/s by 60% to 0.06 cm/s. Expression of AQP1 in oocytes increases P(CO2) 2-fold and doubles the alkaline pH(S) gradient. 2) pCMBS, an inhibitor of the AQP1 water channel, reduces P(CO2) of RBCs solely by action on AQP1 as it has no effect in AQP1-deficient RBCs. 3) P(CO2) determinations of RBCs and pH(S) measurements of oocytes indicate that DIDS inhibits the CO2 pathway of AQP1 by half. 4) RBCs have at least one other DIDS-sensitive pathway for CO2. We conclude that AQP1 is responsible for 60% of the high P(CO2) of red cells and that another, so far unidentified, CO2 pathway is present in this membrane that may account for at least 30% of total P(CO2).

Facilitative UT-A urea transporters play a central role in the urinary concentrating mechanism. There are three major UT-A isoforms found in the mouse kidney: mUT-A1, mUT-A2, and mUT-A3. The major aim of this study was to identify the location and function of mUT-A3. UT-A proteins were investigated using three novel mouse UT-A-targeted antibodies: ML446, MQ2, and ML194. ML446 detected mUT-A1 and mUT-A3. ML194 detected mUT-A1 and mUT-A2. Importantly, MQ2 was found to be selective for mUT-A3. MQ2 detected a 45- to 65-kDa signal in the mouse kidney inner medulla, which was deglycosylated to a 40-kDa protein band. Immunolocalization studies showed that mUT-A3 was strongly detected in the papillary tip, mainly in the basolateral regions of inner medullary collecting duct (IMCD) cells. Immunoblotting of subcellular fractions of inner medullary protein suggested that in mouse kidney mUT-A3 was present in plasma membranes. Consistent with this, immunoelectron microscopy demonstrated that mUT-A3 was predominantly localized at the basal plasma membrane domains of the IMCD cells in mouse kidney. Heterologous expression of mUT-A3-enhanced green fluorescent protein in Madin-Darby canine kidney cells showed that the protein localized to the basolateral membrane. In conclusion, our study indicates that mUT-A3 is a basolateral membrane transporter expressed in IMCD cells.

During maturation of oocytes, Cl− conductance ( G Cl) oscillates and intracellular pH (pHi) increases. Elevating pHi permits the protein synthesis essential to maturation. To examine whether changes in G Cl and pHi are coupled, the Cl− channel ClC-0 was heterologously expressed. Overexpressing ClC-0 elevates pHi, decreases intracellular Cl− concentration ([Cl−]i), and reduces volume. Acute acidification with butyrate does not activate acid extrusion in ClC-0-expressing or control oocytes. The ClC-0-induced pHichange increases after overnight incubation at extracellular pH 8.5 but is unaltered after incubation at extracellular pH 6.5. Membrane depolarization did not change pHi. In contrast, hyperpolarization elevates pHi. Thus neither membrane depolarization nor acute activation of acid extrusion accounts for the ClC-0-dependent alkalinization. Overnight incubation in low extracellular Cl− concentration increases pHiand decreases [Cl−]i in control and ClC-0 expressing oocytes, with the effect greater in the latter. Incubation in hypotonic, low extracellular Cl− solutions prevented pHi elevation, although the decrease in [Cl−]i persisted. Taken together, our observations suggest that KCl loss leads to oocyte shrinkage, which transiently activates acid extrusion. In conclusion, expressing ClC-0 in oocytes increases pHi and decreases [Cl−]i. These parameters are coupled via shrinkage activation of proton extrusion. Normal, cyclical changes of oocyte G Cl may exert an effect on pHi via shrinkage, thus inducing meiotic maturation.

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.

For almost a century it was generally assumed that the lipid phases of all biological membranes are freely permeable to gases. However, recent observations challenge this dogma. The apical membranes of epithelial cells exposed to hostile environments, such as gastric glands, have no demonstrable permeability to the gases CO2 and NH3. Additionally, the water channel protein aquaporin 1 (AQP1), expressed at high levels in erythrocytes, can increase membrane CO2 permeability when expressed in Xenopus oocytes. Similarly, nodulin-26, which is closely related to AQP1, can act as a conduit for NH3. A key question is whether aquaporins, which are abundant in virtually every tissue that transports O2 and CO2 at high levels, ever play a physiologically significant role in the transport of small volatile molecules. Preliminary data are consistent with the hypothesis that AQP1 enhances the reabsorption of HCO3- by the renal proximal tubule by increasing the CO2 permeability of the apical membrane. Other preliminary data on Xenopus oocytes heterologously expressing the electrogenic Na+-HCO3- cotransporter (NBC), AQP1 and carbonic anhydrases are consistent with the hypothesis that the macroscopic cotransport of Na+ plus two HCO3- occurs as NBC transports Na+ plus CO3(2-) and AQP1 transports CO2 and H2O. Although data - obtained on AQP1 reconstituted into liposomes or on materials from AQP1 knockout mice - appear inconsistent with the model that AQP1 mediates substantial CO2 transport in certain preparations, the existence of unstirred layers or perfusion-limited conditions may have masked the contribution of AQP1 to CO2 permeability.

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.

This study aimed to identify genes or gene products associated with high lean muscle mass in bovines that may serve as potential markers for selection. An animal with a genetic predisposition to high lean muscle mass, the Belgium Blue, was chosen as a model to compare with the Holstein Friesian, a model that does not. Two-dimensional polyacrylamide gel electrophoresis analysis was utilized to compare the exhibited skeletal muscle proteome between the two animal types at two stages of foetal development. A previously uncharacterized polymorphism of a high expression myofibrillar protein, myosin light chain 1 fast (MLC-1f), was observed. The characterization of this polymorphism revealed a two amino acid insertion in a part of the protein that has been implicated in modulating myosin S1 ATPase activity. Furthermore, this polymorphism was shown to be the product of two alleles that are codominant. Screening studies were carried out on selected herds and showed a very high frequency of one allele. Both isoforms of MLC-1f were produced by recombinant means and purified. The recombinant proteins were exchanged into purified myosin hexamers that were then subject to assays measuring ATP consumption. The sensitivity of the assay utilized could not reveal any significant difference in ATPase activity between hexamers containing one or the other isoform.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder accounting for about 50% of all dementias, yet its pathogenic mechanisms remain poorly understood. In order to provide a more complete picture of pathogenesis in AD, we analysed six human brain regions for alterations in their proteomes. Quantitative proteome analysis was used to compare signals corresponding to individual proteins between post mortem brain tissues from persons with AD, and those from age-matched nondemented control (NC) tissues. In severely injured brain regions, 76 proteins were differentially expressed in AD hippocampus compared with NC, 62 proteins were differentially expressed in temporal cortex, and 39 proteins were differentially expressed in entorhinal cortex. Significant differences were also present in relatively spared regions. Thus, 34 proteins were differentially expressed in AD cerebellum compared with NC, 125 proteins were differentially expressed in cingulate gyrus, and 75 proteins were differentially expressed in sensorimotor cortex. The identity of 37 of these proteins was determined, and the possible relevance of changes in key pathogenic pathways analysed. These studies provide a unique snapshot illustrating the complexity of interrelated disease mechanisms at work in a complex, multifactorial disease, and show that comparative proteome analysis is a method with the power to develop important new insights into pathogenic mechanisms in the dementias.

Pancreatic islet beta-cells secrete the hormones insulin, amylin and pancreastatin. To search for further beta-cell hormones, we purified peptides from secretory granules isolated from cultured murine beta TC6-F7 beta-cells. We identified a 34-amino-acid peptide (3948 Da), corresponding to Asp(69)-Leu(102) of the proinsulin-like growth factor II E-peptide, which we have termed 'preptin'. Preptin, is present in islet beta-cells and undergoes glucose-mediated co-secretion with insulin. Synthetic preptin increases insulin secretion from glucose-stimulated beta TC6-F7 cells in a concentration-dependent and saturable manner. Preptin infusion into the isolated, perfused rat pancreas increases the second phase of glucose-mediated insulin secretion by 30%, while anti-preptin immunoglobulin infusion decreases the first and second phases of insulin secretion by 29 and 26% respectively. These findings suggest that preptin is a physiological amplifier of glucose-mediated insulin secretion.

The major intrinsic protein (MIP) of lens fiber cells is a member of the aquaporin (AQP) water channel family. The protein is expressed at very high levels in lens fiber cells, but its physiological function is unclear. By homology to known AQPs, we have cloned a full-length cDNA encoding an MIP from the lens of killifish ( Fundulus heteroclitus). The predicted protein (263 amino acids; GenBank accession no. AF191906 ) shows 77% identity to amphibian MIPs, 70% identity to mammalian MIPs, and 46% identity to mammalian AQP1. Expression of MIPfun in Xenopus laevis oocytes causes an ∼40-fold increase in oocyte water permeability. This stimulation is comparable to that seen with AQP1 and substantially larger than that seen with other MIPs. The mercurials HgCl2and p-chloromercuribenzenesulfonate inhibit the water permeability of MIPfun by ∼25%. MIPfun is not permeable to glycerol, urea, or formic acid but is weakly permeable to CO2.

One possible factor resulting in delays in using reperfusion therapy in patients with acute myocardial infarction (AMI) is the failure to recognize cardiac symptomatology early in certain subgroups of patients. These patients may undergo extensive evaluation for gastrointestinal or musculoskeletal complaints before it is recognized that they are suffering from an AMI.The records of patients (52% Black and 48% White; 49% male and 51% female) presenting to an urban teaching hospital with enzyme documented myocardial infarctions were retrospectively examined for traditional elements of atypia in their initial chest pain descriptions to the emergency department (ED). The rate of reperfusion therapy utilization was also determined for this group.Of the patients meeting the study criteria (166 total), 43% were found to have atypical elements in the character of their pain description. This high prevalence of atypia also coincided with a low reperfusion intervention rate of 38%. In examining the subgroups, it appears that African Americans and women had the highest rates of atypical pain (56% and 46%, respectively) while also having the lowest utilization rates for reperfusion therapies (31% and 33%, respectively). This compares to rates of 48% for Whites and 60% for White males who had more typical chest pain.While no direct correlation can be drawn from the data, it has been suggested that atypical presentations may result in early failure to recognize myocardial infarction and cause delay in or prevent appropriate therapy. It is thought that chest pain should be de-emphasized as a part of the indication criteria for thrombolytics and emergent angioplasty.