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PubMed2006

Vasa recta voltage-gated Na+ channel Nav1.3 is regulated by calmodulin.

Rat descending vasa recta (DVR) express a tetrodotoxin (TTX)-sensitive voltage-operated Na(+) (Na(V)) conductance. We examined expression of Na(V) isoforms in DVR and tested for regulation of Na(V) currents by calmodulin (CaM). RT-PCR in isolated permeabilized DVR using degenerate primers targeted to TTX-sensitive isoforms amplified a product whose sequence identified only Na(V)1.3. Immunoblot of outer medullary homogenate verified Na(V)1.3 expression, and fluorescent immunochemistry showed Na(V)1.3 expression in isolated vessels. Immunochemistry in outer medullary serial sections confirmed that Na(V)1.3 is confined to alpha-smooth muscle actin-positive vascular bundles. Na(V)1.3 possesses a COOH-terminal CaM binding motifs. Using pull-down assays and immunoprecipitation experiments, we verified that CaM binds to either full-length Na(V)1.3 or a GST-Na(V)1.3 COOH-terminal fusion protein. In patch-clamp experiments, Na(V) currents were suppressed by calmodulin inhibitory peptide (CIP; 100 nM) or the CaM inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalene-sulphonamide hydrochloride (W7). Neither CIP nor W7 altered the voltage dependence of pericyte Na(V) currents; however, raising electrode free Ca(2+) from 20 to approximately 2,000 nM produced a depolarizing shift of activation. In vitro binding of CaM to GST-Na(V)1.3C was not affected by Ca(2+) concentration. We conclude that Na(V)1.3 is expressed by DVR, binds to CaM, and is regulated by CaM and Ca(2+). Inhibition of CaM binding suppresses pericyte Na(V) currents.

Animals↗
PubMed2004

Expression of endothelial nitric oxide synthase in developing rat kidney.

Endothelium-derived nitric oxide (NO) is synthesized within the developing kidney and may play a crucial role in the regulation of renal hemodynamics. The purpose of this study was to establish the expression and intrarenal localization of the NO-synthesizing enzyme endothelial NO synthase (eNOS) during kidney development. Rat kidneys from 14 (E14)-, 16 (E16)-, 18 (E18)-, and 20-day-old (E20) fetuses and 1 (P1)-, 3 (P3)-, 7 (P7)-, 14 (P14)-, and 21-day-old (P21) pups were processed for immunocytochemical and immunoblot analysis. In fetal kidneys, expression of eNOS was first observed in the endothelial cells of the undifferentiated intrarenal capillary network at E14. At E16, strong eNOS immunoreactivity was observed in the endothelial cells of renal vesicles, S-shaped bodies (stage II glomeruli), and stage III glomeruli at the corticomedullary junction. At E18-20, early-stage developing glomeruli located in the subcapsular region showed less strong eNOS immunoreactivity than those of E16. The eNOS-positive immature glomeruli were observed in the nephrogenic zone until 7 days after birth. In fetal kidneys, eNOS was also expressed in the medulla in the endothelial cells of the capillaries surrounding medullary collecting ducts. After birth, eNOS immunostaining gradually increased in the developing vascular bundles and peritubular capillaries in the medulla and was highest at P21. Surprisingly, eNOS was also expressed in proximal tubules, in the endocytic vacuolar apparatus, only at P1. The strong expression of eNOS in the early stages of developing glomeruli and vasculature suggests that eNOS may play a role in regulating renal hemodynamics of the immature kidney.

Age Factors↗
PubMed2002

Expression of urea transporters in the developing rat kidney.

Urea transport in the kidney is mediated by a family of transporter proteins that includes renal urea transporters (UT-A) and erythrocyte urea transporters (UT-B). Because newborn rats are not capable of producing concentrated urine, we examined the time of expression and the distribution of UT-A and UT-B in the developing rat kidney by light and electron microscopic immunocytochemistry. Kidneys from 16-, 18-, and 20-day-old fetuses, 1-, 4-, 7-, 14-, and 21-day-old pups, and adult animals were studied. In the adult kidney, UT-A was expressed intensely in the inner medullary collecting duct (IMCD) and terminal portion of the short-loop descending thin limb (DTL) and weakly in long-loop DTL in the outer part of the inner medulla. UT-A immunoreactivity was not present in the fetal kidney but was observed in the IMCD and DTL in 1-day-old pups. The intensity of UT-A immunostaining in the IMCD gradually increased during postnatal development. In 4- and 7-day-old pups, UT-A immunoreactivity was present in the DTL at the border between the outer and inner medulla. In 14- and 21-day-old pups, strong UT-A immunostaining was observed in the terminal part of short-loop DTL in the outer medulla, and weak labeling remained in long-loop DTL descending into the outer part of the inner medulla. In the adult kidney, there was intense staining for UT-B in descending vasa recta (DVR) and weak labeling of glomeruli. In the developing kidney, UT-B was first observed in the DVR of a 20-day-old fetus. After birth there was a striking increase in the number of UT-B-positive DVR, in association with the formation of vascular bundles. The intensity of immunostaining remained strong in the outer medulla but gradually decreased in the inner medulla. We conclude that the expression of urea transporters in short-loop DTL and DVR coincides with the development of the ability to produce a concentrated urine.

Age Factors↗
PubMed2003

Physiology of the renal medullary microcirculation.

Perfusion of the renal medulla plays an important role in salt and water balance. Pericytes are smooth muscle-like cells that impart contractile function to descending vasa recta (DVR), the arteriolar segments that supply the medulla with blood flow. DVR contraction by ANG II is mediated by depolarization resulting from an increase in plasma membrane Cl(-) conductance that secondarily gates voltage-activated Ca(2+) entry. In this respect, DVR may differ from other parts of the efferent microcirculation of the kidney. Elevation of extracellular K(+) constricts DVR to a lesser degree than ANG II or endothelin-1, implying that other events, in addition to membrane depolarization, are needed to maximize vasoconstriction. DVR endothelial cytoplasmic Ca(2+) is increased by bradykinin, a response that is inhibited by ANG II. ANG II inhibition of endothelial Ca(2+) signaling might serve to regulate the site of origin of vasodilatory paracrine agents generated in the vicinity of outer medullary vascular bundles. In the hydropenic kidney, DVR plasma equilibrates with the interstitium both by diffusion and through water efflux across aquaporin-1. That process is predicted to optimize urinary concentration by lowering blood flow to the inner medulla. To optimize urea trapping, DVR endothelia express the UT-B facilitated urea transporter. These and other features show that vasa recta have physiological mechanisms specific to their role in the renal medulla.

Animals↗
PubMed2005

A region-based mathematical model of the urine concentrating mechanism in the rat outer medulla. I. Formulation and base-case results.

We have developed a highly detailed mathematical model for the urine concentrating mechanism (UCM) of the rat kidney outer medulla (OM). The model simulates preferential interactions among tubules and vessels by representing four concentric regions that are centered on a vascular bundle; tubules and vessels, or fractions thereof, are assigned to anatomically appropriate regions. Model parameters, which are based on the experimental literature, include transepithelial transport properties of short descending limbs inferred from immunohistochemical localization studies. The model equations, which are based on conservation of solutes and water and on standard expressions for transmural transport, were solved to steady state. Model simulations predict significantly differing interstitial NaCl and urea concentrations in adjoining regions. Active NaCl transport from thick ascending limbs (TALs), at rates inferred from the physiological literature, resulted in model osmolality profiles along the OM that are consistent with tissue slice experiments. TAL luminal NaCl concentrations at the corticomedullary boundary are consistent with tubuloglomerular feedback function. The model exhibited solute exchange, cycling, and sequestration patterns (in tubules, vessels, and regions) that are generally consistent with predictions in the physiological literature, including significant urea addition from long ascending vasa recta to inner-stripe short descending limbs. In a companion study (Layton AT and Layton HE. Am J Physiol Renal Physiol 289: F1367-F1381, 2005), the impact of model assumptions, medullary anatomy, and tubular segmentation on the UCM was investigated by means of extensive parameter studies.

Animals↗
PubMed2005

A region-based mathematical model of the urine concentrating mechanism in the rat outer medulla. II. Parameter sensitivity and tubular inhomogeneity.

In a companion study (Layton AT and Layton HE. Am J Physiol Renal Physiol 289: F1346-F1366, 2005), a region-based mathematical model was formulated for the urine concentrating mechanism (UCM) in the outer medulla (OM) of the rat kidney. In the present study, we quantified the sensitivity of that model to several structural assumptions, including the degree of regionalization and the degree of inclusion of short descending limbs (SDLs) in the vascular bundles of the inner stripe (IS). Also, we quantified model sensitivity to several parameters that have not been well characterized in the experimental literature, including boundary conditions, short vasa recta distribution, and ascending vasa recta (AVR) solute permeabilities. These studies indicate that regionalization elevates the osmolality of the fluid delivered into the inner medulla via the collecting ducts; that model predictions are not significantly sensitive to boundary conditions; and that short vasa recta distribution and AVR permeabilities significantly impact concentrating capability. Moreover, we investigated, in the context of the UCM, the functional significance of several aspects of tubular segmentation and heterogeneity: SDL segments in the IS that are likely to be impermeable to water but highly permeable to urea; a prebend segment of SDLs that may be functionally like thick ascending limb (TAL); differing IS and outer stripe Na(+) active transport rates in TAL; and potential active urea secretion into the proximal straight tubules. Model calculations predict that these aspects of tubular of segmentation and heterogeneity generally enhance solute cycling or promote effective UCM function.

Animals↗
PubMed1985

Physiological and morphological responses of the rat kidney to reduced dietary protein.

Renal physiological and morphological adjustments to a reduced protein diet were studied in young Munich-Wistar rats. Two groups of animals were used for the correlative physiological-morphological studies: normal protein (NP, 24% dietary protein) rats and reduced protein (LP, 8% dietary protein) rats. Both groups were fed their respective diets for 4-5 wk and had free access to drinking water. Physiological measurements of GFR and urea clearance were made on five animals from each group. These data showed that the changes in renal function specifically and almost exclusively affected the handling of urea. There was no difference in GFR between the NP and LP rats. Urea clearance was substantially reduced in LP rats. Morphological analyses were made on perfusion-fixed kidneys of five animals from each group. Selected slices were examined and photographed by light and electron microscopy. These data showed no difference in size and number of elements within the vascular bundles but showed significantly smaller lumina of the thin limbs of the short-looped nephrons and a significant thinning of the wall of the thin descending limbs of the long-looped nephrons. These morphological changes may in part be responsible for the observed physiological adjustments to a reduced protein diet. An additional group of rats (6 NP and 5 LP, all dehydrated) were analyzed for distribution of solutes within the inner medulla. The data showed that the concentration of urea, but not that of Na+, was reduced at the papillary tip in LP rats.

Adaptation, Physiological↗
PubMed1991

Three-dimensional anatomy and renal concentrating mechanism. I. Modeling results.

Simulations were performed to test the hypothesis that the three-dimensional organization of the renal medulla is essential for formation of hypertonic urine. As in previous models, representations of loops of Henle, distal tubules, collecting ducts, and vasa recta and recent estimates of tubule characteristics were included in a simulation of NaCl, urea, and fluid transport. In addition, this model specifies the relative positions of the medullary structures. By assuming that the structure of the minimum functional unit is a vascular bundle surrounded by tubules and ascending vessels, we have represented the three-dimensional organization of the medulla by a cylindrically symmetric two-dimensional model. The resulting set of equations gives rise to a nonlinear boundary value problem with linear boundary conditions, which was solved numerically via quasi linearization. Compared with previous simulations, the concentrations predicted by this model more accurately match measured quantities in two regards. First, papillary tip concentrations of NaCl and urea are significantly higher, and, second, a monotonic increase in osmolarity is observed in the inner medulla. The three-dimensional organization permitted development of local concentration gradients, which are essential to the final result.

Absorption↗
PubMed1992

Distribution of angiotensin II receptor subtypes in rat and human kidney.

Angiotensin II initiates a variety of physiological effects in the kidney by binding to high-affinity receptors on plasma membranes. Recently, two subtypes of angiotensin II receptors have been distinguished on the basis of differences in signal transduction mechanisms, binding affinity to agonists and antagonists, and inhibition of binding by dithiothreitol. To evaluate the density and distribution of these receptor subtypes in the kidney, we performed an in situ autoradiographic study on frozen tissue sections obtained from rat and human kidneys. Sections were incubated with 125I-[Sar1,Ile8]angiotensin II and binding specificity was verified by competition with unlabeled [Sar1]angiotensin II. Angiotensin II receptor subtypes were characterized by competition with the nonpeptide receptor antagonists, DuP 753 (type 1) and PD123177 (type 2). Both rat and human kidney exhibited a high concentration of angiotensin II receptors in glomeruli and in the longitudinal bands traversing the outer portion of the medulla, corresponding to the medullary vascular bundles. Binding affinity (Kd = 0.6 +/- 0.4 nM), determined in rat kidney, was similar to that reported previously in isolated glomeruli and membrane vesicles prepared from renal tubules. Angiotensin II binding was almost completely inhibited by DuP 753, whereas PD123177 had little effect. Thus the predominant angiotensin II receptor subtype in both rat and human kidney is type 1. The distribution of angiotensin II receptors correlates well with the intrarenal sites at which the peptide has its major physiological effects.

1-Sarcosine-8-Isoleucine Angiotensin II↗
PubMed1994

Vasoconstriction of outer medullary vasa recta by angiotensin II is modulated by prostaglandin E2.

Vasa recta were dissected from outer medullary vascular bundles in the rat and perfused in vitro. Examination by transmission electron microscopy reveals them to be only outer medullary descending vasa recta (OM-DVR). To establish a method for systematic examination of vasoconstriction, OMDVR were perfused at 5 nl/min with collection pressure increased to 5 mmHg. Under these conditions, transmembrane volume flux was found to be near zero, and the transmural hydraulic pressure gradient was found to be < 15 mmHg. Over a concentration range of 10(-12) to 10(-8) M, abluminal application of angiotensin II (ANG II) caused graded focal vasoconstriction of OMDVR that is blocked by saralasin. Luminal application of ANG II over the same concentration range was much less effective. Abluminal application of prostaglandin E2 (PGE2) shifted the vasoconstrictor response of OMDVR to higher ANG II concentrations. PGE2 reversibly dilated OMDVR that had been preconstricted by ANG II. These results demonstrate that OMDVR are vasoactive segments. Their anatomical arrangement suggests that they play a key role in the regulation of total and regional blood flow to the renal medulla.

Angiotensin II↗
PubMed1994

The microtubule network of renal epithelial cells is disrupted by ischemia and reperfusion.

Ischemia results in alterations in the integrity of the plasma membrane of renal epithelial cells, changes in cell polarity, and initiation of cell division. Because microtubules are implicated in these processes, we examined the effects of ischemia and reperfusion on the microtubular cytoskeleton in rat kidney. Major alterations in the microtubule network of S3 proximal tubules were detected after 40 min of ischemia followed by 1 h of reperfusion. There was fragmentation of microtubules and considerably less intense staining with antitubulin antibodies than with normal kidneys. Some thick ascending limbs of Henle close to medullary vascular bundles showed a variable loss of tubulin staining. After 24 and 48 h of reperfusion, tubulin labeling was again present in most proximal tubule cells in contact with the basement membrane but was not detectable in exfoliated cells. Numerous mitotic figures were present in kidneys 48 h after reperfusion. Kidneys subjected to 40 min of ischemia without reperfusion and contralateral kidneys studied after 1 h of reperfusion showed only mild microtubular disruption. Because of the established role of microtubules in the generation and maintenance of epithelial cell polarity, their loss may contribute to structural changes that occur after ischemia and reperfusion.

Animals↗
PubMed1995

Inner medullary external osmotic driving force in a 3-D model of the renal concentrating mechanism.

The mechanism by which the renal medulla establishes and maintains a gradient of osmolarity along the corticomedullary axis, especially in the inner medulla, where there is no active transmural flux out of the ascending limbs of Henle, remains a source of controversy. We show here that, if realistic values of urea permeability in the inner medullary descending limbs and water permeability in the upper inner medullary section of the collecting ducts are taken into account, even a model including the three-dimensional vascular bundle structures [A. S. Wexler, R. E. Kalaba, and D. J. Marsh. Am. J. Physiol. 260 (Renal Fluid Electrolyte Physiol. 29): F368-F383, 1991] fails to explain the experimentally observed inner medullary osmolality gradient. We show here that this failure can be overcome by application of an external osmotic driving force, an idea recently revived by J. F. Jen and J. L. Stephenson (Bull. Math. Biol. 56: 491-514, 1994) in the context of a single-solute, single-loop central core model. We show that inclusion of such an external driving force with a value equivalent to at least 100 mosM of inner medullary interstitial osmolytes in the three-dimensional model of Wexler et al. accounts for a physiological osmolality gradient, even in the face of realistic permeability values. Furthermore, inclusion of the external driving force makes the model less dependent on the positions of descending and ascending limbs of Henle with respect to the collecting ducts. In an effort to assess whether there is any experimental basis for osmolytes, we show that a significant amount of extra inner medullary interstitial osmolytes is plausible, based on extrapolation from existing experimental data.

Animals↗
PubMed2001

Magnetic resonance imaging in the preoperative determination of tumor resectability in previously irradiated groin tumor.

We present an unusual case in which magnetic resonance (MR) imaging was utilized in the preoperative determination of resectability of a large inguinal tumor. The patient had previously presented with a large fungating, surgically nonresectable mass, which impinged upon the anterior ramus of the pubic bone. Following chemotherapy and radiation, the mass appeared more mobile and surgically resectable. Due to fibrosis and induration, the relationship of the tumor and underlying femoral vascular bundle were unclear. MR imaging clearly depicted that the tumor was sufficiently distant from the vessels, to allow safe resection with an adequate surgical margin. Resection was performed with clear lateral and deep margins. Histopathology demonstrated squamous cell carcinoma of the inguinal skin replacing subcutaneous tissue.

Carcinoma, Squamous Cell↗
PubMed2006

Emergency peripartum hysterectomy in a tertiary obstetric center: 8-year evaluation.

OBJECTIVE: The aim of the study was to present the incidence, indications, and operative morbidity and mortality in pregnant women undergoing emergency peripartum hysterectomy (EPH) at a tertiary obstetric institution. METHODS: In this retrospective clinical study, performed during the period 1995-2003, 17 EPH procedures were recorded in a total of 21,659 deliveries carried out at Department of Gynecology and Obstetrics, Osijek Clinical Hospital in Osijek, Croatia. Data on the incidence of EPH in total number of deliveries, rate of EPH in vaginal delivery and cesarean section, indications for EPH, and maternal and fetal/early neonatal morbidity and mortality were derived from operative protocols and medical records of hospitalized patients. RESULTS: During the 8-year study period, the incidence of EPH in total number of deliveries was 0.078%. Out of 17 EPH procedures, 5 (29.41%) were performed after vaginal delivery and 12 (70.59%) during cesarean section, elective in five and urgent in seven cases. The indications for EPH included severe peripartum hemorrhage with placenta previa in four, placenta previa percreta in four, various forms of invasive malplacentation (placenta accreta, increta, percreta) in five, uterine rupture in two cases, and atony along with massive retroperitoneal hematoma due to rupture of periuterine vascular bundle during cesarean section in one multipara. EPH was carried out in 12 multipara and five primipara. Lesions of urinary bladder occurred in three cases and were managed by suture. Twelve patients received blood transfusion, whereas development of hemorrhagic shock necessitated transfer to Intensive Care Unit in three patients. No late complications or maternal mortality were recorded. Sixteen total hysterectomies and one supracervical hysterectomy were performed. One case of intrauterine fetal death was caused by total abruptio placenta and uterine rupture during the patient's transport from a primary obstetric institution. CONCLUSION: Invasive malplacentation is a major isolated risk factor for EPH, as shown in the present study. Other risk factors for EPH are massive hemorrhage because placenta previa, uterine atony and uterine rupture associated with multiparity, and previous cesarean section. A great proportion of EPH procedures can be prevented by the introduction of compressive operative methods such as B-Lynch suture in the obstetric algorithms, which will certainly favorably reflect in future fertility and genital health of the female population.

Algorithms↗
PubMed1999

Salt-sensitive hypertension develops after short-term exposure to Angiotensin II.

We hypothesized that short-term exposure to angiotensin II (Ang II) could result in structural and functional changes in the kidney that would favor sodium retention and the development of sustained hypertension. To test this hypothesis, rats were exposed to pressor doses (435 ng. kg-1. min-1) of Ang II for 2 weeks. The infusion of Ang II was associated with acute hypertension, renal dysfunction, proteinuria, and focal tubulointerstitial and vascular damage. At sites of the tubulointerstitial damage, there was a reduction in peritubular capillary endothelial cell staining. By use of immunostaining, we found focal loss of endothelial nitric oxide synthase (eNOS) in the peritubular capillaries at sites of injury and a generalized reduction in eNOS in collecting ducts, thin loops of Henle, and vascular bundles in the medulla. When the Ang II infusion ended, the rats became normotensive and renal function returned toward normal. However, exposure of the rats to high salt diet (4% NaCl) resulted in the redevelopment of hypertension after 3 to 4 weeks. Rats maintained on a high salt diet with no prior exposure to Ang II and rats placed on low salt diet (0.1% NaCl) after exposure to Ang II remained normotensive. Thus, we report a new model of salt-sensitive hypertension induced by transient exposure to pressor doses of Ang II. The mechanism may relate to microvascular injury with peritubular capillary loss coupled with functional changes, such as a loss in intrarenal nitric oxide formation, that could alter the ability of the kidney to excrete a salt load.

Angiotensin II↗
PubMed1995

Tubulointerstitial injury induced in rats by a monoclonal antibody that inhibits function of a membrane inhibitor of complement.

The kidney widely expresses membrane-associated complement regulatory proteins (membrane inhibitors of complement). The aim of this work was to evaluate the roles of these molecules in rat kidneys in vivo. To suppress functions of rat membrane inhibitors of complement, two mAbs, 512 and 6D1, were used. 5I2 and 6D1 inhibit functions of membrane inhibitors of complement at C3 level (rat Crry/p65) and C8/9 level (rat CD59), respectively. F(ab')2 fragment of 5I2 or 6D1 was perfused in the left kidneys, and perfusate was discarded from the renal vein. After perfusion, the left kidneys were connected to systemic circulation. In rats perfused with 5I2, mouse IgG was found in glomeruli, peritubular capillaries, vascular bundles, and tubules 15 min after recirculation. Binding of C3 and C5b-9 was evident in these areas. 1 d after perfusion with 5I2, cast formation, dilatation of tubular lumen, and tubular cell degeneration were observed. At day 4 through day 7, significant mononuclear cell infiltration and proximal tubule damage were observed. These changes were completely prevented by complement depletion. Rats perfused with 6D1 showed the binding of mouse IgG in the similar areas as 5I2, but C3 or C5b-9 deposition was not observed. Rats perfused with 6D1 or vehicle only did not show any pathology in the left kidneys. These results suggest that rat Crry/p65 plays protective roles against spontaneously occurring indiscriminate attack to tubulointerstitial tissues by autologous complement and that rat Crry/p65 is one of the important factors to maintain normal integrity of the kidney in rats.

Animals↗
PubMed1991

Magnetic resonance imaging of the calcaneus: normal anatomy and application in calcaneal fractures.

Images obtained from normal volunteers demonstrate highly detailed anatomy of the soft tissue and bony structures near the calcaneus and subtalar joint. Cortical bone, marrow, articular cartilage, ligaments, tendons, muscles, fibrous tissue, vascular bundles, and nerves can be identified. However, images obtained of acute calcaneal fractures were found to offer unsatisfactory depiction of bony anatomy. The presence of small fragments was obscured by a change in normal marrow signal by contusion, hemorrhage, and edema, and by the inability of magnetic resonance (MR) to image small pieces of cortical bone. Only in rare instances might MR be helpful in the acute setting when the location or displacement of tendons cannot be clearly ascertained with computed tomography (CT). MR may prove more useful in the long-term follow-up of healed fractures with persistent pain. In this setting it might be used in the diagnosis of complications such as residual or recurrent tendon displacement, tenosynovitis, heel fat integrity, and tarsal tunnel evaluation. However, this paper did not directly compare the efficacy of MR with that of CT in the long term. Therefore, the degree to which MR may eventually supplement or supplant CT is unclear and further study is required before the use of MR can be recommended in the routine clinical follow-up of calcaneal fractures.

Ankle↗
PubMed2005

Analysis of xylem sap proteins from Brassica napus.

BACKGROUND: Substance transport in higher land plants is mediated by vascular bundles, consisting of phloem and xylem strands that interconnect all plant organs. While the phloem mainly allocates photoassimilates, the role of the xylem is the transport of water and inorganic nutrients from roots to all aerial plant parts. Only recently it was noticed that in addition to mineral salts, xylem sap contains organic nutrients and even proteins. Although these proteins might have important impact on the performance of above-ground organs, only a few of them have been identified so far and their physiological functions are still unclear. RESULTS: We used root-pressure xylem exudate, collected from cut Brassica napus stems, to extract total proteins. These protein preparations were then separated by high-resolution two-dimensional gel electrophoresis (2-DE). After individual tryptic digests of the most abundant coomassie-stained protein spots, partial peptide sequence information was deduced from tandem mass spectrometric (MS/MS) fragmentation spectra and subsequently used for protein identifications by database searches. This approach resulted in the identification of 69 proteins. These identifications include different proteins potentially involved in defence-related reactions and cell wall metabolism. CONCLUSION: This study provides a comprehensive overview of the most abundant proteins present in xylem sap of Brassica napus. A number of 69 proteins could be identified from which many previously were not known to be localized to this compartment in any other plant species. Since Brassica napus, a close relative of the fully sequenced model plant Arabidopsis thaliana, was used as the experimental system, our results provide a large number of candidate proteins for directed molecular and biochemical analyses of the physiological functions of the xylem under different environmental and developmental conditions. This approach will allow exploiting many of the already established functional genomic resources, like i.e. the large mutant collections, that are available for Arabidopsis.

Brassica napus↗