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Biomedical subjects

D Brown

Publications and source records attributed to D Brown.

At least 289 records · Page 16Linked to original sources
PubMed1998

Immunological detection and mutational analysis of the RNA2-encoded nematode transmission proteins of pea early browning virus.

Pea early browning virus (PEBV) is transmitted between plants by root-feeding trichodorid nematodes. Mutagenesis studies have implicated two non-structural viral proteins in the transmission process. These two proteins [the 29 kDa ('29K') protein and the 23K protein] were expressed in bacteria and used to raise antibodies. In Western blotting experiments, the antibodies detected both of these virus proteins in leaves and roots of infected Nicotiana bethamiana and N. clevelandii plants. Periodate treatment of proteins transferred to nitrocellulose membranes suggested that the PEBV 23K protein may be glycosylated. A PEBV mutant was constructed lacking the complete 23K coding sequence. The mutant was able systemically to infect Nicotiana spp. but caused striking chlorotic ringspot leaf symptoms and stunting of both leaves and roots. These symptoms were absent in plants doubly-infected with the mutant and wild-type PEBV. The 23K gene deletion mutant was transmitted by nematodes at a much reduced frequency compared to wild-type virus, indicating that the 23K protein is involved in but not essential for vector transmission. Western immuno-blot and ELISA experiments revealed that the reduction in the nematode-transmissibility of PEBV carrying mutations in the 23K gene did not result from interference in the expression of the 29K transmission protein or from gross changes in the titre of virus in the roots of infected plants.

Animals↗
PubMed1998

Expression of an AQP2 Cre recombinase transgene in kidney and male reproductive system of transgenic mice.

A transgenic mouse approach was used to examine the mechanism of principal cell-specific expression of aquaporin-2 (AQP2) within the renal collecting duct. RT-PCR and immunocytochemistry revealed that murine AQP2 was expressed in principal cells in the renal collecting duct, epithelial cells of the vas deferens, and seminiferous tubules within testis. The vas deferens expression was confirmed in rats. RT-PCR and immunocytochemistry showed that 14 kb of the human 5'-flanking region confers specific expression of a nucleus-targeted and epitope-tagged Cre recombinase in the principal cells within the renal collecting duct, in the epithelial cells of the vas deferens, and within the testis of transgenic mice. These results suggest that cell-specific expression of AQP2 is mediated at the transcriptional level and that 14 kb of the human AQP2 5'-flanking region contain cis elements that are sufficient for cell-specific expression of AQP2. Finally, renal principal cell expression of Cre recombinase is the first step in achieving cell-specific gene knockouts, thereby allowing focused examination of gene function in this cell type.

Animals↗
PubMed1998

Polarity, integrin, and extracellular matrix dynamics in the postischemic rat kidney.

Acute renal failure (ARF) as a consequence of ischemic injury is a common disease affecting 5% of the hospitalized population. Despite the fact that mortality from ARF is high, there has been little improvement in survival rates over the last 40 years. The pathogenesis of ARF may be related to substantial changes in cell-cell and cell-extracellular matrix interactions mediated by beta1-integrins. On the basis of in vitro and in vivo studies, reorganization of beta1-integrins from basal to apical surfaces of injured tubular epithelia has been suggested to facilitate epithelial detachment, contributing to tubular obstruction and backleak of glomerular filtrate. In this study, we examine integrin and extracellular matrix dynamics during epithelial injury and repair using an in vivo rat model of unilateral ischemia. We find that, soon after reperfusion, beta1-integrins newly appear on lateral borders in epithelial cells of the S3 segment but are not on the apical surface. At later times, as further injury and regeneration coordinately occur, epithelia adherent to the basement membrane localize beta1 predominantly to basal surfaces even while the polarity of other marker proteins is lost. At the same time, amorphous material consisting of depolarized exfoliated cells fills the luminal space. Notably, beta1-integrins are not detected on exfoliated cells. A novel finding is the presence of fibronectin, a glycoprotein of plasma and the renal interstitium, in tubular spaces of the distal nephron and to a lesser extent S3 segments. These results indicate that beta1-integrins dramatically change their distribution during ischemic injury and epithelial repair, possibly contributing to cell exfoliation initially and to epithelial regeneration at later stages. Together with the appearance of large amounts of fibronectin in tubular lumens, these alterations may play a significant role in the pathophysiology of ARF.

Acute Kidney Injury↗
PubMed1998

Proton secretion in the male reproductive tract: involvement of Cl--independent HCO-3 transport.

The lumen of the epididymis is the site where spermatozoa undergo their final maturation and acquire the capacity to become motile. An acidic luminal fluid is required for the maintenance of sperm quiescence and for the prevention of premature activation of acrosomal enzymes during their storage in the cauda epididymis and vas deferens. We have previously demonstrated that a vacuolar H+-ATPase [proton pump (PP)] is present in the apical pole of apical and narrow cells in the caput epididymis and of clear cells in the corpus and cauda epididymis and that this PP is responsible for the majority of proton secretion in the proximal vas deferens. We now show that PP-rich cells in the vas deferens express a high level of carbonic anhydrase type II (CAII) and that acetazolamide markedly inhibits the rate of proton secretion by 46.2 +/- 6.1%. The rate of acidification was independent of Cl- and was strongly inhibited by SITS under both normal and Cl--free conditions (50.6 +/- 5.0 and 57. 5 +/- 6.0%, respectively). In the presence of Cl-, diphenylamine-2-carboxylate (DPC) had no effect, whereas SITS inhibited proton secretion by 63.7 +/- 11.3% when applied together with DPC. In Cl--free solution, DPC markedly inhibited proton efflux by 45.1 +/- 7.6%, SITS produced an additional inhibition of 18.2 +/- 6.6%, and bafilomycin had no additive effect. In conclusion, we propose that CAII plays a major role in proton secretion by the proximal vas deferens. Acidification does not require the presence of Cl-, but DPC-sensitive Cl- channels might contribute to basolateral extrusion of HCO-3 under Cl--free conditions. The inhibition by SITS observed under both normal and Cl--free conditions indicates that a Cl-/HCO-3 exchanger is not involved and that an alternative HCO-3 transporter participates in proton secretion in the proximal vas deferens.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1998

Endotoxin-induced alteration in the expression of leptin and beta3-adrenergic receptor in adipose tissue.

Cytokines, such as tumor necrosis factor (TNF) and interleukin-6, may contribute to the anorexia and cachexia of infection, cancer, and AIDS. The present study tests the hypothesis that endotoxin alters the expression of two key fat cell proteins, leptin and beta3-adrenergic receptor (beta3-AR), through a mechanism involving TNF-alpha. Increasing doses of Escherichia coli endotoxin (lipopolysaccharide, LPS) resulted in dose-dependent elevations of plasma leptin (maximal response approximately 7-fold, half-maximal effective dose of approximately 16 microg/100 g body wt) and white fat leptin mRNA in C3/HeOUJ mice. LPS also produced a large decrease in adipose tissue beta3-AR mRNA and a parallel reduction in beta-agonist-induced activation of adenylyl cyclase. Changes in plasma leptin and beta3-AR mRNA were preceded by an approximately threefold increase in white fat TNF mRNA. TNF administration resulted in changes similar to those seen with LPS. We conclude that endotoxemia results in an induction of leptin mRNA and a decrease in beta3-AR mRNA in adipose tissue, an effect that may be mediated by alterations in TNF-alpha.

Adenylyl Cyclases↗
PubMed1998

Cellular mechanisms of aquaporin trafficking.

Aquaporins (AQPs) are a family of functionally important water channel proteins that are of special cell biological interest because of their diverse intracellular targeting and trafficking properties. AQPs have been found in many different cells and tissues. This short review summarizes recent work that addresses the regulation of AQP2 trafficking in response to vasopressin.

Animals↗
PubMed1998

Basolateral distribution of caveolin-1 in the kidney. Absence from H+-atpase-coated endocytic vesicles in intercalated cells.

In kidney epithelial cells, a variety of physiological processes are dependent on the active recycling of membrane proteins between intracellular vesicles and the cell surface. Although clathrin-mediated endocytosis occurs in several renal cell types, endocytosis can also occur by non-clathrin-coated vesicles, including pinocytotic structures known as caveolae that contain a novel coat protein, caveolin. Exo- and endocytosis of a vacuolar H+-ATPase in intercalated cells also occurs via specialized "coated" vesicles that do not contain clathrin. The aim of this study was to localize caveolin in the kidney and, in addition, to determine whether it could be a component of the H+-ATPase recycling process. Using an antibody against the alpha- and beta-isoforms of caveolin-1, our immunocytochemical data show a marked heterogeneity in the cellular expression of this isoform of caveolin in kidney. In contrast, caveolin-3 was not detectable in renal epithelial cells. Caveolin-1 was abundant in endothelial cells and smooth muscle cells and was present in the parietal cells of Bowman's capsule. Distal tubule cells, connecting tubule cells, and collecting duct principal cells exhibited marked punctate basolateral staining, corresponding to the presence of caveolae detected by electron microscopy, whereas all intercalated cells were negative in both cortex and medulla. These data indicate that although caveolin-1 may participate in basolateral events in some kidney epithelial cell types, it does not appear to be involved in the regulated recycling of H+-ATPase in intercalated cells. Therefore, these cells recycle H+-ATPase by a mechanism that involves neither clathrin nor caveolin-1.

Animals↗
PubMed1998

Immunoexpression of aquaporin-1 in the efferent ducts of the rat and marmoset monkey during development, its modulation by estrogens, and its possible role in fluid resorption.

Recent data suggest that estrogens play a role in regulating fluid resorption from the efferent ducts, though the biochemical mechanisms involved are unknown. The present study has used immunocytochemistry to localize a water channel protein, Aquaporin-1 (AQP-1), to the efferent ducts of male rats and marmoset monkeys from perinatal life through to adulthood and has then investigated its potential hormonal regulation in neonatal/peripubertal life, via administration of a GnRH antagonist (GnRHa) or diethylstilbestrol (DES) to rats. AQP-1 was immunoexpressed intensely in the apical brush border of the epithelium lining the efferent ducts at all ages studied, from late fetal life through puberty to adulthood. In the marmoset, but not the rat, AQP-1 was also expressed in the epithelium of the rete testis. Once the cell types within the efferent duct epithelium had differentiated, it was clear that only nonciliated cells of the rat localized AQP-1. When gonadotropin secretion was suppressed in rats by neonatal administration of GnRHa, immunoexpression of AQP-1 at age 18 and 25 days was virtually unchanged in intensity, though the efferent ducts were reduced in size. In contrast, when DES was administered neonatally to rats (up to day 12), immunoexpression of AQP-1 was reduced at day 10, virtually abolished at day 18, reduced markedly at day 25 and to a small extent at day 35; these findings were confirmed by Western blot analysis at day 18. The DES-induced decrease in immunoexpression of AQP-1 was accompanied by pronounced distension of the efferent ducts and rete, consistent with reduced fluid resorption. The epithelial cells of the efferent ducts in DES-treated rats were cuboidal rather than columnar in shape as in controls and were reduced significantly in height compared with controls at all ages through to adulthood. These findings suggest that estrogens may play a role in regulating fluid resorption from the efferent ducts during fetal/neonatal development and/or a role in the gross and functional development of the efferent ducts and rete testis. The present data also suggest that AQP-1 is one of the elements involved in the regulation of fluid resorption in the efferent ducts. The importance of fluid flow in fetal/neonatal development of the excurrent duct system of the male is also suggested by these observations.

Absorption↗
PubMed1998

Value of a low-cost telemedicine link in the remote echocardiographic diagnosis of congenital heart defects.

We established a low-cost telemedicine link from a district general hospital to the regional paediatric cardiology department about 120 km away. The link was used to transmit echocardiographic images of newborn infants suspected of having congenital heart disease (CHD) to the referral centre, with simultaneous video and audio contact for consultation. Echocardiograms were transmitted for 61 patients suspected of having CHD, aged from 1 to 42 days. The transmitted images were of adequate quality for the paediatric cardiologist to make a diagnosis in 59 (97%). Congenital heart abnormalities were diagnosed in 38 (64%). Twelve patients (20%) had major CHD diagnosed on the transmitted scan and required transfer to the regional cardiology unit either urgently or electively after initial measures to stabilize the patient. Our findings suggest that, for babies suspected of having CHD, ultrasound images of diagnostic quality can be obtained and transmitted using a low-cost telemedicine system.

Cardiology↗
PubMed1998

Cold-induced microtubule disruption and relocalization of membrane proteins in kidney epithelial cells.

Cold preservation of kidneys is commonly used in human transplantation and in vitro studies. However, although disruption of the cytoskeleton by cold has been demonstrated in cultured cells, the effect of cold treatment on intact kidney is poorly understood. In this study, specific antibodies were used to examine the effect of hypothermia on the cytoskeletal network and the trafficking of some membrane proteins in the urinary tubule. Rat kidneys were cut into thin slices (approximately 0.5 mm) that were divided into several groups: (1) some were immediately fixed in paraformaldehyde, sodium periodate, and lysine (PLP); (2) some were stored at 4 degrees C for 15 min or 4 h before being fixed in cold PLP; or (3) after 4 h cold treatment, some slices were rewarmed to 37 degrees C for 15, 30, and 60 min in a physiologic solution, pH 7.4, and were then fixed in warm PLP. Immunofluorescence staining revealed an almost complete disruption of the microtubule network in proximal tubules after 15 min cold treatment, whereas microtubules in other segments were affected after 4 h. A partial recovery of the microtubule network was observed after 60 min rewarming. In contrast, actin filaments seemed to be resistant to cold treatment. gp330, aquaporin-2, H+ ATPase, and the AE1 anion exchanger were all relocated into numerous vesicles that were distributed throughout the cytoplasm after hypothermia followed by rewarming, whereas Na-K-ATPase retained its basolateral localization. The vasopressin-stimulated insertion of aquaporin-2 water channels into the apical membrane was inhibited during the initial rewarming period after cold exposure. Thus, cold preservation of tissues might impair, at least transiently, the polarized membrane expression and function of some transport proteins in renal epithelial cells.

Actins↗
PubMed1998

Immunolocalization and tissue-specific splicing of AE2 anion exchanger in mouse kidney.

In this study, an epitope-unmasking technique was used to immunolocalize AE2 anion exchanger polypeptide to basolateral plasma membranes of tubular epithelial cells in mouse kidney. Kidney AE2 immunostaining in mouse kidney was less prominent than in rat, consistent with the relative levels of AE2 mRNA and polypeptide in these two species. Glomeruli showed faint but consistent AE2 immunostaining, whereas proximal tubules were generally unstained. Macula densa epithelial cells displayed bright AE2 immunostaining, and cortical thick limbs were stained at a lower intensity. AE2 immunostaining was weak or absent in type B intercalated cells and principal cells of the cortical collecting duct, but increased in intensity in principal cells of the inner stripe of the outer medulla. AE2 staining in medullary thick limbs was also of greater intensity than in cortical thick limbs. AE2 staining was strong and uniform in the epithelial cells of the inner medullary collecting duct, and in epithelial cells of the papillary surface, the ureter, and the urinary bladder. Extratubular and epithelial cells of the inner medulla also showed punctate intracellular AE2 staining in a Golgi-like distribution that, in contrast to cell surface staining, was sodium dodecyl sulfate-sensitive. Golgi localization of AE2 epitope was confirmed by immunoperoxidase electron microscopy. Reverse transcription-PCR analysis of mouse kidney RNA detected AE2a, AE2b, and an AE2c2 transcript, but an AE2c1 transcript was absent. Unlike in rat, the mouse AE2c2 mRNA splice variant encoded a polypeptide with a novel predicted N-terminal amino acid sequence.

Amino Acid Sequence↗
PubMed1998

Caveat emptor.

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Ethics, Medical↗
PubMed1998

Head and neck patients. An innovative, cost-effective approach.

An otolaryngology (OTO) acute care unit was developed to provide nursing care for head and neck patients who required intensive respiratory monitoring but did not need to be in an intensive care unit because they were hemodynamically stable. This unit allowed better utilization of intensive care unit beds and contributed to a more cost-effective system for all patients while meeting the special needs of these patients and their families.

Cost-Benefit Analysis↗
PubMed1997

Onset of bursting in oxytocin cells in suckled rats.

1. We tested whether firing characteristics are associated with the onset of bursting in oxytocin cells during suckling. Extracellular electrical activity of paraventricular and supraoptic oxytocin cells was recorded in lactating rats from the beginning of suckling up to the first milk-ejection burst, which occurred either within less than 1 h of suckling (bursting cells) or after injecting facilitatory drugs (non-bursting cells). 2. Significant differences in the distributions of firing rate (using low, intermediate and high categories, < or = 1, 1-3 and > 3 spikes s-1, respectively) of bursting and non-bursting cells were observed during suckling. Thirty minutes after pup application, most bursting cells (56%) had an intermediate firing rate, whereas non-bursting cells had either a low (36%) or high (40%) firing rate. 3. Thirty minutes after applying the pups, probability of bursting was highest for cells firing at 1-4 spikes s-1, and lowest for those firing above 5 spikes s-1. 4. Bursting cells with high initial firing rate decreased their firing rate substantially during suckling while most of those with low initial firing rate showed increases. For non-bursting cells, activity was maintained. 5. There were marked differences in firing rate and its evolution between paired bursting cells. The firing rates for non-bursting cell pairs were similar (mostly either low or high), and remained stable during suckling. 6. In conclusion, we suggest that, as suckling proceeds, probability of bursting is related to the firing rate of oxytocin cells within the whole population, more specifically to the proportion of cells within the animal initially or subsequently displaying a critical firing rate (between 1 and 3 spikes s-1). The firing rates of cells which eventually burst and which are firing outside this range change in a direction which brings them into the preferred range for bursting. 7. We suggest that when bursting occurs unaided, a majority of oxytocin cells fire in the preferred bursting range and facilitate the bursting of cells outside this range. Since such cooperativity does not develop between non-bursting cells, it might be due to centrally released oxytocin.

Action Potentials↗
PubMed1997

Systolic and diastolic effects of variable atrioventricular delay in patients with complete heart block and normal ventricular function.

This study was designed to demonstrate the effects of varying the atrioventricular delay (AVD) on ventricular diastolic filling dynamics and the resultant stroke volume in patients with complete heart block and normal cardiac function. We studied 7 patients with normal cardiac function in whom a dual chamber pacemaker had been implanted because of complete heart block. Doppler and M-mode echocardiography was performed at 70, 100, 140, 180, and 220 ms, AVD with the device in DDD mode at a rate of 80 beats/min. The effects of these variable intervals on the contribution of the E and A waveform to the diastolic filling, on the stroke volume, and on the systolic intervals were evaluated. Optimization of this interval, with a 19% increase in stroke volume was achieved in the group of patients at an AVD of 140 ms. When considered individually, the AVD associated with the largest stroke volume, was 100 ms in 2 patients and 140 ms in the remaining 5. At this individual optimal AVD the ventricular septal contraction occurred 31 +/- 14 ms, before the end of the transmitral flow. The optimal AVD is, therefore, the one which synchronizes the ventricular and atrial systole so that the first ventricular septal contraction occurs after the peak of the A wave, just before the end of the transmitral flow. Because of the different functional cardiovascular status of the single patient, this parameter should be individualized; this can be clinically important as it may lead, in this patient population, to an improvement of the stroke volume up to 42%.

Adult↗