bluuebunny/crossref_metadata_2025 huggingface dataset:
Tactual temporal-onset order discrimination: Effects of frequency and site of stimulation
Authors: Yuan Hanfeng , Reed Charlotte M. , Durlach Nathaniel I.
Published: February 2004

This research is concerned with measurements of temporal onset-order discrimination through the tactual sensory system. Sinusoidal signals were delivered through a multi-finger stimulating device that operates over a frequency range of roughly 0 to 300 Hz and an amplitude range of roughly 0 to 50 dB SL. Measurements were obtained using a one-interval two-alternative forced-choice procedure in which each interval consisted of the random-order presentation of two different sinusoidal signals whose amplitude and duration were roved independently from trial to trial. Thresholds were estimated from psychometric functions of d′ as a function of stimulus-onset asynchrony. Performance was studied as a function of the frequency separation between the two sinusoids presented in a given run for presentation across two different fingers and for presentation to the same finger. Conditions were also included to examine the effects of redundancy of frequency and site of stimulation on performance. Results will be compared across conditions and will be discussed with regard to their implications for the design of tactual displays of speech for persons with profound hearing impairment. [Research supported by Grant No. 5 R01-DC00126 from NIDCD, NIH.]
Development of context dependent sequential K-nearest neighbor classifier for usable speech classification
Authors: Shah J. K. , Iyer A. N. , Smolenski B. Y. , Yantorno R. E.
Published: February 2004

The accuracy of speech processing applications degrades when operating in co-channel environment. Co-channel speech occurs when more than one person is talking at the same time. The idea of usable speech segmentation is to identify and extract those portions of co-channel speech that are minimally degraded but still useful for speech processing applications (such as speaker identification or speech recognition) which do not work in co-channel environments. Usable speech measures are features that are extracted from the co-channel signal to distinguish between usable and unusable speech. Several usable speech extraction methods have recently been developed based on a single feature of the speech signal being considered. In this paper, however, a new usable speech extraction technique, which sequentially and contextually selects several features of the given signal using the K-nearest neighbor classifier, is being investigated. This new approach considers periodicity and structure based features simultaneously in order to achieve the maximum classification rate, and by observing all the incoming frames, avoids the problem of deciding the amount of data needed to make accurate decisions. A 100% accuracy can be achieved in speech processing applications by using this extracted usable speech segment.
Cross-Linking Cellular Prion Protein Triggers Neuronal Apoptosis in Vivo
Authors: Solforosi Laura , Criado Jose R. , McGavern Dorian B. , Wirz Sebastian , Sánchez-Alavez Manuel , Sugama Shuei , DeGiorgio Lorraine A. , Volpe Bruce T. , Wiseman Erika , Abalos Gil , Masliah Eliezer , Gilden Donald , Oldstone Michael B. , Conti Bruno , Williamson R. Anthony
Published: February 2004

Neuronal death is a prominent, but poorly understood, pathological hallmark of prion disease. Notably, in the absence of the cellular prion protein (PrP
C
), the disease-associated isoform, PrP
Sc
, appears not to be intrinsically neurotoxic, suggesting that PrP
C
itself may participate directly in the prion neurodegenerative cascade. Here, cross-linking PrP
C
in vivo with specific monoclonal antibodies was found to trigger rapid and extensive apoptosis in hippocampal and cerebellar neurons. These findings suggest that PrP
C
functions in the control of neuronal survival and provides a model to explore whether cross-linking of PrP
C
by oligomeric PrP
Sc
can promote neuronal loss during prion infection.
Current helmet and protective equipment usage among previously injured ATV and motorcycle riders
Authors: Mangus R S , Simons C J , Jacobson L E , Streib E W , Gomez G A
Published: February 2004

Injury recidivism among trauma patients may be related to an individual pattern of high risk behaviors. The extent to which an injury episode modifies this behavior pattern is unknown. A self report, voluntary, anonymous, cross sectional survey was administered to motorcycle and all-terrain vehicle (ATV) riders at a popular recreation site. Data included demographics, injury history, and current usage of helmet and protective gear. Two hundred eighty surveys were completed. History of ATV/motorcycle related minor and major injury were reported by 21% and 9%, respectively. Persons with a history of minor ATV/motorcycle injury only were less likely to use a helmet or protective equipment (78%v74%, p = 0.58 and 49%v41%, p = 0.29). Persons with a history of any major ATV/motorcycle injury were also less likely to use a helmet or protective equipment (77%v56%, p = 0.03 and 48%v40%, p = 0.53). These findings suggest a pattern of persistent high risk behavior among previously injured persons.
Influence of an injury reduction program on injury and fitness outcomes among soldiers
Authors: Knapik J J , Bullock S H , Canada S , Toney E , Wells J D , Hoedebecke E , Jones B H
Published: February 2004

Objective:This study evaluated the influence of a multiple injury control intervention on injury and physical fitness outcomes among soldiers attending United States Army Ordnance School Advanced Individual Training.

Methods:The study design was quasiexperimental involving a historical control group (n = 2559) that was compared to a multiple intervention group (n = 1283). Interventions in the multiple intervention group included modified physical training, injury education, and a unit based injury surveillance system (UBISS). The management responsible for training independently formed an Injury Control Advisory Committee that examined surveillance reports from the UBISS and recommended changes to training. On arrival at school, individual soldiers completed a demographics and lifestyle questionnaire and took an army physical fitness test (APFT: push-ups, sit-ups, and two mile run). Injuries among soldiers were tracked by a clinic based injury surveillance system that was separate from the UBISS. Soldiers completed a final APFT eight weeks after arrival at school.

Results:Cox regression (survival analysis) was used to examine differences in time to the first injury while controlling for group differences in demographics, lifestyle characteristics, and physical fitness. The adjusted relative risk of a time loss injury was 1.5 (95% confidence interval 1.2 to 1.8) times higher in the historical control men and 1.8 (95% confidence interval 1.1 to 2.8) times higher in the historical control women compared with the multiple intervention men and women, respectively. After correcting for the lower initial fitness of the multiple intervention group, there were no significant differences between the multiple intervention and historical control groups in terms of improvements in push-ups, sit-ups, or two mile run performance.

Conclusions:This multiple intervention program contributed to a reduction in injuries while improvements in physical fitness were similar to a traditional physical training program previously used at the school.
Motor vehicle driver injury and marital status: a cohort study with prospective and retrospective driver injuries
Authors: Whitlock G , Norton R , Clark T , Jackson R , MacMahon S
Published: February 2004

Objective:To investigate the association of marital status with risk of motor vehicle driver injury.

Design:A cohort study with prospective and retrospective outcomes.

Setting:New Zealand.

Participants:A total of 10 525 adults (a volunteer sample of a multi-industry workforce, n  =  8008; and a random sample of urban electoral rolls, n  =  2517).

Exposure variable:Self reported marital status, assessed from a questionnaire administered in 1992–93 (baseline).

Main outcome measure:Motor vehicle driver injury resulting in admission of the driver to hospital and/or the driver’s death, during the period 1988–98; hospitalisation and mortality data were obtained by record linkage to national health databases.

Results:During 108 741 person-years of follow up, 139 driver injury cases occurred (85 before baseline, 54 after). After adjustment for age, sex, and study cohort, never married participants had twice the risk of driver injury (hazard ratio [HR] 2.06, 95% confidence interval [CI] 1.35 to 3.16) as married participants (HR 1.00). The relative risk for never married participants was slightly higher (HR 2.29), though less precise (95% CI 1.39 to 3.76), after further adjustment for alcohol intake, driving exposure, area of residence, body mass index, and occupational status.

Conclusions:After taking age, sex, and other variables into account, never married people had a substantially higher risk of driver injury than married people. While requiring corroboration, these findings imply that it may be appropriate for driver injury countermeasures to be targeted to never married people.
How well do we investigate patients with suspected subarachnoid haemorrhage? The continuing need for cerebrospinal fluid investigations
Authors: Schofield M L A , Lorenz E , Hodgson T J , Yates S , Griffiths P D
Published: February 2004

Objective

To demonstrate the extent of compliance with established guidelines for the investigation of suspected subarachnoid haemorrhage (SAH) and the implications of non-compliance.

Design

Prospective observational study of practice in three hospitals in the Trent region.

Setting

One teaching hospital with a tertiary neuroscience referral centre and two large district general hospitals.

Participants

50 consecutive patients from each centre referred for suspected SAH with negative computed tomography.

Main outcome measures

Diagnosis of SAH confirmed or excluded according to guidelines.

Results

When the data from the three centres were combined (n = 150 computed tomography negative cases) cerebrospinal fluid (CSF) investigation was not performed in 60/150 (40%). In the 90 cases where CSF studies were performed SAH was confirmed in 11 (12%).

Conclusion

There is significant non-compliance in following the established guidelines for the investigation of SAH at the centres studied. As the primary cause of non-traumatic SAH is ruptured aneurysm, which is associated with high morbidity and mortality from second haemorrhage, this highlights a major source of concern for clinical governance.
Drug-eluting stents: new era and new concerns
Authors: Bhatia V , Bhatia R , Dhindsa M
Published: February 2004

At present there is much excitement about drug-eluting stents, which hold promise for the treatment of coronary artery disease. This ingenious therapy involves coating the outside of a standard coronary stent with a thin polymer containing medication that can prevent scarring at the site of coronary intervention. Early trials with sirolimus coated stents showed that they might prevent coronary artery restenosis, but later studies, involving more complex coronary lesions, did not show a complete absence of restenosis. Recent studies have demonstrated the long term cost effectiveness of drug-eluting stents as they have reduced the need for revascularisation procedures. At present there are few data on the safety and effectiveness of stents over follow up periods exceeding two years, and data obtained from animal models of stenting might not be completely applicable to humans. There are concerns that drug-eluting stents might delay, rather than inhibit, restenosis. Also there is concern regarding the inflammation caused by the polymer substrate. This article reviews the present data on drug-eluting stents and their benefits, shortcomings, and concerns.
NUMERICAL SIMULATION OF COUPLED WAVES IN BOREHOLE
Authors: TINIVELLA UMBERTA
Published: February 2004

Borehole drillstring waves play an important role in geotechnical and exploration applications. Coupled waves are simulated inside and outside a real drillstring in borehole. The drillstring is considered a system with variable geometry. We simulated coupled waves in three domains: inner mud, drillstring, and annular mud. Each mode has a characteristic wave propagation velocity. A consequence of this coupling effect is that waves from the downhole tool can be partially reflected and converted to other modes at a discontinuity in the formation stiffness or in the annular cross section that can cause a significant reduction of the transmitted wave energy. Examples of application are reported.
All three ryanodine receptor isoforms generate rapid cooling responses in muscle cells
Authors: Protasi Feliciano , Shtifman Alexander , Julian Fred J. , Allen Paul D.
Published: February 2004

The rapid cooling (RC) response in muscle is an increase in cytoplasmic Ca2+ concentration ([Ca2+]i) that is probably caused by Ca2+ release from the sarcoplasmic reticulum (SR). However, the molecular bases of this response have not been completely elucidated. Three different isoforms of the SR Ca2+ release channels, or ryanodine receptors (RyRs), have been isolated (RyR1, RyR2, and RyR3). In the current investigation, the RC response was studied in RyR-null muscle cells (1B5) before and after transduction with HSV-1 virions containing the cDNAs encoding for RyR1, RyR2, or RyR3. Cells were loaded with fluo 4-AM to monitor changes in [Ca2+]i and perfused with either cold (∼0°C), room temperature (RT), or RT buffer containing 40 mM caffeine. Control cells showed no significant response to cold or caffeine, whereas robust Ca2+ transients were recorded in response to both RC and caffeine in transduced cells expressing any one of the three RyR isoforms. Our data demonstrate directly that RyRs are responsible for the RC response and that all three isoforms respond in a similar manner. Ca2+ release from RyRs is likely caused by a RC-induced conformational change of the channel from the closed to the open state.
Endothelia of Schlemm's canal and trabecular meshwork: distinct molecular, functional, and anatomic features
Authors: Alvarado Jorge A. , Betanzos Abigail , Franse-Carman Linda , Chen Janet , González-Mariscal Lorenza
Published: February 2004

The purpose of this study was to compare human endothelial cells from Schlemm's canal (SCEs) and the trabecular meshwork (TMEs) in terms of ZO-1 isoform expression, hydraulic conductivity (HC) properties, and “giant” vacuole (GV) formation. The principal study methods were Western blot, RT-PCR, immunofluorescence, and perfusion chambers. Blot signals for α+-and α--isoforms were similar in SCEs but less intense for the α+-relative to the α--signal in TMEs. With the anti-α+ antibody used at 1/50 dilution, binding occurred at cell borders of both cell types, but only to SCEs when used at a ≥1/200 dilution in vitro and in vivo. SCEs were more resistive than TMEs (HC = 0.66 vs. 1.32 μl·min-1·mmHg-1·cm-2; P < 0.001) when perfused from apex to base. When perfused in the other direction, SCEs were again more resistive (5.23 vs. 9.04 μl·min-1·mmHg-1·cm-2; P < 0.01). GV formation occurred only in SCEs as a function of flow direction, perfusion pressure, and time. We conclude that SCEs and TMEs have distinctive phenotypic properties involving their content of ZO-1 isoforms, barrier function, and GV formation.
Regulation of MDCK cell-substratum adhesion by RhoA and myosin light chain kinase after ATP depletion
Authors: Prahalad Priya , Calvo Ignacio , Waechter Holly , Matthews Jeffrey B. , Zuk Anna , Matlin Karl S.
Published: February 2004

The attachment of epithelial cells to the extracellular matrix substratum is essential for their differentiation and polarization. Despite this, the precise adhesion mechanism and its regulation are poorly understood. In the kidney, an ischemic insult causes renal tubular epithelial cells to detach from the basement membrane, even though they remain viable. To understand this phenomenon, and to probe the regulation of epithelial cell attachment, we used a model system consisting of newly adherent Madin-Darby canine kidney (MDCK) cells subjected to ATP depletion to mimic ischemic injury. We found that MDCK cells detach from collagen I after 60 min of ATP depletion but reattach when resupplied with glucose. Detachment is not caused by degradation or endocytosis of β1-integrins, which mediate attachment to collagen I. Basal actin filaments and paxillin-containing adhesion complexes are disrupted by ATP depletion and quickly reform on glucose repletion. However, partial preservation of basal actin by overexpression of constitutively active RhoA does not significantly affect cell detachment. Furthermore, Y-27632, an inhibitor of the RhoA effector Rho-kinase, does not prevent reattachment of cells on glucose addition, even though reformation of central stress fibers and large adhesion complexes is blocked. In contrast, reattachment of ATP-depleted cells and detachment of cells not previously subjected to ATP depletion are prevented by ML-7, an inhibitor of myosin light chain kinase (MLCK). We conclude that initial adherence of MDCK cells to a collagen I substratum is mediated by peripheral actin filaments and adhesion complexes regulated by MLCK but not by stress fibers and adhesion complexes controlled by RhoA.
Kv1.1 and Kv1.3 channels contribute to the delayed-rectifying K+ conductance in rat choroid plexus epithelial cells
Authors: Speake Tracey , Kibble Jonathan D. , Brown Peter D.
Published: February 2004

The choroid plexuses secrete, and maintain the composition of, the cerebrospinal fluid. K+ channels play an important role in these processes. In this study the molecular identity and properties of the delayed-rectifying K+ (Kv) conductance in rat choroid plexus epithelial cells were investigated. Whole cell K+ currents were significantly reduced by 10 nM dendrotoxin-K and 1 nM margatoxin, which are specific inhibitors of Kv1.1 and Kv1.3 channels, respectively. A combination of dendrotoxin-K and margatoxin caused a depolarization of the membrane potential in current-clamp experiments. Western blot analysis indicated the presence of Kv1.1 and Kv1.3 proteins in the choroid plexus. Furthermore, the Kv1.3 and Kv1.1 proteins appear to be expressed in the apical membrane of the epithelial cells in immunocytochemical studies. The Kv conductance was inhibited by 1 μM serotonin (5-HT), with maximum inhibition to 48% of control occurring in 8 min ( P < 0.05 by Student's t-test for paired data). Channel inhibition by 5-HT was prevented by the 5-HT2C antagonist mesulergine (300 nM). It was also attenuated in the presence of calphostin C (a protein kinase C inhibitor). The conductance was partially inhibited by 1,2-dioctanoyl- sn-glycerol and phorbol 12-myristate 13-acetate, both of which activate protein kinase C. These data suggest that 5-HT acts at 5-HT2C receptors to activate protein kinase C, which inhibits the Kv channels. In conclusion, Kv1.1 and Kv1.3 channels make a significant contribution to K+ efflux at the apical membrane of the choroid plexus.
Gastric parietal cell secretory membrane contains PKA- and acid-activated Kir2.1 K+ channels
Authors: Malinowska Danuta H. , Sherry Ann M. , Tewari Kirti P. , Cuppoletti John
Published: February 2004

Our objective was to identify and localize a K+ channel involved in gastric HCl secretion at the parietal cell secretory membrane and to characterize and compare the functional properties of native and recombinant gastric K+ channels. RT-PCR showed that mRNA for Kir2.1 was abundant in rabbit gastric mucosa with lesser amounts of Kir4.1 and Kir7.1, relative to β-actin. Kir2.1 mRNA was localized to parietal cells of rabbit gastric glands by in situ RT-PCR. Resting and stimulated gastric vesicles contained Kir2.1 by Western blot analysis at ∼50 kDa as observed with in vitro translation. Immunoconfocal microscopy showed that Kir2.1 was present in parietal cells, where it colocalized with H+-K+-ATPase and ClC-2 Cl- channels. Function of native K+ channels in rabbit resting and stimulated gastric mucosal vesicles was studied by reconstitution into planar lipid bilayers. Native gastric K+ channels exhibited a linear current-voltage relationship and a single-channel slope conductance of ∼11 pS in 400 mM K2SO4. Channel open probability (Po) in stimulated vesicles was high, and that of resting vesicles was low. Reduction of extracellular pH plus PKA treatment increased resting channel Po to ∼0.5 as measured in stimulated vesicles. Full-length rabbit Kir2.1 was cloned. When stably expressed in Chinese hamster ovary (CHO) cells, it was activated by reduced extracellular pH and forskolin/IBMX with no effects observed in nontransfected CHO cells. Cation selectivity was K+ = Rb+ >> Na+ = Cs+ = Li+ = NMDG+. These findings strongly suggest that the Kir2.1 K+ channel may be involved in regulated gastric acid secretion at the parietal cell secretory membrane.
Expression of the AQP-1 water channel in normal human tissues: a semiquantitative study using tissue microarray technology
Authors: Mobasheri A. , Marples D.
Published: February 2004

Aquaporin water channels are a family of membrane proteins that facilitate water movement across biological membranes. Aquaporin-1 (AQP-1) has been found to be important in osmotic water movement across cell membranes of epithelial and endothelial barriers. However, the distribution of AQP-1 in many normal human tissues is still unknown. The aim of this study was to use immunohistochemistry and semiquantitative histomorphometric analysis to determine the tissue distribution and relative expression of AQP-1 in normal human tissues using tissue microarray (TMA) technology. The normal human TMAs employed in this study included cardiovascular, respiratory, gastrointestinal, hepatic and pancreatobiliary, oral, salivary, nasal, mammary, fetal, endocrine, genital tract, central and peripheral nervous systems, urinary tract, skin, cartilage, and other soft connective tissues. Immunohistochemistry and semiquantitative histomorphometric analysis confirmed the presence of AQP-1 in endothelial barriers of almost all tissues and in many epithelial barriers. AQP-1 was highly expressed in the renal cortex, choroid plexus, and pancreatic ducts. AQP-1 expression levels were surprisingly high in the anus, gallbladder, and liver; moderate expression was also detected in the hippocampus and ependymal cells of the central nervous system. This is the first report of AQP-1 protein distribution in normal human TMAs. These findings confirm the presence of AQP-1 in human endothelia and selected water-transporting epithelia and several new locations, including mammary epithelium, articular chondrocytes, synoviocytes, and synovial microvessels where AQP-1 may be involved in milk production, chondrocyte volume regulation, synovial fluid secretion, and homeostasis, respectively.
Induction of control genes in intestinal gluconeogenesis is sequential during fasting and maximal in diabetes
Authors: Mithieux Gilles , Bady Isabelle , Gautier Amandine , Croset Martine , Rajas Fabienne , Zitoun Carine
Published: February 2004

We studied in rats the expression of genes involved in gluconeogenesis from glutamine and glycerol in the small intestine (SI) during fasting and diabetes. From Northern blot and enzymatic studies, we report that only phospho enolpyruvate carboxykinase (PEPCK) activity is induced at 24 h of fasting, whereas glucose-6-phosphatase (G-6-Pase) activity is induced only from 48 h. Both genes then plateau, whereas glutaminase and glycerokinase strikingly rebound between 48 and 72 h. The two latter genes are fully expressed in streptozotocin-diabetic rats. From arteriovenous balance and isotopic techniques, we show that the SI does not release glucose at 24 h of fasting and that SI gluconeogenesis contributes to 35% of total glucose production in 72-h-fasted rats. The new findings are that 1) the SI can quantitatively account for up to one-third of glucose production in prolonged fasting; 2) the induction of PEPCK is not sufficient by itself to trigger SI gluconeogenesis; 3) G-6-Pase likely plays a crucial role in this process; and 4) glutaminase and glycerokinase may play a key potentiating role in the latest times of fasting and in diabetes.
Excess body fat in men decreases plasma fatty acid availability and oxidation during endurance exercise
Authors: Mittendorfer Bettina , Fields David A. , Klein Samuel
Published: February 2004

The effect of relative body fat mass on exercise-induced stimulation of lipolysis and fatty acid oxidation was evaluated in 15 untrained men (5 lean, 5 overweight, and 5 obese with body mass indexes of 21 ± 1, 27 ± 1, and 34 ± 1 kg/m2, respectively, and %body fat ranging from 12 to 32%). Palmitate and glycerol kinetics and substrate oxidation were assessed during 90 min of cycling at 50% peak aerobic capacity (V̇o2 peak) by use of stable isotope-labeled tracer infusion and indirect calorimetry. An inverse relationship was found between %body fat and exercise-induced increase in glycerol appearance rate relative to fat mass ( r2= 0.74; P < 0.01). The increase in total fatty acid uptake during exercise [(μmol/kg fat-free mass) × 90 min] was ∼50% smaller in obese (181 ± 70; P < 0.05) and ∼35% smaller in overweight (230 ± 71; P < 0.05) than in lean (354 ± 34) men. The percentage of total fatty acid oxidation derived from systemic plasma fatty acids decreased with increasing body fat, from 49 ± 3% in lean to 39 ± 4% in obese men ( P < 0.05); conversely, the percentage of nonsystemic fatty acids, presumably derived from intramuscular and possibly plasma triglycerides, increased with increasing body fat ( P < 0.05). We conclude that the lipolytic response to exercise decreases with increasing adiposity. The blunted increase in lipolytic rate in overweight and obese men compared with lean men limits the availability of plasma fatty acids as a fuel during exercise. However, the rate of total fat oxidation was similar in all groups because of a compensatory increase in the oxidation of nonsystemic fatty acids.
5′-AMP-activated protein kinase activity and protein expression are regulated by endurance training in human skeletal muscle
Authors: Frøsig Christian , Jørgensen Sebastian B. , Hardie D. Grahame , Richter Erik A. , Wojtaszewski Jørgen F. P.
Published: February 2004

The 5′-AMP-activated protein kinase (AMPK) is proposed to be involved in signaling pathways leading to adaptations in skeletal muscle in response to both a single exercise bout and exercise training. This study investigated the effect of endurance training on protein content of catalytic (α1, α2) and regulatory (β1, β2 and γ1, γ2, γ3) subunit isoforms of AMPK as well as on basal AMPK activity in human skeletal muscle. Eight healthy young men performed supervised one-legged knee extensor endurance training for 3 wk. Muscle biopsies were obtained before and 15 h after training in both legs. In response to training the protein content of α1, β2 and γ1 increased in the trained leg by 41, 34, and 26%, respectively (α1 and β2 P < 0.005, γ1 P < 0.05). In contrast, the protein content of the regulatory γ3-isoform decreased by 62% in the trained leg ( P = 0.01), whereas no effect of training was seen for α2, β1, and γ2. AMPK activity associated with the α1- and the α2-isoforms increased in the trained leg by 94 and 49%, respectively (both P < 0.005). In agreement with these observations, phosphorylation of α-AMPK-(Thr172) and of the AMPK target acetyl-CoA carboxylase-β(Ser221) increased by 74 and 180%, respectively (both P < 0.001). Essentially similar results were obtained in four additional subjects studied 55 h after training. This study demonstrates that protein content and basal AMPK activity in human skeletal muscle are highly susceptible to endurance exercise training. Except for the increase in γ1 protein, all observed adaptations to training could be ascribed to local contraction-induced mechanisms, since they did not occur in the contralateral untrained muscle.
Amino acid ingestion improves muscle protein synthesis in the young and elderly
Authors: Paddon-Jones Douglas , Sheffield-Moore Melinda , Zhang Xiao-Jun , Volpi Elena , Wolf Steven E. , Aarsland Asle , Ferrando Arny A. , Wolfe Robert R.
Published: February 2004

We recently demonstrated that muscle protein synthesis was stimulated to a similar extent in young and elderly subjects during a 3-h amino acid infusion. We sought to determine if a more practical bolus oral ingestion would also produce a similar response in young (34 ± 4 yr) and elderly (67 ± 2 yr) individuals. Arteriovenous blood samples and muscle biopsies were obtained during a primed (2.0 μmol/kg) constant infusion (0.05 μmol·kg-1·min-1) of l-[ ring-2H5]phenylalanine. Muscle protein kinetics and mixed muscle fractional synthetic rate (FSR) were calculated before and after the bolus ingestion of 15 g of essential amino acids (EAA) in young ( n = 6) and elderly ( n = 7) subjects. After EAA ingestion, the rate of increase in femoral artery phenylalanine concentration was slower in elderly subjects but remained elevated for a longer period. EAA ingestion increased FSR in both age groups by ∼0.04%/h ( P < 0.05). However, muscle intracellular (IC) phenylalanine concentration remained significantly higher in elderly subjects at the completion of the study (young: 115.6 ± 5.4 nmol/ml; elderly: 150.2 ± 19.4 nmol/ml). Correction for the free phenylalanine retained in the muscle IC pool resulted in similar net phenylalanine uptake values in the young and elderly. EAA ingestion increased plasma insulin levels in young (6.1 ± 1.2 to 21.3 ± 3.1 μIU/ml) but not in elderly subjects (3.0 ± 0.6 to 4.3 ± 0.4 μIU/ml). Despite differences in the time course of plasma phenylalanine kinetics and a greater residual IC phenylalanine concentration, amino acid supplementation acutely stimulated muscle protein synthesis in both young and elderly individuals.
Reduced expression of renal Na+transporters in rats with PTH-induced hypercalcemia
Authors: Wang Weidong , Li Chunling , Kwon Tae-Hwan , Miller R. Tyler , Knepper Mark A. , Frøkiær Jørgen , Nielsen Søren
Published: February 2004

The purpose of this study was to evaluate whether the natriuresis and polyuria seen in parathyroid hormone (PTH)-induced hypercalcemia are associated with dysregulation of renal Na transporters. Rats were infused with three different doses of human PTH [PTH ( 1 - 34 ); 7.5, 10, and 15 μg·kg-1·day-1sc] or vehicle for 48 h using osmotic minipumps. The rats treated with PTH developed significant hypercalcemia (plasma total calcium levels: 2.71 ± 0.03, 2.77 ± 0.02, and 3.42 ± 0.06 mmol/l, respectively, P < 0.05 compared with corresponding controls). The rats with severe hypercalcemia induced by high-dose PTH developed a decreased glomerular filtration rate (GFR), increased urine output, reduced urinary osmolality, increased urinary Na excretion, and fractional excretion of Na. This was associated with downregulation (calculated as a fraction of control levels) of whole kidney expression of type 2 Na-Picotransporter (NaPi-2; 16 ± 6%), type 3 Na/H exchanger (NHE3; 42 ± 7%), Na-K-ATPase (55 ± 2%), and bumetanide-sensitive Na-K-2Cl cotransporter (BSC-1; 25 ± 4%). In contrast, an upregulation of the Ca2+-sensing receptor (CaR) was observed. Rats treated with moderate-dose PTH exhibited unchanged GFR but decreased urinary concentration. The whole kidney expression of NHE3 (52 ± 8%) and NaPi-2 (26 ± 5%) was persistently decreased, whereas BSC-1 and Na-K-ATPase protein levels were not altered. CaR expression was also increased. Moreover, rats treated with low-dose PTH showed very mild hypercalcemia but unchanged GFR, normal urinary concentration, and unchanged expression of Na transporters and CaR. In conclusion, the reduced expression of major renal Na transporters is likely to play a role in the increased urinary Na excretion and decreased urinary concentration in rats with PTH-induced hypercalcemia. Moreover, the increase in the CaR in the thick ascending limb (TAL) may indicate a potential role of the CaR in inhibiting Na transport in the TAL.
Characterization of a murine renal distal convoluted tubule cell line for the study of transcellular calcium transport
Authors: Diepens Robin J. W. , den Dekker Els , Bens Marcelle , Weidema A. Freek , Vandewalle Alain , Bindels René J. M. , Hoenderop Joost G. J.
Published: February 2004

To unravel the molecular regulation of renal transcellular Ca2+transport, a murine distal convoluted tubule (mpkDCT) cell line derived from distal convoluted tubules (DCT) microdissected from a SV-PK/Tag transgenic mouse was characterized. This cell line originated from DCT only, as mRNA encoding for the DCT marker thiazide-sensitive Na+/Cl-cotransporter was expressed, whereas mRNA encoding for the connecting tubule and collecting duct marker aquaporin-2 was not detected, as determined by reverse-transcriptase PCR. mpkDCT cells expressed mRNA encoding the Ca2+channels TRPV5 and TRPV6 and other key players necessary for transcellular Ca2+transport, i.e., calbindin-D9k, calbindin-D28k, plasma membrane Ca2+-ATPase isoform 1b, and Na+/Ca2+exchanger 1. Primary cultures of DCT cells exhibited net transcellular Ca2+transport of 0.4 ± 0.1 nmol·h-1·cm-2, whereas net transcellular Ca2+transport across mpkDCT cells was significantly higher at 2.4 ± 0.4 nmol·h-1·cm-2. Transcellular Ca2+transport across mpkDCT cells was completely inhibited by ruthenium red, an inhibitor of TRPV5 and TRPV6, but not by the voltage-operated Ca2+channel inhibitors felodipine and verapamil. With the use of patch-clamp analysis, the IC50of ruthenium red on Na+currents was between the values measured for TRPV5- and TRPV6-expressing HEK 293 cells, suggesting that TRPV5 and/or TRPV6 is possibly active in mpkDCT cells. Forskolin in combination with IBMX, 1,25-dihydroxyvitamin D3, and 1-deamino-8-d-arginine vasopressin increased transcellular Ca2+transport, whereas PMA and parathyroid hormone had no significant effect. In conclusion, the murine mpkDCT cell line provides a unique cell model in which to study the molecular regulation of transcellular Ca2+transport in the kidney in vitro.
Effect of UUO on D1aR expression reveals a link among dopamine, transforming growth factor-β, and nitric oxide
Authors: Stern Joshua M. , Chen Jie , Silver Randi B. , Poppas Dix P. , Vaughan E. Darracott , Felsen Diane
Published: February 2004

Interactions between transforming growth factor-β (TGF-β) and nitric oxide (NO) are important in the pathophysiology of unilateral ureteral obstruction (UUO). Dopamine (DA) is a vasoactive renal mediator active at the D1Areceptor (D1AR), which has not been studied in UUO; therefore, we examined the interactions among DA, TGF-β, and NO in UUO. In vivo, UUO was carried out in rats with or without concurrent treatment with 1D11, a monoclonal antibody to TGF-β, for 14 days. In vitro, NRK-52E cells (normal rat kidney tubules) were treated with DA, and NO and TGF-β release were examined. UUO resulted in a 70% decrease in the expression of renal D1AR, confirmed by both Western blot analysis and immunohistochemistry. 1D11 treatment restored expression to 60% of control values. DA treatment decreased NRK-52E release of TGF-β by 80%; conversely, DA significantly increased NO release from NRK-52E cells. These results suggest that DA modulates the release of cytokines, which are involved in the fibrotic and apoptotic sequelae of UUO, and that these effects are independent of DA's known vasoactive properties.
Sodium-dependent methotrexate carrier-1 is expressed in rat kidney: cloning and functional characterization
Authors: Kneuer Carsten , Honscha Kerstin U. , Honscha Walther
Published: February 2004

Previous Northern blot studies suggested strong expression of a homolog to the sodium-dependent hepatocellular methotrexate transporter in the kidneys. Here, we report on the cloning of the cDNA for the renal methotrexate carrier isoform-1 (RK-MTX-1) and its functional characterization. Sequencing revealed 97% homology to the rat liver methotrexate carrier with an identical open reading frame. Differences were located in the 5′-untranslated region and resulted in the absence of putative regulatory elements (Barbie box, Ah/ARNT receptor) identified in the cDNA for the hepatocellular carrier. For functional characterization, MTX-1 cDNA was stably expressed in Madin-Darby canine kidney (MDCK) cells. A sodium-dependent transport of methotrexate with a Kmof 41 μM and a Vmaxof 337 pmol·mg protein-1·min-1was observed. This uptake was blocked by the reduced folates dihydro- and tetrahydrofolate as well as by methotrexate itself. Folate was inhibiting only weakly, whereas 5-methyltetrahydrofolate was a strong inhibitor. Further inhibitors of the methotrexate transport included the bile acids cholate and taurocholate and xenobiotics like bumetanide and BSP. PAH, ouabain, bumetanide, cholate, taurocholate, and acetyl salicylic acid were tested as potential substrates. However, none of these substances was transported by MTX-1. Furthermore, expression of RK-MTX-1 in MDCK cells enhanced methotrexate toxicity in these cells fivefold. Analysis of a fusion protein of RK-MTX-1 and the influenza virus hemagglutinin epitope by immunoblotting revealed a major band at 72 kDa within the cell membrane but not in the soluble fraction of transfected MDCK. Indirect immunofluorescence staining revealed an exclusive localization of the carrier in the plasma membrane, and by confocal laser-scanning microscopy we were able to demonstrate that the protein is expressed in the serosal region of MDCK tubules grown in a morphogenic collagen gel model.
The actin cytoskeleton facilitates complement-mediated activation of cytosolic phospholipase A2
Authors: Cybulsky Andrey V. , Takano Tomoko , Papillon Joan , Khadir Abdelkrim , Bijian Krikor , Le Berre Ludmilla
Published: February 2004

Cytosolic PLA2-α (cPLA2) and metabolites of arachidonic acid (AA) are key mediators of complement-dependent glomerular epithelial cell (GEC) injury. Assembly of C5b-9 increases cytosolic Ca2+concentration and results in transactivation of receptor tyrosine kinases and activation of PLC-γ1 and the 1,2-diacylglycerol (DAG)-PKC pathway. Ca2+and PKC are essential for membrane association and increased catalytic activity of cPLA2. This study addresses the role of the actin cytoskeleton in cPLA2activation. Depolymerization of F-actin by cytochalasin D or latrunculin B reduced complement-dependent [3H]AA release, as well as the complement-induced increase in cPLA2activity. These effects were due to inhibition of [3H]DAG production and PKC activation, implying interference with PLC. Complement-dependent [3H]AA release was also reduced by jasplakinolide, a compound that stabilizes F-actin and organizes actin filaments at the cell periphery, and calyculin A, which induces condensation of actin filaments at the plasma membrane. The latter drugs did not affect [3H]DAG production, suggesting their inhibitory actions were downstream of PKC. Neither cytochalasin D, latrunculin B, nor calyculin A affected association of cPLA2with microsomal membranes, and cytochalasin D and latrunculin B did not alter the localization of the endoplasmic reticulum. Stable transfection of constitutively active RhoA induced formation of stress fibers, stabilized F-actin, and attenuated the complement-induced increase in [3H]AA. Thus in GEC, cPLA2activation is dependent, in part, on actin remodeling. By regulating complement-mediated activation of cPLA2, the actin cytoskeleton may contribute to the pathophysiology of GEC injury.
Kidney injury molecule-1: a tissue and urinary biomarker for nephrotoxicant-induced renal injury
Authors: Ichimura Takaharu , Hung Cheng Chieh , Yang Soon Ae , Stevens James L. , Bonventre Joseph V.
Published: February 2004

Nephrotoxicity is a common side effect of therapeutic interventions, environmental insults, and exposure to toxicants in the workplace. Although biomarkers for nephrotoxicity are available, they often lack sensitivity and are not specific as indicators of epithelial cell injury. Kidney injury molecule-1 (Kim-1) is a type 1 membrane protein with extracellular immunoglobulin and mucin domains. The mRNA and protein for Kim-1 are expressed at very low levels in normal rodent kidney, but expression increases dramatically after injury in proximal tubule epithelial cells in postischemic rodent kidney and in humans during ischemic acute renal failure. To evaluate the utility of Kim-1 as a biomarker for other types of renal injury, we analyzed tissue and urinary expression in response to three different types of nephrotoxicants in the rat: S-(1,1,2,2-tetrafluoroethyl)-l-cysteine (TFEC), folic acid, and cisplatin. Marked increases in Kim-1 expression were confirmed by immunoblotting in all three models. The protein was shown to be localized to the proximal tubule epithelial cell by immunofluorescence. Furthermore, Kim-1 protein was detected in urine of toxicant-treated rats. The temporal pattern of expression in response to TFEC is similar to the Kim-1 expression pattern in the postischemic kidney. In folic acid-treated kidneys, Kim-1 is clearly localized to the apical brush border of the well-differentiated proximal tubular epithelial cells. After folic acid treatment, expression of Kim-1 is present in the urine despite no significant increase in serum creatinine. Cisplatin treatment results in early detection of urinary Kim-1 protein and diffuse Kim-1 expression in S3 cells of the proximal tubule. Kim-1 can be detected in the tissue and urine on days 1 and 2 after cisplatin administration, occurring before an increase in serum creatinine. The upregulation of expression of Kim-1 and its presence in the urine in response to exposure to various types of nephrotoxicants suggest that this protein may serve as a general biomarker for tubular injury and repair processes.
Serial determination of glomerular filtration rate in conscious mice using FITC-inulin clearance
Authors: Qi Zhonghua , Whitt Irene , Mehta Amit , Jin Jianping , Zhao Min , Harris Raymond C. , Fogo Agnes B. , Breyer Matthew D.
Published: February 2004

Two nonradioactive methods for determining glomerular filtration rate (GFR) in conscious mice using FITC-labeled inulin (FITC-inulin) were evaluated. The first method measured GFR using clearance kinetics of plasma FITC-inulin after a single bolus injection. Based on a two-compartment model, estimated GFR was 236.69 ± 16.55 and 140.20 ± 22.27 μl/min in male and female C57BL/6J mice, respectively. Total or ⅚ nephrectomy reduced inulin clearance to 0 or 32.80 ± 9.32 μl/min, respectively. Conversely, diabetes mellitus induced by streptozotocin was associated with increased GFR. The other approach measured urinary inulin clearance using intraperitoneal microosmotic pumps to deliver FITC-inulin and metabolic cages to collect timed urine samples. This approach yielded similar GFR values of 211.11 ± 26.56 and 157.36 ± 20.02 μl/min in male and female mice, respectively. These studies demonstrate the feasibility of repeated nonisotopic measurement of inulin clearance in conscious mice.
Development of a porcine brain cDNA library, EST database, and microarray resource
Authors: Nobis William , Ren Xiaoning , Suchyta Steven P. , Suchyta Thomas R. , Zanella Adroaldo J. , Coussens Paul M.
Published: February 2004

Recent developments in expressed sequence tag (EST) and cDNA microarray technology have had a dramatic impact on the ability of scientists to study responses of thousands of genes to internal and external stimuli. In neurobiology, studies of the human brain have been expanding rapidly by use of functional genomics techniques. To enhance these studies and allow use of a porcine brain model, a normalized porcine brain cDNA library (PBL) has been generated and used as a base for EST discovery and microarray generation. In this report, we discuss initial sequence analysis of 965 clones from this resource. Our data revealed that library normalization successfully reduced the number of clones representing highly abundant cDNA species and overall clone redundancy. Cluster analysis revealed over 800 unique cDNA species representing a redundancy rate for the normalized library of 6.9% compared with 29.4% before normalization. Sequence information, BLAST results, and TIGR cluster matches for these ESTs are publicly available via a web-accessible database ( http://nbfgc.msu.edu ). A cDNA microarray was created using 877 unique porcine brain EST amplicons spotted in triplicate on glass slides. This microarray was assessed by performing a series of experiments designed to test hybridization efficiency and false-positive rate. Our results indicate that the PBL cDNA microarray is a robust tool for studies of brain gene expression using swine as a model system.
Serial analysis of gene expression in mouse kidney following angiotensin II administration
Authors: Schwartz Faina , Duka Arvi , Triantafyllidi Elena , Johns Conrado , Duka Irena , Cui Jing , Gavras Haralambos
Published: February 2004

As a new line of inquiry into the molecular mechanisms underlying pathophysiological processes associated with angiotensin (ANG II)-dependent hypertension, we applied the method of serial analysis of gene expression (SAGE) to examine genome-wide transcription changes in the kidneys of mice that developed hypertension in response to chronic ANG II administration. Mice were infused subcutaneously via osmotic minipumps with ANG II for 7 days, and systolic blood pressure was measured by tail-cuff plethysmography. Subsequently, mice were euthanized, and the total RNA isolated from the kidneys was used to construct SAGE libraries. Comparison of 11,447 SAGE tags from the hypertensive kidneys, representing 5,740 unique transcripts, and 11,273 tags from the control kidneys, corresponding to 5,619 different transcripts, identified genes that are significantly ( P < 0.05) down- or upregulated in the hypertensive kidney. Our assessment of the genome-wide influence of ANG II resulted in the detection of several novel genes and in a recognition of potential new roles for the previously characterized genes, thus providing new probes with which to further explore the ANG II effects in normal and disease states.
Microarray-based discovery of highly expressed olfactory mucosal genes: potential roles in the various functions of the olfactory system
Authors: Genter Mary Beth , Van Veldhoven Paul P. , Jegga Anil G. , Sakthivel Bhuvana , Kong Sue , Stanley Kristin , Witte David P. , Ebert Catherine L. , Aronow Bruce J.
Published: February 2004

We sought to gain a global view of tissue-specific gene expression in the olfactory mucosa (OM), the major site of neurogenesis and neuroregeneration in adult vertebrates, by examination of its overexpressed genes relative to that in 81 other developing and adult mouse tissues. We used a combination of statistical and fold-difference criteria to identify the top 269 cloned cDNAs from an array of 8,734 mouse cDNA elements on the Incyte Mouse GEM1 array. These clones, representing known and poorly characterized gene transcripts, were grouped according to their relative expression patterns across the other tissues and then further examined with respect to gene ontology categories. Approximately one-third of the 269 genes were also highly expressed in developing and/or adult central nervous system tissues. Several of these have been suggested or demonstrated to play roles in neurogenesis, neuronal differentiation, and/or neuronal migration, further suggesting that many of the unknown genes that share this expression pattern may play similar roles. Highly OM-specific genes included a palate, lung, and nasal epithelium carcinoma-associated gene ( Plunc); sphingosine phosphate lyase ( Sgpl1), and paraoxonase 1 ( Pon1). Cell-type-specific expression within OM was established using in situ hybridization for several representative expression pattern clusters. Using the ENSEMBL-assembled mouse genome and comparative genomics analyses to the human genome, we assigned many of the unknown expressed sequence tags (ESTs) and poorly characterized genes to either novel or known gene products and provided predictive classification. Further exploration of this database will provide additional insights into genes and pathways critical for olfactory neurogenesis, neuronal differentiation, olfaction, and mucosal defense.