Linear Time Course Research Articles

TRPV6 channel mediates alcohol-induced gut barrier dysfunction and systemic response.

SUMMARYIntestinal epithelial tight junction disruption is a primary contributing factor in alcohol-associated endotoxemia, systemic inflammation, and multiple organ damage. Ethanol and acetaldehyde disrupt tight junctions by elevating intracellular Ca2+. Here we identify TRPV6, a Ca2+-permeable channel, as responsible for alcohol-induced elevation of intracellular Ca2+, intestinal barrier dysfunction, and systemic inflammation. Ethanol and acetaldehyde elicit TRPV6 ionic currents in Caco-2 cells. Studies in Caco-2 cell monolayers and mouse intestinal organoids show that TRPV6 deficiency or inhibition attenuates ethanol- and acetaldehyde-induced Ca2+ influx, tight junction disruption, and barrier dysfunction. Moreover, Trpv6−/− mice are resistant to alcohol-induced intestinal barrier dysfunction. Photoaffinity labeling of 3-azibutanol identifies a histidine as a potential alcohol-binding site in TRPV6. The substitution of this histidine, and a nearby arginine, reduces ethanol-activated currents. Our findings reveal that TRPV6 is required for alcohol-induced gut barrier dysfunction and inflammation. Molecules that decrease TRPV6 function have the potential to attenuate alcohol-associated tissue injury.

Pattern of the Heart Rate Performance Curve in Subjects with Beta-Blocker Treatment and Healthy Controls.

(1): Heart rate performance curve (HRPC) in incremental exercise was shown to be not uniform, causing false intensity estimation applying percentages of maximal heart rate (HRmax). HRPC variations are mediated by β-adrenergic receptor sensitivity. The aim was to study age and sex dependent differences in HRPC patterns in adults with β-blocker treatment (BB) and healthy controls (C). (2): A total of 535 (102 female) BB individuals were matched 1:1 for age and sex (male 59 ± 11 yrs, female 61 ± 11 yrs) in C. From the maximum incremental cycle ergometer exercise a first and second heart rate (HR) threshold (Th1 and Th2) was determined. Based on the degree of the deflection (kHR), HRPCs were categorized as regular (downward deflection (kHR > 0.1)) and non-regular (upward deflection (kHR < 0.1), linear time course). (3): Logistic regression analysis revealed a higher odds ratio to present a non-regular curve in BB compared to C (females showed three times higher odds). The odds for non-regular HRPC in BB versus C decreased with older age (OR interaction = 0.97, CI = 0.94–0.99). Maximal and submaximal performance and HR variables were significantly lower in BB (p < 0.05). %HRmax was significantly lower in BB versus C at Th2 (male: 77.2 ± 7.3% vs. 80.8 ± 5.0%; female: 79.2 ± 5.1% vs. 84.0 ± 4.3%). %Pmax at Th2 was similar in BB and C. (4): The HRPC pattern in incremental cycle ergometer exercise is different in individuals receiving β-blocker treatment compared to healthy individuals. The effects were also dependent on age and sex. Relative HR values at Th2 varied substantially depending on treatment. Thus, the percentage of Pmax seems to be a stable and independent indicator for exercise intensity prescription.

A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors.

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would not occur spontaneously. These proteins are crucial for essentially all aspects of cellular life, including metabolism, cell division, responses to environmental changes and movement. The protocol presented here describes a nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay that has been adapted to semi-high throughput screening of small molecule ATPase inhibitors. The assay has been applied to cardiac and skeletal muscle myosin II's, two actin-based molecular motor ATPases, as a proof of principle. The hydrolysis of ATP is coupled to the oxidation of NADH by enzymatic reactions in the assay. First, the ADP generated by the ATPase is regenerated to ATP by pyruvate kinase (PK). PK catalyzes the transition of phosphoenolpyruvate (PEP) to pyruvate in parallel. Subsequently, pyruvate is reduced to lactate by lactate dehydrogenase (LDH), which catalyzes the oxidation of NADH in parallel. Thus, the decrease in ATP concentration is directly correlated to the decrease in NADH concentration, which is followed by change to the intrinsic fluorescence of NADH. As long as PEP is available in the reaction system, the ADP concentration remains very low, avoiding inhibition of the ATPase enzyme by its own product. Moreover, the ATP concentration remains nearly constant, yielding linear time courses. The fluorescence is monitored continuously, which allows for easy estimation of the quality of data and helps to filter out potential artifacts (e.g., arising from compound precipitation or thermal changes).

Abstract 2252: Biomarkers for staging melanoma, a search at transcriptome level

Abstract Background: Melanoma represents one of the most aggressive malignancies and has a high tendency to metastasize. Metastatic potential may not be predicted from the morphologic features alone, and the use of routine histologic prognostic factors is insufficient for melanoma staging. Because metastatic melanoma remains extremely difficult to cure, there has been an urgent need to find new markers to identify tumors capable of metastasizing. Here, we used a computational strategy based on transcriptome meta-analysis for melanoma staging. We aimed to identify biomarkers that differentiate between non-metastatic and metastatic melanomas. Methods: Affymetrix HU 133A Plus 2.0 microarray data were obtained from the GEO database for the meta-analysis, representing benign nevi (N), stages I, II and III melanoma (EI-III), stage IV melanoma from primary site (EIV) and stage IV melanoma from metastatic site (Mm). Differential expression was analyzed using linear models (118 files) and time course (64 files). Transcripts with a log fold change of &amp;lt;-1/&amp;gt;1 and B-statistic &amp;gt;3 using linear models and evaluate by MB-statistic for Time Course were included in the study. The analysis focused on genes that encode secreted proteins, whose expression was upregulated throughout the progression of melanomas, starting at N and increasing gradually from EI-III to EIV and finally to Mm. Results: We obtained 6137 differentially expressed genes with linear models (between EI-III vs. N, EIV vs. EI-III, Mm vs. EIV, EIV vs. N and Mm vs. N contrasts) and took the top 6137 genes ranked with Time Course. Our results show 17 possible biomarkers that grouped into three types: 1) genes capable of differentiating between EIV/Mm and EI-III/N (SPP1); 2) genes capable of differentiating between Mm and EIV/EI-III/N (CXCR4, FCGR1B, NGRN, PHTF1, RAB8B, ARHGEF2, ARMC9, BCL2A1, CALU, DLGAP5, DMXL2, FKBP11 and STARD3NL); and 3) genes capable of differentiating between Mm/EIV/EI-III and N (ADAM10, CLIC4 and STK36). Conclusions: Our analyses, in agreement with previously reported results, identify SPP1 and CXCR4 as biomarkers of metastatic disease and poor clinical outcome, supporting the quality of our analysis. We also identified new biomarkers that may be used to predict undetectable metastasis. In order to provide a more precise evaluation, experimental validation or functional exploration of the reported gene expression differences is warranted. Citation Format: Daniel Ortega-Bernal, Claudia Rangel-Escareño, Elena Arechaga-Ocampo, Claudia H. Gonzalez-De la Rosa. Biomarkers for staging melanoma, a search at transcriptome level [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2252.

Impact of early life adversity on EMG stress reactivity of the trapezius muscle.

Human and animal research indicates that exposure to early life adversity increases stress sensitivity later in life. While behavioral markers of adversity-induced stress sensitivity have been suggested, physiological markers remain to be elucidated. It is known that trapezius muscle activity increases during stressful situations. The present study examined to what degree early life adverse events experienced during early childhood (0-11 years) and adolescence (12-17 years) moderate experimentally induced electromyographic (EMG) stress activity of the trapezius muscles, in an experimental setting. In a general population sample (n = 115), an anticipatory stress effect was generated by presenting a single unpredictable and uncontrollable electrical painful stimulus at t = 3 minutes. Subjects were unaware of the precise moment of stimulus delivery and its intensity level. Linear and nonlinear time courses in EMG activity were modeled using multilevel analysis. The study protocol included 2 experimental sessions (t = 0 and t = 6 months) allowing for examination of reliability.Results show that EMG stress reactivity during the stress paradigm was consistently stronger in people with higher levels of early life adverse events; early childhood adversity had a stronger moderating effect than adolescent adversity. The impact of early life adversity on EMG stress reactivity may represent a reliable facet that can be used in both clinical and nonclinical studies.

The progression of corrected myopia.

This study seeks to demonstrate the existence of a feedback loop controlling myopia by comparing the prediction of a feedback model to the actual progression of corrected myopia. In addition to theoretical results, confirming clinical data are presented. The refraction of 13 continuously corrected myopic eyes was collected over a period of time ranging from 4 to 9 years from the time of their first correction. Refractive data was collected in an optometry office from myopic young subjects from the general population in Boston. Subjects were myopes, ages 2 to 22 at the time of first correction selected randomly from a larger population. All individuals were fully corrected with lenses; new lenses were prescribed every time that their myopia increased by 0.25 diopters or more. Subjects wore their spectacle lenses during the followed period. Subjects exhibit a linear time course of myopia progression when corrected with lenses. The observed rate of myopia increase is 0.2 to 1.0 diopters/year, with a mean correlation coefficient r = -0.971, p < 0.005. This report establishes that feedback control theory applies to the clinical phenomenon of progressive myopia. Continuous correction of myopia results in a linear progression that increases myopia. The Laplace transformation of temporal refractive data to the s-domain simplifies the study of myopia and emmetropia. The feedback transfer function predicts that continuous correction of myopia results in a linear progression because continuous correction opens the feedback loop. This prediction is confirmed with all subjects.

Experimentally induced stress validated by EMG activity.

Experience of stress may lead to increased electromyography (EMG) activity in specific muscles compared to a non-stressful situation. The main aim of this study was to develop and validate a stress-EMG paradigm in which a single uncontrollable and unpredictable nociceptive stimulus was presented. EMG activity of the trapezius muscles was the response of interest. In addition to linear time effects, non-linear EMG time courses were also examined. Taking into account the hierarchical structure of the dataset, a multilevel random regression model was applied. The stress paradigm, executed in N = 70 subjects, consisted of a 3-minute baseline measurement, a 3-minute pre-stimulus stress period and a 2-minute post-stimulus phase. Subjects were unaware of the precise moment of stimulus delivery and its intensity level. EMG activity during the entire experiment was conform a priori expectations: the pre-stimulus phase showed a significantly higher mean EMG activity level compared to the other two phases, and an immediate EMG response to the stimulus was demonstrated. In addition, the analyses revealed significant non-linear EMG time courses in all three phases. Linear and quadratic EMG time courses were significantly modified by subjective anticipatory stress level, measured just before the start of the stress task. Linking subjective anticipatory stress to EMG stress reactivity revealed that subjects with a high anticipatory stress level responded with more EMG activity during the pre-stimulus stress phase, whereas subjects with a low stress level showed an inverse effect. Results suggest that the stress paradigm presented here is a valid test to quantify individual differences in stress susceptibility. Further studies with this paradigm are required to demonstrate its potential use in mechanistic clinical studies.

Evolution of radiographic damage in ankylosing spondylitis: a 12 year prospective follow-up of the OASIS study

ObjectivesTo describe the evolution of radiographic abnormalities of the spine in patients with ankylosing spondylitis (AS).MethodsPatients with AS were followed prospectively with 2 yearly radiographs for 12 years. The modified...

SAT0248 Evolution of Radiographic Damage in Ankylosing Spondylitis Over 12 Years of Follow-Up

BackgroundRadiographic damage is one of the core outcomes recommended by the ASAS for follow-up of patients with AS. So far, the evolution of radiographic damage over a long period has...

Biochemical and molecular characterization of a thermostable chitosanase produced by the strain Paenibacillus sp. 1794 newly isolated from compost

Chitosan raises a great interest among biotechnologists due to its potential for applications in biomedical or environmental fields. Enzymatic hydrolysis of chitosan is a recognized method allowing control of its molecular size, making possible its optimization for a given application. During the industrial hydrolysis process of chitosan, viscosity is a major problem; which can be circumvented by raising the temperature of the chitosan solution. A thermostable chitosanase is compatible with enzymatic hydrolysis at higher temperatures thus allowing chitosan to be dissolved at higher concentrations. Following an extensive micro-plate screening of microbial isolates from various batches of shrimp shells compost, the strain 1794 was characterized and shown to produce a thermostable chitosanase. The isolate was identified as a novel member of the genus Paenibacillus, based on partial 16S rDNA and rpoB gene sequences. Using the chitosanase (Csn1794) produced by this strain, a linear time course of chitosan hydrolysis has been observed for at least 6 h at 70 °C. Csn1794 was purified and its molecular weight was estimated at 40 kDa by SDS-PAGE. Optimum pH was about 4.8, the apparent Km and the catalytic constant kcat were 0.042 mg/ml and 7,588 min⁻¹, respectively. The half-life of Csn1794 at 70 °C in the presence of chitosan substrate was >20 h. The activity of chitosanase 1794 varied little with the degree of N-acetylation of chitosan. The enzyme also hydrolyzed carboxymethylcellulose but not chitin. Chitosan or cellulose-derived hexasaccharides were cleaved preferentially in a symmetrical way ("3+3") but hydrolysis rate was much faster for (GlcN)₆ than (Glc)₆. Gene cloning and sequencing revealed that Csn1794 belongs to family 8 of glycoside hydrolases. The enzyme should be useful in biotechnological applications of chitosan hydrolysis, dealing with concentrated chitosan solutions at high temperatures.

Task-related component analysis for functional neuroimaging and application to near-infrared spectroscopy data

Reproducibility of experimental results lies at the heart of scientific disciplines. Here we propose a signal processing method that extracts task-related components by maximizing the reproducibility during task periods from neuroimaging data. Unlike hypothesis-driven methods such as general linear models, no specific time courses are presumed, and unlike data-driven approaches such as independent component analysis, no arbitrary interpretation of components is needed. Task-related components are constructed by a linear, weighted sum of multiple time courses, and its weights are optimized so as to maximize inter-block correlations (CorrMax) or covariances (CovMax). Our analysis method is referred to as task-related component analysis (TRCA). The covariance maximization is formulated as a Rayleigh-Ritz eigenvalue problem, and corresponding eigenvectors give candidates of task-related components. In addition, a systematic statistical test based on eigenvalues is proposed, so task-related and -unrelated components are classified objectively and automatically. The proposed test of statistical significance is found to be independent of the degree of autocorrelation in data if the task duration is sufficiently longer than the temporal scale of autocorrelation, so TRCA can be applied to data with autocorrelation without any modification. We demonstrate that simple extensions of TRCA can provide most distinctive signals for two tasks and can integrate multiple modalities of information to remove task-unrelated artifacts. TRCA was successfully applied to synthetic data as well as near-infrared spectroscopy (NIRS) data of finger tapping. There were two statistically significant task-related components; one was a hemodynamic response, and another was a piece-wise linear time course. In summary, we conclude that TRCA has a wide range of applications in multi-channel biophysical and behavioral measurements.

Estimation of disease onset for patients with geographic atrophy due to age-related macular degeneration

Introduction: Patients of the longitudinal FAM Study receive a follow-up after the diagnosis of AMD. The patient's natural course of geographic atrophy (GA) is observed and related to lifestyle factors. Since patients are recruited after disease onset it is of interest to estimate the patient's age at disease onset and its link to risk factors. Material and Methods: Individual GA time courses are modelled by linear mixed effect models which allow to fit linear and non-linear curves and to extrapolate the individual GA time course to the time of onset for each patient eye. Linear or quadratic time course models result in specific onset scenarios and age of onset with regard to putative risk factors (e.g. smoking history, BMI, gender, hypertension). These onset distributions can be compared with data from other studies and used for the appropriate choice of a time course model for the FAM data. GAMLSS models assess differences between the onset distributions with respect to underlying risk factors. Results: 179 patients and 279 eyes with GA were observed over a follow-up time with IQR 1.01–3.57 years. Given the GA size at baseline and the individual linear (quadratic) growth curve, the onset of the disease process was estimated to start on average 4.75 y (5.31 y) before time of first GA measurement. The median age at onset is 69.80 (69.85) (IQR: 63.95–75.15; 64.33–75.55). In the linear model the density functions of age at onset in the exposed vs. non exposed showed differences with respect to gender (p=0.008) and BMI>/≤30 (p=0.088). Discussion: We try to validate onset scenarios by comparing the estimated distribution with data of the Munster Aging and Retina Study and the Rotterdam Study. Risk factors measured at baseline are considered as surrogate parameters for lifestyle factors in the past.

Parameters Affecting Cell Binding to Lectin Beads in Carbohydrate‐Based Drug Development

Using a model system of washed yeast (Saccharomyces cerevisiae) that possess mannose‐rich cell surfaces and washed concanavalin A derivatized agarose beads that preferentially bind to glycans containing mannose/glucose residues, we tested 4 concentrations of alpha methyl mannose and the same 4 concentrations of D‐melezitose (0.025 mM – 25 mM) and a 50/50 mixture of these 2 sugars at the same final concentrations, over a 30 minute time course, assaying how much yeast remained bound to the beads at 10 minute intervals with and without sugar at each sugar concentration tested. In 15,379 replicate experiments, averaging about 300 trials for each sugar at each time tested, alpha methyl mannose and D‐melezitose caused dissociation of yeast from the Con A beads in a concentration dependent manner with an almost linear time course with little dissociation occurring immediately and incrementally increased dissociation occurring after 10,20 and 30 minutes. The highest sugar concentration (25 mM) tested here was most effective in causing dissociation and the 50/50 mixture of sugars was less effective than the single sugars in causing dissociation at the lower concentrations (less than 25 mM). The results suggest that careful attention should be paid to sugar concentration and incubation times in experiments using lectin derivatized beads in molecular purification protocols and in development of carbohydrate‐based drugs and diagnostic tests (Supported by NIH NIGMS SCORE (S06‐48680, NIH MBRS RISE, NIH MARC and the Joseph Drown Foundation).

Age-Related Hearing Loss in C57BL/6J Mice has both Frequency-Specific and Non-Frequency-Specific Components that Produce a Hyperacusis-Like Exaggeration of the Acoustic Startle Reflex

Auditory brainstem-evoked response (ABR) thresholds were obtained in a longitudinal study of C57BL/6J mice between 10 and 53 weeks old, with repeated testing every 2 weeks. On alternate weeks, acoustic startle reflex (ASR) amplitudes were measured, elicited by tone pips with stimulus frequencies of 3, 6, 12, and 24 kHz, and intensities from subthreshold up to 110 dB sound pressure level. The increase in ABR thresholds for 3 and 6 kHz test stimuli followed a linear time course with increasing age from 10 to 53 weeks, with a slope of about 0.7 dB/week, and for 48 kHz a second linear time course, but beginning at 10 weeks with a slope of about 2.3 dB/week. ABR thresholds for 12, 24, and 32 kHz increased after one linear segment with a 0.7 dB slope, then after a variable delay related to the test frequency, shifted to a second segment having slopes of 3-5 dB/week. Hearing loss initially reduced the ASR for all eliciting stimuli, but at about 6 months of age, the response elicited by intense 3 and 6 kHz stimuli began to increase to reach values about three times above normal, and previously subthreshold stimuli came to elicit vigorous responses seen at first only for the intense stimuli. This hyperacusis-like effect appeared in all mice but was especially pronounced in mice with more serious hearing loss. These ABR data, together with a review of histopathological data in the C57BL/6 literature, suggest that the non-frequency-specific slow time course of hearing loss results from pathology in the lateral wall of the cochlea, whereas the stimulus-specific hearing loss with a rapid time course results from hair cell loss. Delayed exaggeration of the ASR with hearing loss reveals a deficit in centrifugal inhibitory control over the afferent reflex pathways after central neural reorganization, suggesting that this mouse may provide a useful model of age-related tinnitus and associated hyperacusis.

A new method for isolating physiologically active Mg-protoporphyrin monomethyl ester, the substrate of the cyclase enzyme of the chlorophyll biosynthetic pathway

Mg-protoporphyrin monomethyl ester (MPE) is a biosynthetic intermediate of chlorophyll and converted by MPE cyclase to protochlorophyllide. Limited availability of MPE has so far hampered cyclase research. In a new, simplified, method MPE was prepared from freeze dried bchE mutant Rhodobacter capsulatus DB575 cells by extraction with acetone/H 2O/25% NH 3. Isolated MPE was identified by absorption and fluorescence spectroscopy, and its purity was analyzed by HPLC. The extracted MPE was dried and redissolved in buffered DMSO and its substrate activity is shown by enzymatic cyclase assays. A linear time course was observed for MPE conversion to protochlorophyllide by enzymes from barley etioplasts. Our innovation of freeze drying the R. capsulatus cells before extraction provides a high yield method for MPE, which is significantly faster and more reproducible than previous extraction methods.

Poststroke Cognition and the Fight Against the Hard Problem

See related article, pages 198–203 In this issue of Stroke , Snaphaan and de Leeuw report on their results obtained from a systematic review on poststroke memory dysfunction.1 Of the 798 articles their search strategy identified, only 5 mentioned memory testing at different poststroke intervals, only 3 studies followed patients for at least 1 year, whereas the largest study contained only 196 patients. The results of the review allow at least 2 conclusions. One is drawn by the authors, in that memory dysfunction, a key element in the “diagnosis” of poststroke dementia, does not follow a linear time course after stroke. Leys et al came to a similar conclusion with regard to poststroke dementia, which was defined as any dementia after stroke.2 Short-term studies over-diagnose cognitive poststroke dysfunction. The finding warns us against making simple models of reality when it comes to poststroke cognitive impairment, and makes us aware that we should save our patients the embarrassment of early, false-positive predictions in this respect. The second conclusion the review allows is that our current knowledge of poststroke cognitive impairment and its long-term development is rather poor, as illustrated by the review’s data on memory dysfunction, a key element in the diagnostic criteria for dementia. The poststroke dementia studies reviewed by Leys et al, although larger and with longer follow-up by some, did not provide any detail either except for the criteria that they used for the diagnosis.2 One may question the value of studying any cognitive details when we can already make diagnoses such as vascular dementia to characterize the condition. We, physicians, are keen on making a diagnosis because it will tell us something about disease cause, spectrum of clinical manifestation, progression, and hopefully also about treatment. Exactly for those reasons we should not be …

A Transverse Tubule NADPH Oxidase Activity Stimulates Calcium Release from Isolated Triads via Ryanodine Receptor Type 1 S -Glutathionylation

We report here the presence of an NADPH oxidase (NOX) activity both in intact and in isolated transverse tubules and in triads isolated from mammalian skeletal muscle, as established by immunochemical, enzymatic, and pharmacological criteria. Immunohistochemical determinations with NOX antibodies showed that the gp91(phox) membrane subunit and the cytoplasmic regulatory p47(phox) subunit co-localized in transverse tubules of adult mice fibers with the alpha1s subunit of dihydropyridine receptors. Western blot analysis revealed that isolated triads contained the integral membrane subunits gp91(phox) and p22(phox), which were markedly enriched in isolated transverse tubules but absent from junctional sarcoplasmic reticulum vesicles. Isolated triads and transverse tubules, but not junctional sarcoplasmic reticulum, also contained varying amounts of the cytoplasmic NOX regulatory subunits p47(phox) and p67(phox). NADPH or NADH elicited superoxide anion and hydrogen peroxide generation by isolated triads; both activities were inhibited by NOX inhibitors but not by rotenone. NADH diminished the total thiol content of triads by one-third; catalase or apocynin, a NOX inhibitor, prevented this effect. NADPH enhanced the activity of ryanodine receptor type 1 (RyR1) in triads, measured through [3H]ryanodine binding and calcium release kinetics, and increased significantly RyR1 S-glutathionylation over basal levels. Preincubation with reducing agents or NOX inhibitors abolished the enhancement of RyR1 activity produced by NADPH and prevented NADPH-induced RyR1 S-glutathionylation. We propose that reactive oxygen species generated by the transverse tubule NOX activate via redox modification the neighboring RyR1 Ca2+ release channels. Possible implications of this putative mechanism for skeletal muscle function are discussed.

Induction of acid phosphatase activity during germination of maize ( Zea mays) seeds

Acid phosphatase activity (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.2) increased during the first 24 h of maize ( Zea mays) seed germination. The enzyme displayed a pH optimum of 4.5–5.5. Catalytic activity in vitro displayed a linear time course (60 min) and reached its half maximum value at 0.47 mM p-nitrophenyl phosphate (pNPP). Phosphatase activity towards phosphoamino acids was greatest for phosphotyrosine. The phosphatase activity was strongly inhibited by ammonium molybdate, vanadate and NaF and did not require divalent cations for the catalysis. The temperature optimum for pNPP hydrolysis was 37 °C. Under the same conditions, no enzyme activity was detected with phytic acid as substrate. Western blotting of total homogenates during seed germination revealed proteins/polypeptides that were phosphorylated on tyrosine residues; a protein of approximately 14 kDa is potentially a major biological substrate for the phosphatase activity. The results presented in this study suggest that the acid phosphatase characterized under the tested conditions is a member of the phosphotyrosine phosphatase family.

Strongest correlation of HbA 1c with 1-month-earlier glucose levels in type 2 diabetes

We examined the correlations of hemoglobin A 1c (HbA 1c) with each plasma glucose (PG) level obtained at 0 (the same day), 1 and 2 month(s) prior to HbA 1c determination. Data were from glycemic profiles of four patients of type 2 diabetes mellitus treated with tablets whose HbA 1c and pre- and post-breakfast PG levels were monitored each month. There was no significant difference in the correlation coefficients in cases 1 and 2, who presented with linear glycemic time courses. In contrast, HbA 1c correlated with 1-month-earlier pre-breakfast PG level more strongly than 2-month-earlier post-breakfast PG level in cases 3 and 4, and than same-day post-breakfast PG level in case 3 ( P<0.05, ANOVA). The cases 3 and 4 presented with fluctuating glycemic time courses. Samples were separated into upslope’s and downslope’s sections according to HbA 1c fluctuation in the latter two cases. Reflecting around the 1-month lag between HbA 1c and PG, the two sections’ regression lines for PG versus HbA 1c corresponded in the only samples related to 1-month-earlier pre- and post-breakfast PG ( t-test). In conclusion, it appears that pre- and post-breakfast PG levels are the most reliable predictors of 1-month-later HbA 1c in type 2 diabetic outpatients who undergo medical examinations every month.

Time-course of the internalization and recycling of neurokinin 1 receptors in rat dorsal horn neurons

Neurokinin 1 receptor (NK1R) internalization in dorsal horn neurons is important for intracellular signaling in nociception. Since the rates of NK1R internalization and recycling vary substantially, particularly between cultured and native cells, it is imperative to characterize them in dorsal horn neurons. When rat spinal cord slices were incubated at 35 °C with 1 μM substance P (SP), NK1Rs in lamina I neurons internalized rapidly following apparent exponential association kinetics (half-life=71 s). Confocal images of neuronal somas at different incubation times revealed that NK1Rs were uniformly distributed at the cell surface up to 30 s and formed aggregates at the membrane by 60 s. NK1R-containing endosomes migrated to the cell interior at 90–120 s, and were found throughout the cytoplasm at 300 s and thereafter. Upon elimination of SP, NK1Rs recycled back to the cell surface following an apparent linear time-course. Recycling was slower than internalization, being completed in 60–90 min. Confocal microscopy revealed that NK1R-containing endosomes docked at the cell surface 45 min after the elimination of SP. NK1Rs still formed aggregates at the cell surface at 60 min, but were once again uniformly distributed along the membrane by 90 min. NK1R internalization and recycling also occurred in lamina I dendrites. NK1R-containing endosomes in dendrites did not migrate to the cytoplasm. These results show that NK1R internalization and recycling are considerably faster in dorsal horn neurons than in cultured cells, and that most NK1Rs in dorsal horn neurons are internalized when NK1R-mediated hyperalgesia is more severe.