Cytokine regulation of facilitated glucose transport in human articular chondrocytes.

Glucose serves as the major energy substrate and the main precursor for the synthesis of glycosaminoglycans in chondrocytes. Facilitated glucose transport represents the first rate-limiting step in glucose metabolism. This study examines molecular regulation of facilitated glucose transport in normal human articular chondrocytes by proinflammatory cytokines. IL-1beta and TNF-alpha, and to a lesser degree IL-6, accelerate facilitated glucose transport as measured by [(3)H]2-deoxyglucose uptake. IL-1beta induces an increased expression of glucose transporter (GLUT) 1 mRNA and protein, and GLUT9 mRNA. GLUT3 and GLUT8 mRNA are constitutively expressed in chondrocytes and are not regulated by IL-1beta. GLUT2 and GLUT4 mRNA are not detected in chondrocytes. IL-1beta stimulates GLUT1 protein glycosylation and plasma membrane incorporation. IL-1beta regulation of glucose transport in chondrocytes depends on protein kinase C and p38 signal transduction pathways, and does not require phosphoinositide 3-kinase, extracellular signal-related kinase, or c-Jun N-terminal kinase activation. IL-1beta-accelerated glucose transport in chondrocytes is not mediated by endogenous NO or eicosanoids. These results demonstrate that stimulation of glucose transport represents a component of the chondrocyte response to IL-1beta. Two classes of GLUTs are identified in chondrocytes, constitutively expressed GLUT3 and GLUT8, and the inducible GLUT1 and GLUT9.     

南方4种草本植物对铝胁迫生理响应的研究

 不同的植物对铝胁迫的生理响应不同, 因而对铝毒的耐性也不相同。设置5种铝浓度,进行砂培法处理,研究了4种我国南方红壤广泛分布的草本植物——牵牛(Pharbitis nil)、望江南(Cassia occidentlis)、光头稗(Echinochloa colonum)和合萌(Aeschynomene indica)的种子萌发、光合色素、脯氨酸含量、丙二醛(MDA)含量、可溶性糖(SS)含量、质膜透性(MP)、过氧化氢酶 (CAT) 活性以及过氧化物酶 (POD)活性的变化。结果表明铝对4种植物的生理特性都有明显的影响。4种植物的种子在10 000 mg·L－1 Al3+处理条件下都不能萌发。2 000 mg·L－1 Al3+处理都不利于4种植物的生长,与对照相比,2 000 mg·L－1 Al3+处理时4种草本植物叶绿素和叶绿素总含量显著降低(p<0.05);MDA含量和MP显著增加(p<0.05);脯氨酸含量极显著增加(p<0.01);POD和CAT活性极显著降低(p<0.01)。中低铝(80和400 mg·L－1)处理时,牵牛和合萌与对照相比,MP和MDA含量降低,POD和CAT活性升高;望江南的反应与牵牛和合萌的反应相反;光头稗在80 mg·L－1 Al3+处理时,与牵牛和合萌的变化一致,在400 mg·L－1 Al3+处理时,则相反。植物在中低铝处理条件下,通过维持较高的POD和CAT活性和脯氨酸、叶绿素含量,较低的MP和MDA含量来增加其对铝的耐性。     

Platform development for expression and purification of stable isotope labeled monoclonal antibodies in Escherichia coli

The widespread use of monoclonal antibodies (mAbs) as a platform for therapeutic drug development in the pharmaceutical industry has led to an increased interest in robust experimental approaches for assessment of mAb structure, stability and dynamics. The ability to enrich proteins with stable isotopes is a prerequisite for the in-depth application of many structural and biophysical methods, including nuclear magnetic resonance (NMR), small angle neutron scattering, neutron reflectometry, and quantitative mass spectrometry. While mAbs can typically be produced with very high yields using mammalian cell expression, stable isotope labeling using cell culture is expensive and often impractical. The most common and cost-efficient approach to label proteins is to express proteins in Escherichia coli grown in minimal media; however, such methods for mAbs have not been reported to date. Here we present, for the first time, the expression and purification of a stable isotope labeled mAb from a genetically engineered E. coli strain capable of forming disulfide bonds in its cytoplasm. It is shown using two-dimensional NMR spectral fingerprinting that the unlabeled mAb and the mAb singly or triply labeled with ¹³C, ¹⁵N, ²H are well folded, with only minor structural differences relative to the mammalian cell-produced mAb that are attributed to the lack of glycosylation in the Fc domain. This advancement of an E. coli-based mAb expression platform will facilitate the production of mAbs for in-depth structural characterization, including the high resolution investigation of mechanisms of action.     

Application of Reference Data for Assessing and Restoring Headwater Ecosystems

Attributes of 25 headwater streams and their associated wetlands were quantitatively sampled in the inner coastal plain of eastern North Carolina. Data from these sites were used to construct and test one functional assessment model (biogeochemical cycling) using the hydrogeomorphic (HGM) approach. Of the 25 sites sampled, 16 unaltered sites were used to establish standards against which field indicators could be compared (indexed). Nine altered sites were used to examine the sensitivity of the model to assess the types of alterations typically inflicted upon headwater ecosystems in eastern North Carolina: channelization, logging, construction of cross-floodplain ditches to shunt water directly from uplands to the main stream channel, and conversion of stream floodplains and buffer zones to cropland. Of 30 field indicators measured that potentially could be used to model alterations to hydrologic regime and biomass stocks, we found six were robust in assessing conditions related to biogeochemical cycling. Hydrologic indicators used in the model included: (1) presence/absence of channelization, (2) presence/absence of cross-floodplain ditches, and (3) a measure of buffer condition (using width and quality). Biomass indicators included: (4) total basal area of trees, (5) percent litter cover, and (6) volume of coarse woody debris. Our preliminary biogeochemical cycling model using these six variables was sensitive to alterations in nine altered sites and to a suite of hypothetical restorations of the most altered site. However, in order to improve accuracy of our preliminary model, it should be validated with studies designed to measure how alterations of various types and magnitudes affect biogeochemical processes.     

Effects of estradiol on renal cyclic guanosine monophosphate and oxidative stress in spontaneously hypertensive rats

Background: Evidence suggests that estradiol offers protection against the development of cardiovascular and renal pathologies, although the mechanisms involved are still under investigation. The nitric oxide (NO) pathway regulates blood pressure and kidney function, and estradiol is associated with increases in NO bioavailability. We hypothesized that in female spontaneously hypertensive rats (SHRs), estra-diol increases NO bioavailability, activates the NO synthase (NOS) pathway, and suppresses superoxide production compared with rats that underwent ovariectomy (OVX).Objective: The goal of this study was to determine whether estradiol regulates the NO/cyclic guanosine monophosphate (cGMP) pathway and superoxide levels in the kidneys of female SHR.Methods: Three types of SHRs were studied: gonad-intact females, OVX rats, and OVX rats with estra-diol replacement (OVX+E). Renal cortical cGMP levels were measured to assess NO bioavailability. NOS enzymatic activity, NOS protein expression, basal superoxide production, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity were measured in the renal cortex.Results: Fifty-six SHRs were included in the study (17 intact females, 21 OVX rats, 18 OVX+E rats). Mean (SEM) cGMP levels were significantly lower in the renal cortex of OVX rats (0.03 [0.008] pmol/mg, n = 5) than in intact females (0.1 [0.02] pmol/mg, n = 6; P < 0.05), and estradiol restored cGMP levels to those seen in intact females (0.1 [0.01] pmol/mg, n = 5; P < 0.05). Despite a decrease in cGMP following OVX, renal cortical NOS activity, NOS1 and NOS3 protein expression, and the phosphorylation status of NOS3 were comparable among the 3 groups (n = 7–9 per group). However, mean basal superoxide production in the renal cortex was higher in OVX rats (3.2 [0.3] cpm/mg, n = 12) than in intact females (1.9 [0.3] cpm/mg, n = 8; P < 0.05) and lower in OVX+E rats (1.3 [0.3] cpm/mg, n = 9; P < 0.05). Mean NADPH oxidase activity was comparable in the renal cortex of intact females and OVX rats (81 [4] and 83 [12] cpm/35 μg, respectively [n = 5 per group]). OVX+E rats had significantly lower mean renal cortical NADPH oxidase activity than did rats in the other groups (45 [6] cpm/35 μg, n = 6; P < 0.05), and the decrease in activity was accompanied by a decrease in p22^phox protein expression.Conclusions: In vivo manipulations of estradiol levels influenced renal cortical NO bioavailability, as assessed indirectly by cGMP measurements. The decrease in cGMP following OVX was not due to alterations in the activity or expression of NOS.     

Canopy Composition and Forest Structure Provide Restoration Targets for Low-Order Riparian Ecosystems

Many programs are in place to protect and restore low-order streams and riparian zones. However, information on riparian zone forests is sparse for many biogeographical regions, especially compositional and structural data that would provide useful targets for restoration. This study provides quantitative data on riparian zone composition and forest structure from three physiographic provinces of eastern United States. Data from 219 low-order (first- to fourth-order) forested reaches were arranged by three basal area (BA) categories meant to represent successional categories and variations in forest structure. Detrended correspondence analysis (DCA) was used to illustrate differences among successional categories and physiographic provinces. The DCA ordination separated stands into four physiographic subregions, based on the species composition of late-successional stands. Many early to mid-successional stands (<30 m²/ha) were similar in composition to late-successional reference stands (BA ≥ 30 m²/ha) in the same physiographic subregion. In such sites, natural successional processes would likely be sufficient to restore the compositional and structural attributes inherent in late-successional stands if provided long-term protection. Other sites with dissimilar compositions may have been recovering from more intensive types of alterations, such as mechanized land clearing. In such sites, restoration to historic compositions could benefit functionally by planting oaks ( Quercus spp. L.) and other heavy mast species.     

SHAMS: Combining chemical modification of RNA with mass spectrometry to examine polypurine tract-containing RNA/DNA hybrids

Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) has gained popularity as a facile method of examining RNA structure both in vitro and in vivo, exploiting accessibility of the ribose 2′-OH to acylation by N-methylisatoic anhydride (NMIA) in unpaired or flexible configurations. Subsequent primer extension terminates at the site of chemical modification, and these products are fractionated by high-resolution gel electrophoresis. When applying SHAPE to investigate structural features associated with the wild-type and analog-substituted polypurine tract (PPT)–containing RNA/DNA hybrids, their size (20–25 base pairs) rendered primer extension impractical. As an alternative method of detection, we reasoned that chemical modification could be combined with tandem mass spectrometry, relying on the mass increment of RNA fragments containing the NMIA adduct (M_r = 133 Da). Using this approach, we demonstrate both specific modification of the HIV-1 PPT RNA primer and variations in its acylation pattern induced by replacing template nucleotides with a non-hydrogen-bonding thymine isostere. Our selective 2′-hydroxyl acylation analyzed by mass spectrometry strategy (SHAMS) should find utility when examining the structure of small RNA fragments or RNA/DNA hybrids where primer extension cannot be performed.