Functional aspects of the photosynthetic light reactions in heat stressed Arabidopsis deficient in digalactosyl-diacylglycerol

Plants are often submitted, in their natural environment, to various abiotic stresses such as heat stress. However, elevated temperature has a detrimental impact on overall plant growth and development. We have examined the physiological response of the dgd1-2 and dgd1-3 Arabidopsis mutants lacking 30-40% of digalactosyl-diacylglycerol (DGDG) exposed to heat constraint. These mutants, which grow similarly to wild type under normal conditions, were previously reported to be defective in basal thermotolerance as measured by cotyledon development. However their functional properties were not described. Chlorophyll fluorescence measurements and absorbance changes at 820 nm were used to monitor photosystem II (PSII) and PSI activity, respectively. It was observed that both mutants have similar photosystem activities with some differences. The mutants were less able to use near saturation light energy and elicited higher rates of cyclic PSI electron flow compare to wild type. Arabidopsis leaves exposed to short-term (5 min) mild (40 °C) or strong (44 °C) heat treatment have shown a decline in the operating effective quantum yield of PSII and in the proportion of active PSI reaction centers. However, cyclic PSI electron flow was enhanced. The establishment of the energy-dependent non-photochemical quenching of chlorophyll fluorescence was accelerated but its decline under illumination was inhibited. Furthermore, heat stress affected the process implicated in the redistribution of light excitation energy between the photosystems known as the light state transitions. All the effects of heat stress mentioned above were more intense in the mutant leaves with dgd1-3 being even more susceptible. The decreased DGDG content of the thylakoid membranes together with other lipid changes are proposed to influence the thermo-sensitivity of the light reactions of photosynthesis towards heat stress.     

Genomic mRNA profiling reveals compensatory mechanisms for the requirement of the essential splicing factor U2AF

The large subunit of the U2 auxiliary factor (U2AF) recognizes the polypyrimidine tract (Py-tract) located adjacent to the 3' splice site to facilitate U2 snRNP recruitment. While U2AF is considered essential for pre-mRNA splicing, its requirement for splicing on a genome-wide level has not been analyzed. Using Solexa sequencing, we performed mRNA profiling for splicing in the Schizosaccharomyces pombe U2AF(59) (prp2.1) temperature-sensitive mutant. Surprisingly, our analysis revealed that introns show a range of splicing defects in the mutant strain. While U2AF(59) inactivation (nonpermissive) conditions inhibit splicing of some introns, others are spliced apparently normally. Bioinformatics analysis indicated that U2AF(59)-insensitive introns have stronger 5' splice sites and higher A/U content. Most importantly, features that contribute to U2AF(59) insensitivity of an intron unexpectedly reside in its 5'-most 30 nucleotides. These include the 5' splice site, a guanosine at position 7, and the 5' splice site-to-branch point sequence context. A differential requirement (similar to U2AF(59)) for introns may also apply to other general splicing factors (e.g., prp10). Our combined results indicate that U2AF insensitivity is a common phenomenon and that varied intron features support the existence of unrecognized aspects of spliceosome assembly.     

The Ngal reporter mouse detects the response of the kidney to injury in real time

Many proteins have been proposed to act as surrogate markers of organ damage, yet for many candidates the essential biomarker characteristics that link the protein to the injured organ have not yet been described. We generated an Ngal reporter mouse by inserting a double-fusion reporter gene encoding luciferase-2 and mCherry (Luc2-mC) into the Ngal (Lcn2) locus. The Ngal-Luc2-mC reporter accurately recapitulated the endogenous message and illuminated injuries in vivo in real time. In the kidney, Ngal-Luc2-mC imaging showed a sensitive, rapid, dose-dependent, reversible, and organ- and cell-specific relationship with tubular stress, which correlated with the level of urinary Ngal (uNgal). Unexpectedly, specific cells of the distal nephron were the source of uNgal. Cells isolated from Ngal-Luc2-mC mice also revealed both the onset and the resolution of the injury, and the actions of NF-κB inhibitors and antibiotics during infection. Thus, imaging of Ngal-Luc2-mC mice and cells identified injurious and reparative agents that affect kidney damage.     

Elevated expression of stromal palladin predicts poor clinical outcome in renal cell carcinoma

The role that stromal renal cell carcinoma (RCC) plays in support of tumor progression is unclear. Here we sought to determine the predictive value on patient survival of several markers of stromal activation and the feasibility of a fibroblast-derived extracellular matrix (ECM) based three-dimensional (3D) culture stemming from clinical specimens to recapitulate stromal behavior in vitro. The clinical relevance of selected stromal markers was assessed using a well annotated tumor microarray where stromal-marker levels of expression were evaluated and compared to patient outcomes. Also, an in vitro 3D system derived from fibroblasts harvested from patient matched normal kidney, primary RCC and metastatic tumors was employed to evaluate levels and localizations of known stromal markers such as the actin binding proteins palladin, alpha-smooth muscle actin (α-SMA), fibronectin and its spliced form EDA. Results suggested that RCCs exhibiting high levels of stromal palladin correlate with a poor prognosis, as demonstrated by overall survival time. Conversely, cases of RCCs where stroma presents low levels of palladin expression indicate increased survival times and, hence, better outcomes. Fibroblast-derived 3D cultures, which facilitate the categorization of stromal RCCs into discrete progressive stromal stages, also show increased levels of expression and stress fiber localization of α-SMA and palladin, as well as topographical organization of fibronectin and its splice variant EDA. These observations are concordant with expression levels of these markers in vivo. The study proposes that palladin constitutes a useful marker of poor prognosis in non-metastatic RCCs, while in vitro 3D cultures accurately represent the specific patient's tumor-associated stromal compartment. Our observations support the belief that stromal palladin assessments have clinical relevance thus validating the use of these 3D cultures to study both progressive RCC-associated stroma and stroma-dependent mechanisms affecting tumorigenesis. The clinical value of assessing RCC stromal activation merits further study.     

Prostacyclin receptor-mediated ATP release from erythrocytes requires the voltage-dependent anion channel

Erythrocytes have been implicated as controllers of vascular caliber by virtue of their ability to release the vasodilator ATP in response to local physiological and pharmacological stimuli. The regulated release of ATP from erythrocytes requires activation of a signaling pathway involving G proteins (G(i) or G(s)), adenylyl cyclase, protein kinase A, and the cystic fibrosis transmembrane conductance regulator as well as a final conduit through which this highly charged anion exits the cell. Although pannexin 1 has been shown to be the final conduit for ATP release from human erythrocytes in response to reduced oxygen tension, it does not participate in transport of ATP following stimulation of the prostacyclin (IP) receptor in these cells, which suggests that an additional protein must be involved. Using antibodies directed against voltage-dependent anion channel (VDAC)1, we confirm that this protein is present in human erythrocyte membranes. To address the role of VDAC in ATP release, two structurally dissimilar VDAC inhibitors, Bcl-x(L) BH4(4-23) and TRO19622, were used. In response to the IP receptor agonists, iloprost and UT-15C, ATP release was inhibited by both VDAC inhibitors although neither iloprost-induced cAMP accumulation nor total intracellular ATP concentration were altered. Together, these findings support the hypothesis that VDAC is the ATP conduit in the IP receptor-mediated signaling pathway in human erythrocytes. In addition, neither the pannexin inhibitor carbenoxolone nor Bcl-x(L) BH4(4-23) attenuated ATP release in response to incubation of erythrocytes with the β-adrenergic receptor agonist isoproterenol, suggesting the presence of yet another channel for ATP release from human erythrocytes.     

Effect of introducing a short amyloidogenic sequence from the Aβ peptide at the N‐terminus of 18‐residue amphipathic helical peptides

Fibril formation is the hallmark of pathogenesis in Alzheimer's disease and other amyloid disorders caused by conformational alterations leading to the aggregation of soluble monomers. Aβ40 self‐associates to form amyloid fibrils. Its central seven‐residue segment KLVFFAE (Aβ16–22), which is thought to be crucial for fibril formation of the full‐length peptide, forms fibrils even in isolation. Context‐dependent induction of amyloid formation by such sequences in peptides, which otherwise do not have that propensity, is of considerable interest. We have examined the effect of introducing the Aβ16–22 sequence at the N‐terminus of two amphipathic helical 18‐residue peptides Ac‐WYSEMKRNVQRLERAIEE‐am and Ac‐KQLIRFLKRLDRNLWGLA‐am, which have high average hydrophobic moment <μH> values but have net charges of 0 and +4, respectively, at neutral pH. Upon incubation in aqueous buffer, fibril‐like aggregates were discernible by transmission electron microscopy for the peptide with only 0 net charge, which also displayed ThT binding and β‐structure. Although both the sequences have been derived from amphipathic helical segments in globular proteins and possess high average hydrophobic moments, the +4 charge peptide lacks the ability to form fibrils, while the peptide with 0 charge has the tendency to form fibrillar structures. Variation in the net charge and the presence of several glutamic acids in the sequence of the peptide with net charge 0 appear to favor the formation of fibrils when the Aβ16–22 sequence is attached at the N‐terminus. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

NGAL-Siderocalin in kidney disease

Kidney damage induces the expression of a myriad of proteins in the serum and in the urine. The function of these proteins in the sequence of damage and repair is now being studied in genetic models and by novel imaging techniques. One of the most intensely expressed proteins is lipocalin2, also called NGAL or Siderocalin. While this protein has been best studied by clinical scientists, only a few labs study its underlying metabolism and function in tissue damage. Structure-function studies, imaging studies and clinical studies have revealed that NGAL-Siderocalin is an endogenous antimicrobial with iron scavenging activity. This review discusses the "iron problem" of kidney damage, the tight linkage between kidney damage and NGAL-Siderocalin expression and the potential roles that NGAL-Siderocalin may serve in the defense of the urogenital system. This article is part of a Special Issue entitled: Cell Biology of Metals.     

Amino acid sequence divergence of Tat protein (exon1)of subtype B and C HIV-1 strains: Does it have implications for vaccine development?

Functional genes of HIV-1 like the tat express proteins essential for viral survival and propagation. There are variations reported in levels of Tat transactivation among the different subtypes of HIV-1. This study looked at the amino acid differences in the different regions of Tat protein (exon 1) of subtype B and C strains of HIV-1 and tried to observe a molecular basis for protein function. HIV-1 sequences of subtype B (n=30) and C (n=60) strains were downloaded from HIV-1 Los Alamos data base. Among the 60 subtype C strain sequences, 30 each were from India and Africa. A HIV-1 Tat protein (exon 1) sequence, the consensus B and C sequence was obtained from the 'sequence search interface' in the Los Alamos HIV-1 sequence data. The sequences were visualized using Weblogo and the RNA binding regions of the three consensus sequences were also determined using BindN software program. Compared to subtype B, there was a high level of divergence in the auxiliary domain of tat exon 1 (amino acid positions 58- 69). The net charge of the subtype C (Indian) Tat protein (exon 1) auxiliary domain was -1.9 at pH 7 and it had an isoelectric point of 4.1. The net charge of the subtype C (African) auxiliary domain was -2.9 at pH 7 and it had an isoelectric point of 3.7 while the net charge of same region in subtype B was -0.9 at pH 7 with an isoelectric point of 4.9. The ratio of the hydrophilic residues to the total number of residues was 60% in the in both the Indian and African subtype C in the auxiliary domain while this was 50% in subtype B. The consensus subtype B sequence was found to have 36 RNA binding sites while subtype C (India) had 33 and subtype C (Africa) had 32 RNA binding sites. The HIV-1 Tat-TAR interaction is a potential target for inhibitors and being considered for its potential use in HIV-1 vaccines. Development of such inhibitor/vaccines would have to take into consideration the variation in amino acid sequence analyzed in this study as this could determine epitope presentation on MHC class I antigen for afferent immune response.     

A novel gamma -secretase assay based on detection of the putative C-terminal fragment-gamma of amyloid beta protein precursor

Alzheimer's disease is characterized by the deposits of the 4-kDa amyloid beta peptide (A beta). The A beta protein precursor (APP) is cleaved by beta-secretase to generate a C-terminal fragment, CTF beta, which in turn is cleaved by gamma-secretase to generate A beta. Alternative cleavage of the APP by alpha-secretase at A beta 16/17 generates the C-terminal fragment, CTFalpha. In addition to A beta, endoproteolytic cleavage of CTF alpha and CTF beta by gamma-secretase should yield a C-terminal fragment of 57-59 residues (CTF gamma). However, CTF gamma has not yet been reported in either brain or cell lysates, presumably due to its instability in vivo. We detected the in vitro generation of A beta as well as an approximately 6-kDa fragment from guinea pig brain membranes. We have provided biochemical and pharmacological evidence that this 6-kDa fragment is the elusive CTF gamma, and we describe an in vitro assay for gamma-secretase activity. The fragment migrates with a synthetic peptide corresponding to the 57-residue CTF gamma fragment. Three compounds previously identified as gamma-secretase inhibitors, pepstatin-A, MG132, and a substrate-based difluoroketone (t-butoxycarbonyl-Val-Ile-(S)-4-amino-3-oxo-2, 2-difluoropentanoyl-Val-Ile-OMe), reduced the yield of CTF gamma, providing additional evidence that the fragment arises from gamma-secretase cleavage. Consistent with reports that presenilins are the elusive gamma-secretases, subcellular fractionation studies showed that presenilin-1, CTF alpha, and CTF beta are enriched in the CTF gamma-generating fractions. The in vitro gamma-secretase assay described here will be useful for the detailed characterization of the enzyme and to screen for gamma-secretase inhibitors.