Protein and peptide array analysis of autoimmune disease

Molecular cloning, sequencing of the human genome, and other major advances in biomedical research have contributed substantially to our understanding of autoimmune disease. Nevertheless, to date, such advances have failed to reveal the etiology of or yield curative therapies for autoimmune disease. New approaches are needed. Proteomics, the large-scale study of expression and function of proteins that compose our tissues and mediate disease, represents a powerful and promising strategy. We developed protein and peptide arrays to profile autoantibody responses in autoimmune disease. Protein and peptide array analysis of autoimmune samples is revealing human and pathogen proteins involved in initiation and perpetuation of autoimmunity. Proteomic determination of autoantibody profiles can be utilized for diagnosis, prognostication, and guiding tolerizing therapy for autoimmune disease.     

SMN-mediated assembly of RNPs: a complex story

Although many RNA-protein complexes or ribonucleoproteins (RNPs) assemble spontaneously in vitro, little is known about how they form in the environment of a living cell. Insight into RNP assembly has come unexpectedly from functional analyses of the survival motor neuron (SMN) protein, a gene product that is affected in the neuromuscular disease spinal muscular atrophy. These studies show that the assembly of spliceosomal U-rich small nuclear RNPs in vivo depends on the activity of two large protein complexes, one of which contains the SMN protein. These complexes might also facilitate the assembly of other cellular RNPs.     

Protein arrays for autoantibody profiling and fine-specificity mapping

Protein arrays provide a powerful approach to study autoimmune disease. Autoimmune responses activate B cells to produce autoantibodies that recognize self-molecules termed autoantigens, many of which are proteins or protein complexes. Protein arrays enable profiling of the specificity of autoantibody responses against panels of peptides and proteins representing known autoantigens as well as candidate autoantigens. In addition to identifying autoantigens and mapping immunodominant epitopes, proteomic analysis of autoantibody responses will further enable diagnosis, prognosis, and tailoring of antigen-specific tolerizing therapy.     

The fate of U1 snRNP during anti-Fas induced apoptosis: specific cleavage of the U1 snRNA molecule

During apoptosis, the U1-70K protein, a component of the spliceosomal U1 snRNP complex, is specifically cleaved by the enzyme caspase-3, converting it into a C-terminally truncated 40-kDa fragment. In this study, we show that the 40-kDa U1-70K fragment is still associated with the complete U1 snRNP complex, and that no obvious modifications occur with the U1 snRNP associated proteins U1A, U1C and Sm-B/B'. Furthermore, it is described for the first time that the U1 snRNA molecule, which is the backbone of the U1 snRNP complex, is modified during apoptosis by the specific removal of the first 5 - 6 nucleotides including the 2,2, 7-trimethylguanosine (TMG) cap. The observations that U1 snRNA cleavage is very specific (no such modifications were detected for the other U snRNAs tested) and that U1 snRNA cleavage is markedly inhibited in the presence of caspase inhibitors, indicate that an apoptotically activated ribonuclease is responsible for the specific modification of U1 snRNA during apoptosis.     

Impact of ‘Stretch’ Targets for Cardiovascular Disease Management within a Local Pay-for-Performance Programme

Pay-for-performance programs are often aimed to improve the management of chronic diseases. We evaluate the impact of a local pay for performance programme (QOF+), which rewarded financially more ambitious quality targets (‘stretch targets’) than those used nationally in the Quality and Outcomes Framework (QOF). We focus on targets for intermediate outcomes in patients with cardiovascular disease and diabetes. A difference-in-difference approach is used to compare practice level achievements before and after the introduction of the local pay for performance program. In addition, we analysed patient-level data on exception reporting and intermediate outcomes utilizing an interrupted time series analysis. The local pay for performance program led to significantly higher target achievements (hypertension: p-value <0.001, coronary heart disease: p-values <0.001, diabetes: p-values <0.061, stroke: p-values <0.003). However, the increase was driven by higher rates of exception reporting (hypertension: p-value <0.001, coronary heart disease: p-values <0.03, diabetes: p-values <0.05) in patients with all conditions except for stroke. Exception reporting allows practitioners to exclude patients from target calculations if certain criteria are met, e.g. informed dissent of the patient for treatment. There were no statistically significant improvements in mean blood pressure, cholesterol or HbA1c levels. Thus, achievement of higher payment thresholds in the local pay for performance scheme was mainly attributed to increased exception reporting by practices with no discernable improvements in overall clinical quality. Hence, active monitoring of exception reporting should be considered when setting more ambitious quality targets. More generally, the study suggests a trade-off between additional incentive for better care and monitoring costs.     

Broadening the genetic base of European maize heterotic pools with US Cornbelt germplasm using field and molecular marker data

Maize (Zea mays L.) breeders are concerned about the narrowing of the genetic base of elite germplasm. To reverse this trend, elite germplasm from other geographic regions can be introgressed, but due to lack of adaptation it is difficult to assess their breeding potential in the targeted environment. The objectives of this study were to (1) investigate the relationship between European and US maize germplasm, (2) examine the suitability of different mega-environments and measures of performance to assess the breeding potential of exotics, and (3) study the relationship of genetic distance with mid-parent heterosis (MPH). Eight European inbreds from the Dent and Flint heterotic groups, 11 US inbreds belonging to Stiff Stalk (SS), non-Stiff Stalk (NSS), and CIMMYT Pool 41, and their 88 factorial crosses in F₁ and F₂ generations were evaluated for grain yield and dry matter concentration. The experiments were conducted in three mega-environments: Central Europe (target mega-environment), US Cornbelt (mega-environment where donor lines were developed), and Southeast Europe (an intermediate mega-environment). The inbreds were also fingerprinted with 266 SSR markers. Suitable criteria to identify promising exotic germplasm were F₁ hybrid performance in the targeted mega-environment and F₁ and parental performance in the intermediate mega-environment. Marker-based genetic distances reflected relatedness among the inbreds, but showed no association with MPH. Based on genetic distance, MPH, and F₁ performance, we suggest to introgress SS germplasm into European Dents and NSS into European Flints, in order to exploit the specific adaptation of European flint germplasm and the excellent combining ability of US germplasm in European maize breeding programs.     

Temporal and temperature effects on the maximum rate of rewarming from hibernation

During hibernation animals oscillate from near ambient (T(a)) to euthermic body temperatures (T(b)). As animals arouse, the rate of rewarming (RRW) might be expected to simply increase as a function of time. We monitored the T(b) of golden-mantled ground squirrels (Spermophilus lateralis) housed at 4, 8, 12, and 16 degrees C during natural arousals. The maximum RRW, the time required to reach a maximum RRW, and the relative time index all demonstrated negative relationships with T(a). The T(b) corresponding to maximal RRW demonstrated a positive relationship with T(a). Squirrels reached maximal RRW when they had generated 30 to 40% of the heat required to reach a euthermic T(b). These data suggest that arousal is more constrained than expected and that both time and temperature influence the RRW.     

Distribution of neuropeptides in the primary olfactory center of the heliothine moth <Emphasis Type="Italic">Heliothis virescens</Emphasis>

Neuropeptides are a diverse widespread class of signaling substances in the nervous system. As a basis for the analysis of peptidergic neurotransmission in the insect olfactory system, we have studied the distribution of neuropeptides in the antennal lobe of the moth Heliothis virescens. Immunocytochemical experiments with antisera recognizing A-type allatostatins (AST-As), Manduca sexta allatotropin (Mas-AT), FMRFamide-related peptides (FaRPs), and tachykinin-related peptides (TKRPs) have shown that members of all four peptide families are present in local interneurons of the antennal lobe. Whereas antisera against AST-As, Mas-AT, and FaRPs give similar staining patterns characterized by dense meshworks of processes confined to the core of all antennal-lobe glomeruli, TKRPs are present only in neurons with blebby processes distributed throughout each glomerulus. In addition to local neurons, a pair of centrifugal neurons with cell bodies in the lateral subesophageal ganglion, arborizations in the antennal lobe, and projections in the inner antenno-cerebral tracts exhibits tachykinin immunostaining. Double-label immunofluorescence has detected the co-localization of AST-As, Mas-AT, and FaRPs in certain local interneurons, whereas TKRPs occurs in a distinct population. MALDI-TOF mass spectrometry has revealed nearly 50 mass peaks in the antennal lobe. Seven of these masses (four AST-As, two N-terminally extended FLRFamides, and Mas-AT) match known moth neuropeptides. The data thus show that local interneurons of the moth antennal lobe are highly differentiated with respect to their neuropeptide content. The antennal lobe therefore represents an ideal preparation for the future analysis of peptide signaling in insect brain.     

Stn1 is critical for telomere maintenance and long‐term viability of somatic human cells

Disruption of telomere maintenance pathways leads to accelerated entry into cellular senescence, a stable proliferative arrest that promotes aging‐associated disorders in some mammals. The budding yeast CST complex, comprising Cdc13, Stn1, and Ctc1, is critical for telomere replication, length regulation, and end protection. Although mammalian homologues of CST have been identified recently, their role and function for telomere maintenance in normal somatic human cells are still incompletely understood. Here, we characterize the function of human Stn1 in cultured human fibroblasts and demonstrate its critical role in telomere replication, length regulation, and function. In the absence of high telomerase activity, shRNA‐mediated knockdown of hStn1 resulted in aberrant and fragile telomeric structures, stochastic telomere attrition, increased telomere erosion rates, telomere dysfunction, and consequently accelerated entry into cellular senescence. Oxidative stress augmented the defects caused by Stn1 knockdown leading to almost immediate cessation of cell proliferation. In contrast, overexpression of hTERT suppressed some of the defects caused by hStn1 knockdown suggesting that telomerase can partially compensate for hStn1 loss. Our findings reveal a critical role for human Stn1 in telomere length maintenance and function, supporting the model that efficient replication of telomeric repeats is critical for long‐term viability of normal somatic mammalian cells.     

Morphology and morphogenesis of a freshwater ciliate, Epistylis chlorelligerum Shen, 1980 (Ciliophora, Peritrichia)

An oligohalobic peritrichous ciliate, Epistylis chlorelligerum Shen, 1980, was collected from a ditch in Hangzhou, China. The morphology, oral infraciliature, and morphogenesis of the species were studied using living and protargol-impregnated specimens. Zooids of E. chlorelligerum are 160-230 × 50-60 μm in vivo, and characterized by green-colored endoplasm containing symbiotic algae. The oral infraciliature presents a well-developed filamentous reticulum linked to the circular fiber of the cytostome; the outer two rows of P3 extend adstomally over P1 and usually enfold it. During binary fission, one daughter cell inherits most part of the old buccal apparatus and the reorganized haplokinety and germinal kinety (Hk' and G'), and new buccal apparatus of the other daughter cell is mostly developed from the original germinal kinety (G) and haplokinety (Hk): new peniculi 2, 3 (2P2, 2P3), new haplokinety (2Hk), and new germinal kinety (2G) are formed from G, while the new peniculus 1 (2P1) and its peristomial extention (2Pk) originate from Hk. The epistomial membrane can be observed until the two sets of buccal apparatus begin to separate from each other.