A rapid cloning method employing orthogonal end protection

We describe a novel in vitro cloning strategy that combines standard tools in molecular biology with a basic protecting group concept to create a versatile framework for the rapid and seamless assembly of modular DNA building blocks into functional open reading frames. Analogous to chemical synthesis strategies, our assembly design yields idempotent composite synthons amenable to iterative and recursive split-and-pool reaction cycles. As an example, we illustrate the simplicity, versatility and efficiency of the approach by constructing an open reading frame composed of tandem arrays of a human fibronectin type III (FNIII) domain and the von Willebrand Factor A2 domain (VWFA2), as well as chimeric (FNIII)(n)-VWFA2-(FNIII)(n) constructs. Although we primarily designed this strategy to accelerate assembly of repetitive constructs for single-molecule force spectroscopy, we anticipate that this approach is equally applicable to the reconstitution and modification of complex modular sequences including structural and functional analysis of multi-domain proteins, synthetic biology or the modular construction of episomal vectors.     

Field and laboratory studies on the bioconversion of coal to methane in the San Juan Basin

The bioconversion of coal to methane in the San Juan Basin, New Mexico, was investigated. Production waters were analyzed via enrichment studies, metabolite-profiling, and culture-independent methods. Analysis of 16S rRNA gene sequences indicated the presence of methanogens potentially capable of acetoclastic, hydrogenotrophic, and methylotrophic metabolisms, predominantly belonging to the Methanosarcinales and Methanomicrobiales. Incubations of produced water and coal readily produced methane, but there was no correlation between the thermal maturity and methanogenesis. Coal methanogenesis was greater when samples with a greater richness of Firmicutes were utilized. A greater archaeal diversity was observed in the presence of several aromatic and short-chain fatty acid metabolites. Incubations amended with lactate, hydrogen, formate, and short-chain alcohols produced methane above un-amended controls. Methanogenesis from acetate was not observed. Metabolite profiling showed the widespread occurrence of putative aromatic ring intermediates including benzoate, toluic acids, phthalic acids, and cresols. The detection of saturated and unsaturated alkylsuccinic acids indicated n-alkane and cyclic alkane/alkene metabolism. Microarray analysis complemented observations based on hybridization to functional genes related to the anaerobic metabolism of aromatic and aliphatic substrates. These data suggest that coal methanogenesis is unlikely to be limited by methanogen biomass, but rather the activation and degradation of coal constituents.     

Induction of long-term B-cell depletion in refractory rheumatoid arthritis patients preferentially affects autoreactive more than protective humoral immunity

Introduction

B-cell depletion has become a common treatment strategy in anti-TNF-refractory rheumatoid arthritis (RA). Although the exact mechanism of how B-cell depletion leads to clinical amelioration in RA remains to be elucidated, repetitive treatment with B-cell-depleting agents leading to long-term B-cell depletion has been reported to be beneficial. The latter has led to the hypothesis that the beneficial effects of B-cell depletion might act through their influence on pathogenic autoreactive plasma cells.

Methods

In this study, we investigated the effects of a fixed retreatment regimen with anti-CD20 mAbs on the humoral (auto)immune system in a cohort of therapy-refractory RA patients.

Results

Fixed retreatment led to long-term B-cell depletion in peripheral blood, bone marrow and, to a lesser extent, synovium. Also, pathologic autoantibody secretion (that is, anticitrullinated peptide antibodies (ACPAs)) was more profoundly affected by long-term depletion than by physiological protective antibody secretion (that is, against measles, mumps and rubella). This was further illustrated by a significantly shorter estimated life span of ACPA-IgG secretion compared to total IgG secretion as well as protective antibody secretion.

Conclusion

By studying plasma cell function during an extensive 2-year period of B-cell depletion, autoantibody secretion was significantly shorter-lived than physiologically protective antibody secretion. This suggests that the longevity of autoreactive plasma cells is different from protective long-lived plasma cells and might indicate a therapeutic window for therapies that target plasma cells.     

"Rheumatoid Arthritis": A balanced overview of current research

This is a brief review of the third volume of the series edited by AN Theofilopoulos, "Current Directions in Autoimmunity". This hard-cover volume comprises 282 pages, 46 figures and 14 tables. The book has a cover price of 196 Swiss Francs, or 255 Deutschmarks, or 170.50 US dollars.     

The myogenic component in distention-induced peristalsis in the guinea pig small intestine

In an in vitro model for distention-induced peristalsis in the guinea pig small intestine, the electrical activity, intraluminal pressure, and outflow of contents were studied simultaneously to search for evidence of myogenic control activity. Intraluminal distention induced periods of nifedipine-sensitive slow wave activity with superimposed action potentials, alternating with periods of quiescence. Slow waves and associated high intraluminal pressure transients propagated aborally, causing outflow of content. In the proximal small intestine, a frequency gradient of distention-induced slow waves was observed, with a frequency of 19 cycles/min in the first 1 cm and 11 cycles/min 10 cm distally. Intracellular recording revealed that the guinea pig small intestinal musculature, in response to carbachol, generated slow waves with superimposed action potentials, both sensitive to nifedipine. These slow waves also exhibited a frequency gradient. In addition, distention and cholinergic stimulation induced high-frequency membrane potential oscillations (~55 cycles/min) that were not associated with distention-induced peristalsis. Continuous distention produced excitation of the musculature, in part neurally mediated, that resulted in periodic occurrence of bursts of distally propagating nifedipine-sensitive slow waves with superimposed action potentials associated with propagating intraluminal pressure waves that caused pulsatile outflow of content at the slow wave frequency.     

Allele-specific expression of the IL-1 alpha gene in human CD4+ T cell clones

A number of reports have described the monoallelic expression of murine cytokine genes. Here we describe the monoallelic expression of the human IL-1alpha gene in CD4+ T cells. Analysis of peripheral blood T cell clones derived from healthy individuals revealed that the IL-1alpha gene shows predominantly monoallelic expression. Monoallelic expression was observed in Th0, Th1, and Th2 cell clones. In addition, we demonstrate monoallelic expression in T cell clones from rheumatoid arthritis patients derived from synovial fluid of the knee joint, suggesting that the occurrence of this phenomenon is not different from that in clones derived from healthy individuals. The finding of monoallelic expression of a cytokine gene in human CD4+ T cell clones provides evidence for allele-specific silencing/activation as another layer of regulation of IL-1alpha gene expression.     

A consensus tetrapeptide selected by phage display adopts the conformation of a dominant discontinuous epitope of a monoclonal anti-VWF antibody that inhibits the von Willebrand factor-collagen interaction

Monoclonal antibody (mAb) 82D6A3 is an anti-von Willebrand factor (VWF) mAb directed against the A3-domain of VWF that inhibits the VWF binding to fibrillar collagens type I and III in vitro and in vivo. To identify the discontinuous epitope of this mAb, we used phage display, mutant analysis, and peptide modeling. All 82D6A3-binding phages displayed peptides containing the consensus sequence SPWR that could be aligned with P981W982 in the VWF A3-domain. Next, the binding of mAb 82D6A3 to 27 Ala mutants with mutations in the A3-domain of VWF revealed that amino acids Arg963, Pro981, Asp1009, Arg1016, Ser1020, Met1022, and His1023 are part of the epitope of mAb 82D6A3. Inspection of residues Ser1020, Arg1016, Pro981, and Trp982 in the three-dimensional structure of the A3-domain demonstrated that these residues are close together in space, pointing out that the structure of the SPWR consensus sequence might mimic this discontinuous epitope. Modeling of a cyclic 6-mer peptide containing the consensus sequence and superposition of its three-dimensional structure onto the VWF A3-domain demonstrated that the Ser and Arg in the peptide matched the Ser1020 and Arg1016 in the A3-domain. The Pro residue of the peptide served as a spacer, and the side chain of the Trp pointed in the direction of Trp982. In conclusion, to our knowledge, this is the first report where a modeled peptide containing a consensus sequence could be fitted onto the three-dimensional structure of the antigen, indicating that it might adopt the conformation of the discontinuous epitope.     

Male preponderance in early diagnosed type 2 diabetes is associated with the ARE insertion/deletion polymorphism in the <Emphasis Type="Italic">PPP1R3A</Emphasis> locus

Background  

The ARE insertion/deletion polymorphism of PPP1R3A has been associated with variation in glycaemic parameters and prevalence of diabetes. We have investigated its role in age of diagnosis, body weight and glycaemic control in 1,950 individuals with type 2 diabetes in Tayside, Scotland, and compared the ARE2 allele frequencies with 1,014 local schoolchildren.     

LabPatch, an acquisition and analysis program for patch-clamp electrophysiology

An acquisitionand analysis program, "LabPatch," has been developed for use inpatch-clamp research. LabPatch controls any patch-clamp amplifier,acquires and records data, runs voltage protocols, plots and analyzesdata, and connects to spreadsheet and database programs. Controlswithin LabPatch are grouped by function on one screen, much like anoscilloscope front panel. The software is mouse driven, so that theuser need only point and click. Finally, the ability to copy data toother programs running in Windows 95/98, and the ability to keep trackof experiments using a database, make LabPatch extremely versatile. Thesystem requirements include Windows 95/98, at least a 100-MHz processor and 16 MB RAM, a data acquisition card, digital-to-analogconverter, and a patch-clamp amplifier. LabPatch is available free ofcharge at http://www.fhs.mcmaster.ca/huizinga/.