Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.      

Two silicone nontoxic fouling release coatings: Hydrosilation cured PDMS and CaCO3 filled,ethoxysiloxane cured RTV11

Two silicone coatings have been evaluated for barnacle adhesion. One coating is an unfilled hydrosilation cured polydimethylsiloxane (PDMS) network, while the other is a room temperature vulcanized (RTV), filled, ethoxysiloxane cured PDMS elastomer, RTV11^?. The adhesion strength of one species of barnacle, Balanus eburneus, to the hydrosilation coatings is in the range of 0.37–0.60 kg cm^‐2 while the corresponding range for RTV11 is 0.64–0.90 kg cm^‐2. The easier release of B. eburneus from the hydrosilation cured network compared to RTV11 is discussed in relationship to differences in bulk and surface properties. Preliminary results suggest bulk modulus may be the most important parameter in determining barnacle adhesion strength. In light or mechanical property analysis, a re‐evaluation of surface properties and chemical stability is presented.     

F1-ATPase of Escherichia coli: THE ?-INHIBITED STATE FORMS AFTER ATP HYDROLYSIS,IS DISTINCT FROM THE ADP-INHIBITED STATE,AND RESPONDS DYNAMICALLY TO CATALYTIC SITE LIGANDS*

F₁-ATPase is the catalytic complex of rotary nanomotor ATP synthases. Bacterial ATP synthases can be autoinhibited by the C-terminal domain of subunit ϵ, which partially inserts into the enzyme''s central rotor cavity to block functional subunit rotation. Using a kinetic, optical assay of F₁·ϵ binding and dissociation, we show that formation of the extended, inhibitory conformation of ϵ (ϵ_X) initiates after ATP hydrolysis at the catalytic dwell step. Prehydrolysis conditions prevent formation of the ϵ_X state, and post-hydrolysis conditions stabilize it. We also show that ϵ inhibition and ADP inhibition are distinct, competing processes that can follow the catalytic dwell. We show that the N-terminal domain of ϵ is responsible for initial binding to F₁ and provides most of the binding energy. Without the C-terminal domain, partial inhibition by the ϵ N-terminal domain is due to enhanced ADP inhibition. The rapid effects of catalytic site ligands on conformational changes of F₁-bound ϵ suggest dynamic conformational and rotational mobility in F₁ that is paused near the catalytic dwell position.     

Regulation of the Rev1–pol ζ complex during bypass of a DNA interstrand cross‐link

DNA interstrand cross‐links (ICLs) are repaired in S phase by a complex, multistep mechanism involving translesion DNA polymerases. After replication forks collide with an ICL, the leading strand approaches to within one nucleotide of the ICL (“approach”), a nucleotide is inserted across from the unhooked lesion (“insertion”), and the leading strand is extended beyond the lesion (“extension”). How DNA polymerases bypass the ICL is incompletely understood. Here, we use repair of a site‐specific ICL in Xenopus egg extracts to study the mechanism of lesion bypass. Deep sequencing of ICL repair products showed that the approach and extension steps are largely error‐free. However, a short mutagenic tract is introduced in the vicinity of the lesion, with a maximum mutation frequency of ~1%. Our data further suggest that approach is performed by a replicative polymerase, while extension involves a complex of Rev1 and DNA polymerase ζ. Rev1–pol ζ recruitment requires the Fanconi anemia core complex but not FancI–FancD2. Our results begin to illuminate how lesion bypass is integrated with chromosomal DNA replication to limit ICL repair‐associated mutagenesis.     

Supported membrane composition analysis by secondary ion mass spectrometry with high lateral resolution

The lateral organization of lipid components within membranes is usually investigated with fluorescence microscopy, which, though highly sensitive, introduces bulky fluorophores that might alter the behavior of the components they label. Secondary ion mass spectroscopy performed with a NanoSIMS 50 instrument also provides high lateral resolution and sensitivity, and many species can be observed in parallel without the use of bulky labels. A tightly focused beam (approximately 100 nm) of Cs ions is scanned across a sample, and up to five of the resulting small negative secondary ions can be simultaneously analyzed by a high-resolution mass spectrometer. Thin layers of (15)N- and (19)F-labeled proteins were microcontact-printed on an oxidized silicon substrate and imaged using the NanoSIMS 50, demonstrating the sensitivity and selectivity of this approach. Supported lipid bilayers were assembled on an oxidized silicon substrate, then flash-frozen and freeze-dried to preserve their lateral organization. Lipid bilayers were analyzed with the NanoSIMS 50, where the identity of each specific lipid was determined through detection of its unique secondary ions, including (12)C(1)H(-), (12)C(2)H(-), (13)C(-), (12)C(14)N(-), and (12)C(15)N(-). Steps toward obtaining quantitative composition analysis of lipid membranes that varied spatially in isotopic composition are presented. This approach has the potential to provide a composition-specific analysis of membrane organization that compliments other imaging modalities.     

Novel functionalized biodegradable polymers for nanoparticle drug delivery systems

We have prepared and screened a library of novel functionalized polymers for development of nanoparticle drug delivery systems. The polymer backbone consisting of two ester-linked, nontoxic, biological monomers, glycerol and adipic acid, was prepared using a hydrolytic enzyme. The specificity of the chosen enzyme yields a linear polymer with one free pendant hydroxyl group per repeat unit, which can be further functionalized. This protocol gives control over the backbone polymer molecular weight, together with the ability to incorporate various amounts of different fatty acyl substituents. These functionalized polymers are able to self-assemble into well-defined small particles of high homogeneity with a very low toxicity. They are able to incorporate a water soluble drug, dexamethasone phosphate, with a high efficiency and drug loading which varies with the polymer specification. The above characteristics strongly suggest that these polymers could be developed into useful nanoparticulate drug delivery systems.     

Targeting tumour necrosis factor alleviates signs and symptoms of inflammatory osteoarthritis of the knee

Introduction

Inflammation associated with synovial expression of TNFα is a recognised feature of osteoarthritis (OA), although no studies have yet reported beneficial effects of anti-TNFα therapy on clinical manifestations of inflammation in OA.

Methods

We conducted an open-label evaluation of adalimumab over 12 weeks in 20 patients with OA of the knee and evidence of effusion clinically. Inclusion criteria included daily knee pain for the month preceding study enrolment and a summed pain score of 125 to 400 mm visual analogue scale on the Western Ontario and McMaster University Osteoarthritis Index (WOMAC) pain subscale. The primary outcome was the Osteoarthritis Research Society International/Outcome Measures in Rheumatology Clinical Trials (OARSI/OMERACT) response criterion at week 12. Secondary outcomes included the WOMAC pain score 20% and 50% improvement, WOMAC stiffness and function scores, patient and physician global visual analogue scale, as well as target joint swelling.

Results

Treatment was well tolerated and completed by 17 patients with withdrawals unrelated to lack of efficacy or adverse events. By intention to treat, an OARSI/OMERACT response was recorded in 14 (70%) patients. WOMAC pain 20% and 50% responses were recorded in 14 (70%) patients and eight (40%) patients, respectively. Significant improvement was observed in mean WOMAC pain, stiffness, function, physician and patient global, as well as target joint swelling at 12 weeks (P < 0.0001 for all). After treatment discontinuation, 16 patients were available for assessment at 22 weeks and OARSI/OMERACT response compared with baseline was still evident in 10 (50%) patients.

Conclusion

Targeting TNFα may be of therapeutic benefit in OA and requires further evaluation in controlled trials.

Trial registration

ClinicalTrials.gov: {"type":"clinical-trial","attrs":{"text":"NCT00686439","term_id":"NCT00686439"}}NCT00686439.     

Can erosions on MRI of the sacroiliac joints be reliably detected in patients with ankylosing spondylitis? - A cross-sectional study

Introduction

Erosions of the sacroiliac joints (SIJ) on pelvic radiographs of patients with ankylosing spondylitis (AS) are an important feature of the modified New York classification criteria. However, radiographic SIJ erosions are often difficult to identify. Recent studies have shown that erosions can be detected also on magnetic resonance imaging (MRI) of the SIJ early in the disease course before they can be seen on radiography. The goals of this study were to assess the reproducibility of erosion and related features, namely, extended erosion (EE) and backfill (BF) of excavated erosion, in the SIJ using a standardized MRI methodology.

Methods

Four readers independently assessed T1-weighted and short tau inversion recovery sequence (STIR) images of the SIJ from 30 AS patients and 30 controls (15 patients with non-specific back pain and 15 healthy volunteers) ≤45 years old. Erosions, EE, and BF were recorded according to standardized definitions. Reproducibility was assessed by percentage concordance among six possible reader pairs, kappa statistics (erosion as binary variable) and intraclass correlation coefficient (ICC) (erosion as sum score) for all readers jointly.

Results

SIJ erosions were detected in all AS patients and six controls by ≥2 readers. The median number of SIJ quadrants affected by erosion recorded by four readers in 30 AS patients was 8.6 in the iliac and 2.1 in the sacral joint portion (P < 0.0001). For all 60 subjects and for all four readers, the kappa value for erosion was 0.72, 0.73 for EE, and 0.63 for BF. ICC for erosion was 0.79, 0.72 for EE, and 0.55 for BF, respectively. For comparison, the kappa and ICC values for bone marrow edema were 0.61 and 0.93, respectively.

Conclusions

Erosions can be detected on MRI to a comparable degree of reliability as bone marrow edema despite the significant heterogeneity of their appearance on MRI.