Use of anti-neoepitope antibodies for the analysis of degradative events in cartilage and the molecular basis for neoepitope specificity

Degradation of the cartilage proteoglycan, aggrecan, is an essential aspect of normal growth and development, and of joint pathology. The roles of different proteolytic enzymes in this process can be determined from the sites of cleavage in the aggrecan core protein, which generates novel termini (neoepitopes). Antibodies specific for the different neoepitopes generated by such cleavage events provide powerful tools with which to analyse these processes. The same approach can be used to differentiate the processed, active forms of proteases from their inactive pro-forms. Since the proteolytic processing of these enzymes requires the removal of the inhibitory pro-region, it also results in the generation of N-terminal neoepitopes. Using the newborn rat long bone as a model system, it was shown that the active form of ADAMTS-4 [ADAM (a disintegrin and metalloproteinase) with thrombospondin motifs-4], but not ADAMTS-5, co-localizes with the aggrecan cleavage neoepitopes known to be produced by this metalloproteinase. Thus, in long bone growth, aggrecan turnover seems to be dependent on ADAMTS-4 activity. To demonstrate the molecular basis of the specificity of anti-neoepitope antibodies, the Fv region of a monoclonal antibody specific for a neoepitope generated by the ADAMTS-4-mediated cleavage of aggrecan has been modelled and the binding of the peptide epitope simulated. In the docked structure, the N-terminus of the peptide antigen is clearly buried in the binding-site cavity. The absence of an open cleft makes it impossible for the intact substrate to pass through the binding site, providing a rationale for the specificity of this class of antibodies.     

Autocatalytic cleavage of ADAMTS-4 (Aggrecanase-1) reveals multiple glycosaminoglycan-binding sites

ADAMTS-4, also referred to as aggrecanase-1, is a glutamyl endopeptidase capable of generating catabolic fragments of aggrecan analogous to those released from articular cartilage during degenerative joint diseases such as osteoarthritis. Efficient aggrecanase activity requires the presence of sulfated glycosaminoglycans (GAGs) attached to the aggrecan core protein, implying the contribution of substrate recognition/binding site(s) to ADAMTS-4 activity. In the present study, we demonstrate that full-length ADAMTS-4 (M(r) approximately 68,000) undergoes autocatalytic C-terminal truncation to generate two discrete isoforms (M(r) approximately 53,000 and M(r) approximately 40,000), which exhibit a marked reduction in affinity of binding to sulfated GAGs. C-terminal sequencing and mass analyses revealed that the GAG-binding thrombospondin type I motif was retained following autocatalysis, indicating that sites present in the C-terminal cysteine (cys)-rich and/or spacer domains also effect binding of full-length ADAMTS-4 to sulfated GAGs. Binding-competition experiments conducted using native and deglycosylated aggrecan provided direct evidence for interaction of the ADAMTS-4 cysteine-rich/spacer domains with aggrecan GAGs. Furthermore, synthetic peptides mimicking putative (consensus) GAG-binding sequences located within the ADAMTS-4 cysteine-rich and spacer domains competitively blocked binding of sulfated GAGs to full-length ADAMTS-4, thereby identifying multiple GAG-binding sites, which may contribute to the regulation of ADAMTS-4 function.     

Leaf temperature of desert sand dune plants: perspectives on the adaptability of leaf morphology

The leaf temperature of six annual and six perennial plant species was monitored during spring and summer on a sand dune ecosystem in the delta Mediterranean coast of Egypt. During winter, leaves of all tested perennial species attained temperatures higher than the air temperature at night and shortly after sunrise, with maximum leaf–air temperature differences reaching up to 8°C. The lowest differences were less than 1°C. Around noon, the leaves of several species attained temperatures lower than that of the air whereas others showed higher temperatures. The opposite was true during summer, when leaf temperatures were lower than air temperature. The maximum leaf–air temperature differences occurred after midnight towards sunrise and reached up to 10°C. The lowest differences were found around noon and were of less than 5°C. The annual plant species have more pronounced variations than perennials in their leaf temperatures during the night and for most of the day. The leaves were heated or cooled a few degrees above or below the air temperature. The results are discussed in relation to the morphological characters of the leaves. The variation in leaf temperature at different times of the day was significantly related to leaf morphology, specific leaf area, thickness, volume, leaf area index and the surrounding environment.     

Histochemical Study of Photoperiodic and Low Temperature Effects on Floral Induction of Spinacia oleracea

Shoot apices of Spinacia oleracea plants have been induced toflower either by: (a) subjecting leaves to 24 h long day, or(b) exposure to a short photoperiod but displaced by 8 h (displacedshort day) in the usual 24 h short-day cycle, or (c) exposureto low temperature (5 °C) during the dark period of thenormal short day. A quantitative cytochemical assay of pentosephosphate pathway activity during floral induction indicatesan approximate doubling of the rate of activity when comparedto that of vegetative apices (short day) (21 °C). Exposure to either low temperature, or a displaced short photoperiodstimulates pentose phosphate pathway activity in the shoot apexin a manner similar to that seen by long-day induction. Thischange in metabolic activity is accompanied by changes in theshape of the shoot apex which resembles that seen at an earlystage during floral induction. Spinacia oleracea, pentose phosphate pathway, shoot apex, glucose-6-phosphate dehydrogenase, floral induction, chilling, displaced short day     

Developmental acclimation of the thylakoid proteome to light intensity in Arabidopsis

Photosynthetic acclimation, the ability to adjust the composition of the thylakoid membrane to optimise the efficiency of electron transfer to the prevailing light conditions, is crucial to plant fitness in the field. While much is known about photosynthetic acclimation in Arabidopsis, to date there has been no study that combines both quantitative label-free proteomics and photosynthetic analysis by gas exchange, chlorophyll fluorescence and P700 absorption spectroscopy. Using these methods we investigated how the levels of 402 thylakoid proteins, including many regulatory proteins not previously quantified, varied upon long-term (weeks) acclimation of Arabidopsis to low (LL), moderate (ML) and high (HL) growth light intensity and correlated these with key photosynthetic parameters. We show that changes in the relative abundance of cytb₆f, ATP synthase, FNR2, TIC62 and PGR6 positively correlate with changes in estimated PSII electron transfer rate and CO₂ assimilation. Improved photosynthetic capacity in HL grown plants is paralleled by increased cyclic electron transport, which positively correlated with NDH, PGRL1, FNR1, FNR2 and TIC62, although not PGR5 abundance. The photoprotective acclimation strategy was also contrasting, with LL plants favouring slowly reversible non-photochemical quenching (qI), which positively correlated with LCNP, while HL plants favoured rapidly reversible quenching (qE), which positively correlated with PSBS. The long-term adjustment of thylakoid membrane grana diameter positively correlated with LHCII levels, while grana stacking negatively correlated with CURT1 and RIQ protein abundance. The data provide insights into how Arabidopsis tunes photosynthetic electron transfer and its regulation during developmental acclimation to light intensity.     

Phenolics Composition and Biological Activities Assessment of Leaves,Flowers and Roots Extracts from Erodium glaucophyllum,Erodium hirtum and Erodium guttatum

Biology Bulletin - Erodium glaucophyllum, Erodium hirtum and Erodium guttatum were medicinal herbs from a southern Mediterranean known for its health benefits. There is a strong demand for the find...     

Decline in diarrhea mortality and admissions after routine childhood rotavirus immunization in Brazil: a time-series analysis

Background

In 2006, Brazil began routine immunization of infants <15 wk of age with a single-strain rotavirus vaccine. We evaluated whether the rotavirus vaccination program was associated with declines in childhood diarrhea deaths and hospital admissions by monitoring disease trends before and after vaccine introduction in all five regions of Brazil with varying disease burden and distinct socioeconomic and health indicators.

Methods and Findings

National data were analyzed with an interrupted time-series analysis that used diarrhea-related mortality or hospitalization rates as the main outcomes. Monthly mortality and admission rates estimated for the years after rotavirus vaccination (2007–2009) were compared with expected rates calculated from pre-vaccine years (2002–2005), adjusting for secular and seasonal trends. During the three years following rotavirus vaccination in Brazil, rates for diarrhea-related mortality and admissions among children <5 y of age were 22% (95% confidence interval 6%–44%) and 17% (95% confidence interval 5%–27%) lower than expected, respectively. A cumulative total of ∼1,500 fewer diarrhea deaths and 130,000 fewer admissions were observed among children <5 y during the three years after rotavirus vaccination. The largest reductions in deaths (22%–28%) and admissions (21%–25%) were among children younger than 2 y, who had the highest rates of vaccination. In contrast, lower reductions in deaths (4%) and admissions (7%) were noted among children two years of age and older, who were not age-eligible for vaccination during the study period.

Conclusions

After the introduction of rotavirus vaccination for infants, significant declines for three full years were observed in under-5-y diarrhea-related mortality and hospital admissions for diarrhea in Brazil. The largest reductions in diarrhea-related mortality and hospital admissions for diarrhea were among children younger than 2 y, who were eligible for vaccination as infants, which suggests that the reduced diarrhea burden in this age group was associated with introduction of the rotavirus vaccine. These real-world data are consistent with evidence obtained from clinical trials and strengthen the evidence base for the introduction of rotavirus vaccination as an effective measure for controlling severe and fatal childhood diarrhea. Please see later in the article for the Editors'' Summary