Reduced transforming growth factor-beta signaling in cartilage of old mice: role in impaired repair capacity

Osteoarthritis (OA) is a common joint disease, mainly effecting the elderly population. The cause of OA seems to be an imbalance in catabolic and anabolic factors that develops with age. IL-1 is a catabolic factor known to induce cartilage damage, and transforming growth factor (TGF)-beta is an anabolic factor that can counteract many IL-1-induced effects. In old mice, we observed reduced responsiveness to TGF-beta-induced IL-1 counteraction. We investigated whether expression of TGF-beta and its signaling molecules altered with age. To mimic the TGF-beta deprived conditions in aged mice, we assessed the functional consequence of TGF-beta blocking. We isolated knee joints of mice aged 5 months or 2 years, half of which were exposed to IL-1 by intra-articular injection 24 h prior to knee joint isolation. Immunohistochemistry was performed, staining for TGF-beta1, -2 or -3, TGF-betaRI or -RII, Smad2, -3, -4, -6 and -7 and Smad-2P. The percentage of cells staining positive was determined in tibial cartilage. To mimic the lack of TGF-beta signaling in old mice, young mice were injected with IL-1 and after 2 days Ad-LAP (TGF-beta inhibitor) or a control virus were injected. Proteoglycan (PG) synthesis (³⁵S-sulfate incorporation) and PG content of the cartilage were determined. Our experiments revealed that TGF-beta2 and -3 expression decreased with age, as did the TGF-beta receptors. Although the number of cells positive for the Smad proteins was not altered, the number of cells expressing Smad2P strongly dropped in old mice. IL-1 did not alter the expression patterns. We mimicked the lack of TGF-beta signaling in old mice by TGF-beta inhibition with LAP. This resulted in a reduced level of PG synthesis and aggravation of PG depletion. The limited response of old mice to TGF-beta induced-IL-1 counteraction is not due to a diminished level of intracellular signaling molecules or an upregulation of intracellular inhibitors, but is likely due to an intrinsic absence of sufficient TGF-beta receptor expression. Blocking TGF-beta distorted the natural repair response after IL-1 injection. In conclusion, TGF-beta appears to play an important role in repair of cartilage and a lack of TGF-beta responsiveness in old mice might be at the root of OA development.     

Water Flow Through Vessel Perforation Plates--A Fluid Mechanical Approach

The effect of scalariform perforation plates on the flow ofwater through plant vessels remains poorly understood. In thisstudy, a new computational tool based on finite element methodsolutions to the Navier-Stokes equation was applied to modellingfluid flow through these structures in plant vessels. Modelsdeveloped for Liriodendron tulipifera vessel elements were solvedfor cells with and without the perforation plate to study effectsof the plate on the pressure drop along the cell. Results indicatethat the pressure gradient was 5-fold greater through the platethan for regions before and after the plate. However, the perforationplate in this species accounts for only about 8% of the resistanceto flow through typical vessels because the plate influencesflow for only a short distance along the cell relative to itslength. Details of the flow characteristics through pores ofthe perforation plate are also described. Key words: Conductance, finite element method, perforation plate, vessel, water flow     

Accumulation of Gliadin and Glutenin Polypeptides during Development of Normal and Sulphur-deficient Wheat Seed: Analysis using Specific Monoclonal Antibodies

A panel of monoclonal antibodies with various specificitiesfor wheat (Triticum aestivum L.) gluten polypeptides has beenused to analyse the accumulation of these polypeptides in theendosperm of developing wheat seeds grown under normal and sulphur-deficientconditions. Immunoblots of polypeptides fractionated by SDS-PAGEallowed a qualitative analysis of gliadin and HMW glutenin accumulationfor high- and low-sulphur seeds 8 d to 30 d after anthesis (d.a.a.).In addition, quantitative analysis of the deposition of variousgluten polypeptides was performed, with a solid-phase radioimmunoassayon extracts of seeds harvested 4–36 d.a.a. The initialaccumulation of HMW glutenin subunits was detectable at an earlierstage of development than that of gliadins for both normal andsulphur-deficient seeds. The initiation of detectable gliadinaccumulation was asynchronous with an order of alpha-gliadins,beta-, gamma- and some omega-gliadins and finally the remainingomega-gliadins. In sulphur deficiency, all gliadins reacheda constant proportion of the dry weight of the endosperm earlierthan in normal wheat, while a more marked increase in the proportionof HMW glutenin occurred late in grain development. The proteinblot studies also identified a putative omega-gliadin polypeptidewhich was detectable late in seed development and only in sulphur-deficientseeds. Key words: Wheat, seed maturation, immunoassay     

Growth distribution in the light-growth response of Phycomyces

Elongation of sporangiophores marked with numerous starch grains was photographically recorded in the steady state and during the light-growth response when the rate is more than doubled. From these records the spatial distribution of growth within the cell's growth zone was derived. Stimulation by a single saturating flash of light speeds growth proportionally in all parts of the growing zone, maintaining the same pattern of growth distribution as in the steady state. This finding implies that light is absorbed and acts locally throughout the length of the cell's growth zone. Cohen and Delbrück's proposal of a partial spatial separation of light reception and growth is discussed.     

Use of chromomycin A3 staining in bovine sperm cells for detection of protamine deficiency

Sperm chromatin integrity is essential for accurate transmission of male genetic information, and normal sperm chromatin structure is important for fertilization. Protamine is a nuclear protein that plays a key role in sperm DNA integrity, because it is responsible for sperm DNA stability and packing until the paternal genome is delivered into the oocyte during fertilization. Our aim was to investigate protamine deficiency in sperm cells of Bos indicus bulls (Nelore) using chromomycin A₃ (CMA₃) staining. Frozen semen from 14 bulls were thawed, then fixed in Carnoy's solution. Smears were prepared and analyzed by microscopy. As a positive control of CMA₃ staining, sperm from one bull was subjected to deprotamination of nuclei. The percentage of CMA₃-positive bovine sperm did not vary among batches. Only two bulls showed a higher percentage of CMA₃-positive sperm cells compared to the others. CMA₃ is a simple and useful tool for detecting sperm protamine deficiency in bulls.