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.     

Antisense inhibition of the sucrose transporter in potato: effects on amount and activity

The sucrose proton-cotransporter gene from potato (StSUT1) is mainly expressed in the phloem of mature, exporting leaves. To study the in vivo role of the protein, potato plants were transformed with antisense constructs of the sucrose transporter cDNA under control of the CaMV35S and the rolC promoters, respectively. Both types of transgenic plant develop symptoms characteristic of an inhibition of phloem loading. To determine the level of inhibition, immunological and transport studies were performed. Purified antibodies directed against a peptide from the central loop of SUT1 recognized a transporter with an apparent molecular mass of 47 kDa in leaf plasma membrane vesicles. Antisense repression under control of the non-specific CaMV35S promoter led to a strong reduction in SUT1 protein, whereas no such reduction could be detected when the companion cell-specific rolC promoter was used. Similarily. sucrose uptake in plasma membrane vesicles was reduced by 50–75% in CaMV35S but not in rolC plants. These data suggest that, unlike the rolC promoter, the sucrose transporter is expressed not only in the companion cells but also in other leaf cells. However, inhibition of the transporter by rolC-controlled antisense repression is sufficient to impair phloem loading.     

Manipulation of sink-source relations in transgenic plants

Since 1980, the use of transgenic plants in modern plant science has become a powerful tool to study whole plant physiology. In this review, we try to summarize the data obtained in the field of photoassimilate partitioning. Attempts to study sink-source interactions concern factors which might limit sink strength and source capacity. Transgenic plants have been used to manipulate the sucrose to starch ratio in order to produce plants with higher sucrose levels in their source leaves. Alterations in partitioning were achieved by manipulating Calvin cycle enzymes, transport proteins and sucrose biosynthetic enzymes. The ability of sink tissues to attract photoassimilates has been altered by either increasing or decreasing sucrose hydrolytic activities. The increase of sucrose hydrolysis was achieved by creating transgenic potato plants with tuber specific yeast-derived invertase. Decreased sucrose utilization was achieved by antisense inhibition of sucrose synthase in potato tubers.     

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.     

Polymorphic microsatellite markers for population analysis of a tephritid pest species, Bactrocera tryoni

To obtain a set of microsatellite markers for the Queensland fruit fly Bactrocera tryoni , a genomic library was screened with a number of simple repeat oligonucleotide probes. Sequencing recovered 22 repeat loci. The microsatellite sequences were short, with repeat numbers ranging from five to 11. Of these, 16 polymerase chain reaction (PCR) primer sets yielded amplifiable products, which were tested on 53 flies from five widely separated sites. All loci showed polymorphism in the population sample, with the number of alleles ranging from two to 16. Several dinucleotide repeats showed alleles separated by single-base differences and multiple steps, suggesting a mutation process more complex than the stepwise mutation model.     

The genome of Rhizobium leguminosarum has recognizable core and accessory components

Background

Rhizobium leguminosarum is an α-proteobacterial N₂-fixing symbiont of legumes that has been the subject of more than a thousand publications. Genes for the symbiotic interaction with plants are well studied, but the adaptations that allow survival and growth in the soil environment are poorly understood. We have sequenced the genome of R. leguminosarum biovar viciae strain 3841.

Results

The 7.75 Mb genome comprises a circular chromosome and six circular plasmids, with 61% G+C overall. All three rRNA operons and 52 tRNA genes are on the chromosome; essential protein-encoding genes are largely chromosomal, but most functional classes occur on plasmids as well. Of the 7,263 protein-encoding genes, 2,056 had orthologs in each of three related genomes (Agrobacterium tumefaciens, Sinorhizobium meliloti, and Mesorhizobium loti), and these genes were over-represented in the chromosome and had above average G+C. Most supported the rRNA-based phylogeny, confirming A. tumefaciens to be the closest among these relatives, but 347 genes were incompatible with this phylogeny; these were scattered throughout the genome but were over-represented on the plasmids. An unexpectedly large number of genes were shared by all three rhizobia but were missing from A. tumefaciens.

Conclusion

Overall, the genome can be considered to have two main components: a 'core', which is higher in G+C, is mostly chromosomal, is shared with related organisms, and has a consistent phylogeny; and an 'accessory' component, which is sporadic in distribution, lower in G+C, and located on the plasmids and chromosomal islands. The accessory genome has a different nucleotide composition from the core despite a long history of coexistence.