The third helix of the homeodomain of paired class homeodomain proteins acts as a recognition helix both for DNA and protein interactions

The transcription factor Pax6 is essential for the development of the eyes and the central nervous system of vertebrates and invertebrates. Pax6 contains two DNA-binding domains; an N-terminal paired domain and a centrally located homeodomain. We have previously shown that the vertebrate paired-less isoform of Pax6 (Pax6ΔPD), and several other homeodomain proteins, interact with the full-length isoform of Pax6 enhancing Pax6-mediated transactivation from paired domain-DNA binding sites. By mutation analyses and molecular modeling we now demonstrate that, surprisingly, the recognition helix for specific DNA binding of the homeodomains of Pax6 and Chx10 interacts with the C-terminal RED subdomain of the paired domain of Pax6. Basic residues in the recognition helix and the N-terminal arm of the homeodomain form an interaction surface that binds to an acidic patch involving residues in helices 1 and 2 of the RED subdomain. We used fluorescence resonance energy transfer assays to demonstrate such interactions between Pax6 molecules in the nuclei of living cells. Interestingly, two mutations in the homeodomain recognition helix, R57A and R58A, reduced protein–protein interactions, but not DNA binding of Pax6ΔPD. These findings suggest a critical role for the recognition helix and N-terminal arm of the paired class homeodomain in protein–protein interactions

Transforming growth factor-β-inducible early response gene 1 is a novel substrate for atypical protein kinase Cs

The protein kinase C (PKC) family of serine/threonine kinases consists of ten different isoforms grouped into three subfamilies, denoted classical, novel and atypical PKCs (aPKCs). The aPKCs, PKCι/λ and PKCζ serve important roles during development and in processes subverted in cancer such as cell and tissue polarity, cell proliferation, differentiation and apoptosis. In an effort to identify novel interaction partners for aPKCs, we performed a yeast two-hybrid screen with the regulatory domain of PKCι/λ as bait and identified the Krüppel-like factors family protein TIEG1 as a putative interaction partner for PKCι/λ. We confirmed the interaction of both aPKCs with TIEG1 in vitro and in cells, and found that both aPKCs phosphorylate the DNA-binding domain of TIEG1 on two critical residues. Interestingly, the aPKC-mediated phosphorylation of TIEG1 affected its DNA-binding activity, subnuclear localization and transactivation potential

Nutritional value of feeds with different physical qualities

In a previously performed study, rainbow trout with mean initial weight 1144 g was fed two experimental feeds with similar chemical composition, but differing in physical properties (high and low water stability, denoted Feed A and B, respectively). The fish was also exposed to stable or fluctuating environment (salinity, temperature and O2 saturation). The feed intake was highest in trout fed Feed B, and when kept at stable environment. The growth tended to follow the same pattern (not significantly though). At termination of the trial, the stomachs of trout fed Feed B contained the largest amounts of feed material. The stomachs of these fish also contained large amounts of free water and oil, most severely in trout kept at stable environment, coinciding with the highest feed intake. In the present experiment, the physical properties of the two feeds were further investigated. Feed A had less dust, more unbroken pellets, higher hardness, larger diameter and shorter pellets than Feed B. The apparent digestibility (ADC, %) of protein (the sum of amino acids) and most individual amino acids, starch, dry matter, energy and phosphorus was highest in Feed A. The environmental treatment also affected the ADC of some amino acids and minerals. In general however, the ADC appeared to be reduced at high feed intake. The plasma concentrations of sodium and chloride ions were not affected by the feed, but were elevated in individuals kept at the constant environment. The results clearly demonstrate that the nutritional value of a feed is affected by its physical properties. Furthermore, several of the treatment effects in the fish caused by physical feed properties, may be related to the feed intake.Nutritional value of feeds with different physical qualitiespublishedVersio

Autologous chondrocyte implantation to repair knee cartilage injury : ultrastructural evaluation at 2 years and long term follow up including muscle strength measurements

Autologous chondrocyte implantation (ACI) usually results in improvement in clinical scores. However, long-term isokinetic muscle strength measurements have not been reported. Biopsies from the repair tissue have shown variable proportions of hyaline-like cartilage. In this study, 21 consecutive patients were treated with autologous cartilage implantations in the knee. Mean size of the lesions was 5.5 cm2. Follow-up arthroscopy with biopsy was performed at 2 years in 19 patients. The biopsies were examined with both light microscopy and transmission electron microscopy (TEM) techniques including immunogold analysis of collagen type 1. Patient function was evaluated with modified 10-point scales of the Cincinnati knee rating system obtained preoperatively and at 1 and 8.1 years. Isokinetic quadriceps and hamstrings muscle strength testing was performed at 1, 2 and 7.4 years. Light microscopy and TEM both showed predominately fibrous cartilage. The immunogold analysis showed a high percentage of collagen type I. At 7.4 years, the total work deficits when compared with the contra-lateral leg for isokinetic extension were 19.1 and 11.4%, and for isokinetic flexion 11.8 and 8.5% for 60 and 2408/s, respectively. Mean pain score improved from 4.3 preoperatively to 6.3 at 1 year (p = 0.031) and 6.6 at 8.1 years (p = 0.013). Overall health condition score improved from 4.1 preoperatively to 6.1 at 1 year (p = 0.004) and 6.5 at 8.1 years (p = 0.008). Three patients later went through revision surgery with other resurfacing techniques and are considered failures. In summary, the formation of fibrous cartilage following ACI was confirmed by TEM with immunogold histochemistry. Although the functional scores were generally good, strength measurements demonstrated that the surgically treated leg remained significantly weaker