The gene encoding human low-molecular weight insulin-like growth-factor binding protein (IGF-BP25): regional localization to 7p12–p13 and description of a DNA polymorphism

The low-molecular weight insulin-like growth-factor binding protein (IGF-BP25) is synthesized by human liver, secretory endometrium and decidua, and is also present in human serum. It binds insulin-like growth factors IGF-I and IGF-II with high affinity, and is proposed to act as a paracrine regulator of cell growth. In situ hybridization studies with a cDNA encompassing the entire protein coding region of IGF-BP25 localized the gene to bands p12-p13 on chromosome 7. Southern blot analysis with the enzyme BglII revealed a common restriction fragment length polymorphism: the presence of the polymorphic BglII site results in the formation of two fragments 4.6 kb and 1.6 kb in size whereas its absence produces a single 6.2 kb fragment. The frequencies of the two alleles were 0.73 and 0.27, respectively. IGF-BP25 constitutes a useful genetic marker for the proximal short arm of chromosome 7.     

Chondroitin sulfate at the plasma membranes of cultured fibroblasts

We have previously shown that in confluent human fibroblast cultures chondroitin sulfate proteoglycan is a component of the fibronectin-containing pericellular matrix fibers. In the present work the distribution of chondroitin sulfate was studied in subconfluent cell cultures using antibodies that bind to a chemically defined carbohydrate fragment of chondroitinase ABC-modified chondroitin sulfate proteoglycan. Using immunofluorescence microscopy, we observed, in addition to the fibrillar matrix staining, chondroitin sulfate diffusely distributed at the cell surface. In indirect immunoferritin electron microscopy this staining corresponded to patchy binding of ferritin close (24 nm) to the outer aspect of the plasma membrane. The patchy organization appeared uniform in all cell surfaces. The cell surface chondroitin sulfate could not be removed from the plasma membrane by agents that dissociate electrostatic interactions. These data show that in fibroblasts chondroitin sulfate is a component of the outer aspect of the plasma membrane, and raise the possibility of an integral plasma membrane chondroitin sulfate proteoglycan.     

COMMD1 is linked to the WASH complex and regulates endosomal trafficking of the copper transporter ATP7A

COMMD1 deficiency results in defective copper homeostasis, but the mechanism for this has remained elusive. Here we report that COMMD1 is directly linked to early endosomes through its interaction with a protein complex containing CCDC22, CCDC93, and C16orf62. This COMMD/CCDC22/CCDC93 (CCC) complex interacts with the multisubunit WASH complex, an evolutionarily conserved system, which is required for endosomal deposition of F-actin and cargo trafficking in conjunction with the retromer. Interactions between the WASH complex subunit FAM21, and the carboxyl-terminal ends of CCDC22 and CCDC93 are responsible for CCC complex recruitment to endosomes. We show that depletion of CCC complex components leads to lack of copper-dependent movement of the copper transporter ATP7A from endosomes, resulting in intracellular copper accumulation and modest alterations in copper homeostasis in humans with CCDC22 mutations. This work provides a mechanistic explanation for the role of COMMD1 in copper homeostasis and uncovers additional genes involved in the regulation of copper transporter recycling.     

Expedited quantification of mutant ribosomal RNA by binary deoxyribozyme (BiDz) sensors

Mutations in ribosomal RNA (rRNA) have traditionally been detected by the primer extension assay, which is a tedious and multistage procedure. Here, we describe a simple and straightforward fluorescence assay based on binary deoxyribozyme (BiDz) sensors. The assay uses two short DNA oligonucleotides that hybridize specifically to adjacent fragments of rRNA, one of which contains a mutation site. This hybridization results in the formation of a deoxyribozyme catalytic core that produces the fluorescent signal and amplifies it due to multiple rounds of catalytic action. This assay enables us to expedite semi-quantification of mutant rRNA content in cell cultures starting from whole cells, which provides information useful for optimization of culture preparation prior to ribosome isolation. The method requires less than a microliter of a standard Escherichia coli cell culture and decreases analysis time from several days (for primer extension assay) to 1.5 h with hands-on time of ∼10 min. It is sensitive to single-nucleotide mutations. The new assay simplifies the preliminary analysis of RNA samples and cells in molecular biology and cloning experiments and is promising in other applications where fast detection/quantification of specific RNA is required.     

Comparison of single-phase isotropic elastic and fibril-reinforced poroelastic models for indentation of rabbit articular cartilage

Classically, single-phase isotropic elastic (IE) model has been used for in situ or in vivo indentation analysis of articular cartilage. The model significantly simplifies cartilage structure and properties. In this study, we apply a fibril-reinforced poroelastic (FRPE) model for indentation to extract more detailed information on cartilage properties. Specifically, we compare the information from short-term (instantaneous) and long-term (equilibrium) indentations, as described here by IE and FRPE models. Femoral and tibial cartilage from rabbit (age 0–18 months) knees (n=14) were tested using a plane-ended indenter (diameter=0.544 mm). Stepwise creep tests were conducted to equilibrium. Single-phase IE solution for indentation was used to derive instantaneous modulus and equilibrium (Young's) modulus for the samples. The classical and modified Hayes’ solutions were used to derive values for the indentation moduli. In the FRPE model, the indentation behavior was sample-specifically described with three material parameters, i.e. fibril network modulus, non-fibrillar matrix modulus and permeability. The instantaneous and fibril network modulus, and the equilibrium Young's modulus and non-fibrillar matrix modulus showed significant (p<0.01) linear correlations of R²=0.516 and 0.940, respectively (Hayes’ solution) and R²=0.531 and 0.960, respectively (the modified Hayes’ solution). No significant correlations were found between the non-fibrillar matrix modulus and instantaneous moduli or between the fibril network modulus and the equilibrium moduli. These results indicate that the instantaneous indentation modulus (IE model) provides information on tensile stiffness of collagen fibrils in cartilage while the equilibrium modulus (IE model) is a significant measure for stiffness of PG matrix. Thereby, this study highlights the feasibility of a simple indentation analysis.