Signaling Cascades Governing Cdc42-Mediated Chondrogenic Differentiation and Mensenchymal Condensation

Endochondral ossification consists of successive steps of chondrocyte differentiation, including mesenchymal condensation, differentiation of chondrocytes, and hypertrophy followed by mineralization and ossification. Loss-of-function studies have revealed that abnormal growth plate cartilage of the Cdc42 mutant contributes to the defects in endochondral bone formation. Here, we have investigated the roles of Cdc42 in osteogenesis and signaling cascades governing Cdc42-mediated chondrogenic differentiation. Though deletion of Cdc42 in limb mesenchymal progenitors led to severe defects in endochondral ossification, either ablation of Cdc42 in limb preosteoblasts or knockdown of Cdc42 in vitro had no obvious effects on bone formation and osteoblast differentiation. However, in Cdc42 mutant limb buds, loss of Cdc42 in mesenchymal progenitors led to marked inactivation of p38 and Smad1/5, and in micromass cultures, Cdc42 lay on the upstream of p38 to activate Smad1/5 in bone morphogenetic protein-2-induced mesenchymal condensation. Finally, Cdc42 also lay on the upstream of protein kinase B to transactivate Sox9 and subsequently induced the expression of chondrocyte differential marker in transforming growth factor-β1-induced chondrogenesis. Taken together, by using biochemical and genetic approaches, we have demonstrated that Cdc42 is involved not in osteogenesis but in chondrogenesis in which the BMP2/Cdc42/Pak/p38/Smad signaling module promotes mesenchymal condensation and the TGF-β/Cdc42/Pak/Akt/Sox9 signaling module facilitates chondrogenic differentiation.     

Histone H1 variants as sperm-specific nuclear proteins of Rana catesbeiana,and their role in maintaining a unique condensed state of sperm chromatin

Amino acid analyses of nuclear basic proteins of an anuran amphibian, Rana catesbeiana, revealed that they are comprised of a full set of core histones and three types of lysine-rich, sperm-specific proteins. On the basis of their amino-acid compositions and partial amino-acid sequences of their trypsin-resistant cores, the sperm-specific proteins could be defined as members of the histone H1 family. Both micrococcal nuclease digestion and electron microscopy indicated that sperm chromatin consists of nucleosomal and fibrillar DNA structures which are irregularly interspersed with each other. When sperm nuclei were incubated with nucleoplasmin, nuclei decondensed to some extent, and the sperm-specific H1s were removed, but not completely. The residual sperm-specific histone H1 variants were also found in reconstituted male pronuclear chromatin, comprising regularly spaced nucleosomes. We conclude that sperm-specific histone H1 variants are essential for chromatin condensation in the sperm nuclei, but that their complete removal is not necessary for the remodeling into somatic chromatin that takes place after fertilization. Mol. Reprod. Dev. 47:181–190, 1997. © 1997 Wiley-Liss, Inc.     

Topologically associating domains and chromatin loops depend on cohesin and are regulated by CTCF,WAPL, and PDS5 proteins

Mammalian genomes are spatially organized into compartments, topologically associating domains (TADs), and loops to facilitate gene regulation and other chromosomal functions. How compartments, TADs, and loops are generated is unknown. It has been proposed that cohesin forms TADs and loops by extruding chromatin loops until it encounters CTCF, but direct evidence for this hypothesis is missing. Here, we show that cohesin suppresses compartments but is required for TADs and loops, that CTCF defines their boundaries, and that the cohesin unloading factor WAPL and its PDS5 binding partners control the length of loops. In the absence of WAPL and PDS5 proteins, cohesin forms extended loops, presumably by passing CTCF sites, accumulates in axial chromosomal positions (vermicelli), and condenses chromosomes. Unexpectedly, PDS5 proteins are also required for boundary function. These results show that cohesin has an essential genome‐wide function in mediating long‐range chromatin interactions and support the hypothesis that cohesin creates these by loop extrusion, until it is delayed by CTCF in a manner dependent on PDS5 proteins, or until it is released from DNA by WAPL.     

Reaction equilibrium for lipase-catalyzed condensation in organic solvent systems

Lipase-catalyzed condensation in an organic solvent is useful for the syntheses of esters. To reasonably design and optimize the reaction conditions, knowledge of the reaction equilibrium is required. The interaction of water with other reactants and the quantitative predictions for adsorption of water by a desiccant are discussed. The solvent effects on the reaction equilibrium are also elucidated in mixtures of nitrile and tert-alcohol.     

Feulgen-DNA response and chromatin condensation in Malpighian tubules of Melipona rufiventris and Melipona quadrifasciata (Hymenoptera, Apoidea)

Melipona quadrifasciata and Melipona rufiventris are stingless bee species which present low and high heterochromatin content, respectively, on their mitotic chromosomes as assessed visually after a C-banding assay. However, these species do not show differences in the C-banding responses of their Malpighian tubule interphase nuclei. In the present study, the Feulgen-DNA response, which could inform on differences in DNA depurination due to differences in chromatin condensation, was compared in the cell nuclei of the Malpighian tubules of these species. It was hypothesized that differences in acid hydrolysis kinetics patterns, as assessed by Feulgen reaction and studied microspectrophotometrically, could discriminate M. quadrifasciata and M. rufiventris interphase nuclei not distinguishable with the C-banding method. Feulgen-DNA values corresponding to more than one ploidy class were found in both species; these values at the hydrolysis time corresponding to the maximal DNA depurination for each ploidy degree were higher in M. quadrifasciata, reflecting a higher DNA content in the Malpighian tubule cell nuclei of this species compared to those of M. rufiventris at the same larval instar. The maximal Feulgen-DNA values of M. quadrifasciata after short (50 min) and long (90 min) hydrolysis times were found to be closer to each other, while those of M. rufiventris occurred sharply at the long hydrolysis time, indicating that DNA depurination in M. quadrifasciata occurred faster. This result is probably related to the involvement of differences in chromatin condensation; it agrees with the idea that M. rufiventris contains more heterochromatin than M. quadrifasciata, which is supported by the analysis of results obtained with the image analysis parameter average absorption ratio. The depurination kinetics studied here with the Feulgen reaction were revealed to be more pertinent than the C-banding technique in establishing differences in levels of chromatin condensation for these cell nuclei.     

Multiple roles for apoptosis facilitating condensation of the Drosophila ventral nerve cord

At the end of embryogenesis, the ventral nerve cord (VNC) of Drosophila undergoes a shape change, termed condensation. During condensation the length of the VNC shortens by 25%, a process dependent on extracellular matrix deposited by hemocytes, an intact cytoskeleton of glia and neurons and neural activity. Here we show that cell death contributes to nerve cord shortening. Firstly, apoptosis occurs at the interface of the epidermis and the nerve cord where it plays a role in the separation of these two tissues. Separation precedes condensation and in conditions where separation is prevented, condensation fails. Secondly, many cells undergo apoptosis within VNC during condensation. This cell death is localized mainly to the posterior part of the nerve cord where more than half of all cell death occurs. Preventing apoptosis either in neurons or glia partially inhibits VNC shortening during condensation. Despite the importance of midline glia in axon tract development, preventing midline glia cell death results in normal hatching and adult formation. We find that undead midline glia are eliminated from the midline and become mispositioned or expelled from the nervous system. We suggest that this represent a form of pattern repair that operates to reduce the impact of the additional cells.     

About mechanism of chitosan cross-linking with glutaraldehyde

The regularities of the reaction of aminopolysaccharide chitosan with glutaraldehyde (GA) have been considered. The equilibrium forms of GA in water have been thoroughly studied by NMR spectroscopy. It has been established that at pH 5.6, the exchange of the protons of O=CHCH₂ groups for deuterium occurs, indicating the presence of an anion, a product of the first stage of the aldol reaction; at pH > 7.2, the formation of the products of an aldol reaction and aldol condensation takes place. The kinetics of the reaction between the amino groups of chitosan and GA, the kinetics of gel formation in chitosan solutions in the presence of GA, and the kinetics of changes in the rigidity of gels formed have been studied by UV spectroscopy. IR spectra of cross-linked chitosan have been obtained. It has been shown that chitosan catalyzes the polymerization of GA to form irregular products; in this process, the length of oligomeric chains in modified or cross-linked chitosan and the concentration of conjugated bonds increase with the GA concentration and pH of the reaction medium.     

Pathogenesis of ER Storage Disorders: Modulating Russell Body Biogenesis by Altering Proximal and Distal Quality Control

In many protein storage diseases, detergent‐insoluble proteins accumulate in the early secretory compartment (ESC). Protein condensation reflects imbalances between entry into (synthesis/translocation) and exit from (secretion/degradation) ESC, and can be also a consequence of altered quality control (QC) mechanisms. Here we exploit the inducible formation of Russell bodies (RB), dilated ESC cisternae containing mutant Ig‐µ chains, as a model to mechanistically dissect protein condensation. Depending on the presence or absence of Ig‐L chains, mutant Ig‐µ chains lacking their first constant domain (Ch 1) accumulate in rough or smooth RB (rRB and sRB), dilations of the endoplasmic reticulum (ER) and ER‐Golgi intermediate compartment (ERGIC), respectively, reflecting the proximal and distal QC stations in the stepwise biogenesis of polymeric IgM. Either weakening ERp44‐dependent distal QC or facilitating ER‐associated degradation (ERAD) inhibits RB formation. Overexpression of PDI or ERp44 inhibits µΔCh 1 secretion. However, PDI inhibits while ERp44 promotes µΔCh 1 condensation. Both Ero1α silencing and overexpression prevent RB formation, demonstrating a strict redox dependency of the phenomenon. Altogether, our findings identify key controllers of protein condensation along the ESC as potential targets to handle certain storage disorders.     

An improved technique for obtaining well-spread metaphases from plants with numerous large chromosomes

Preparations that contain well-spread metaphase chromosomes are critical for plant cytogenetic analyses including chromosome counts, banding procedures, in situ hybridization, karyotyping and construction of ideograms. Chromosome spreading is difficult for plants with large and numerous chromosomes. We report here a technique for obtaining cytoplasm-free, well-spread metaphases from two Amaryllidaceae species: Sprekelia formosissima (2n = 120) and Hymenocallis howardii (2n = 96). The technique has three main steps: 1) pretreatment to cause chromosome condensation, 2) dripping onto tilted slides coated with a thin layer of pure acetic acid and 3) application of steam and acetic acid to produce cytoplasmic hydrolysis, which spreads the chromosomes.