Restriction endonuclease resistant chromatin in human chromosomes

Summary The recent addition of restriction endonucleases in obtaining selective bands in the human genome has added a new dimension to molecular genetics. However, a considerable discrepancy exists in banding patterns produced by AluI in chromosomes 19 and 20, by MboI in chromosomes 4, 5, 8, 21 and 22 and by RsaI in chromosomes 12, 21 and 22. The principal causes of these differences are highlighted.     

Calmodulin supports the force-generating function in desensitized muscle fibers

Externally added calmodulin (CaM) restored Ca2+ regulation for the tension development by skeletal muscle fibers of hamster and rabbit desensitized by the troponin C (TnC) extraction treatment. CaM produced this action by combining with the TnC-denuded sites in the fiber. However, the binding properties differed strikingly from TnC: unlike TnC, CaM binding required the continued presence of Ca2+ and the bound portion was completely released with EGTA in the physiological milieu. The maximal uptake was 1.7 g of CaM/kg of muscle in the present study. The apparent Ca2+ sensitivity for force development with 200 micrograms/ml CaM in the solution was lower than in the native fiber or in the TnC-loaded fiber. The apparent association constant for CaM binding to the TnC-denuded sites was found as 4.9 x 10(5) M-1, and the extrapolated maximum force (Fmax) with CaM was close to PO. The intrinsic CaM level in intact muscle was also measured and was 18.6 mg/kg, amounting to about 1% of the total TnC or the CaM uptake by TnC-denuded fibers. The intrinsic CaM was not dislodged by EDTA treatment, indicating tight binding and suggesting that it exists in a separate pool from the vacated TnC sites adsorbing externally added CaM. The stringent Ca+ dependence of the CaM adsorption to TnC sites in the regulatory complex in the fiber supports the view that the evolutionary replacement of residues in the amino terminus helix portion of the "EF-hand" motif of site IV may be critical for the functional specialization by TnC.     

Chondroid bone arises from mesenchymal stem cells in organ culture of mandibular condyles

Mandibular condyles of fetal mice 19 to 20 days in utero comprising clean cartilage and its perichondrium were cultured for up to 14 days, and their capacity to develop osteoid and to mineralize in vitro was examined. After 3 days in culture the cartilage of the mandibular condyle appeared to have lost its inherent structural characteristics, including its various cell layers: chondroprogenitor, chondroblastic, and hypertrophic cells. At that time interval no chondroblasts could be seen; instead, most of the cartilage consisted of hypertrophic chondrocytes. By that time, the surrounding perichondrium, which contains pluripotential mesenchymal stem cells, revealed the first signs of extracellular matrix enclosing type I collagen, bone alkaline phosphatase, osteonection, fibronectin, and bone sialoprotein as demonstrated by immunofluorescent techniques. Electron microscopic examinations of the newly formed matrix revealed foci of mineralization within and along collagen fibers as is normally observed during bone development. The composition of the latter mineral deposits resembled calcium pyrophosphate crystals. Following 14 days in culture larger portions of the condyle revealed signs of osseous matrix, yet the tissue reacted positively for type II collagen. Hence, the condylar cartilage, a genuine representative of secondary-type cartilage, elaborated in vitro a unique type of bone that would be most appropriately defined as chondroid bone. Biochemical assays indicated that the de novo formation of chondroid bone was correlated with changes in alkaline phosphatase activity and 45Ca incorporation. The findings of the present study imply that mesenchymal stem cells that ordinarily differentiate into cartilage possess the capacity to differentiate into osteogenic cells and form chondroid bone.     

ePAD,an oocyte and early embryo-abundant peptidylarginine deiminase-like protein that localizes to egg cytoplasmic sheets

Selected for its high relative abundance, a protein spot of MW approximately 75 kDa, pI 5.5 was cored from a Coomassie-stained two-dimensional gel of proteins from 2850 zona-free metaphase II mouse eggs and analyzed by tandem mass spectrometry (TMS), and novel microsequences were identified that indicated a previously uncharacterized egg protein. A 2.4-kb cDNA was then amplified from a mouse ovarian adapter-ligated cDNA library by RACE-PCR, and a unique 2043-bp open reading frame was defined encoding a 681-amino-acid protein. Comparison of the deduced amino acid sequence with the nonredundant database demonstrated that the protein was approximately 40% identical to the calcium-dependent peptidylarginine deiminase (PAD) enzyme family. Northern blotting, RT-PCR, and in situ hybridization analyses indicated that the protein was abundantly expressed in the ovary, weakly expressed in the testis, and absent from other tissues. Based on the homology with PADs and its oocyte-abundant expression pattern, the protein was designated ePAD, for egg and embryo-abundant peptidylarginine deiminase-like protein. Anti-recombinant ePAD monospecific antibodies localized the molecule to the cytoplasm of oocytes in primordial, primary, secondary, and Graafian follicles in ovarian sections, while no other ovarian cell type was stained. ePAD was also expressed in the immature oocyte, mature egg, and through the blastocyst stage of embryonic development, where expression levels began to decrease. Immunoelectron microscopy localized ePAD to egg cytoplasmic sheets, a unique keratin-containing intermediate filament structure found only in mammalian eggs and in early embryos, and known to undergo reorganization at critical stages of development. Previous reports that PAD-mediated deimination of epithelial cell keratin results in cytoskeletal remodeling suggest a possible role for ePAD in cytoskeletal reorganization in the egg and early embryo.     

Identification of aspirin and diclofenac binding proteins in the red complex pathogens

Red complex organisms are a group of organisms (Porphyromonas gingivalis ATCC 33277, Treponema denticola ATCC 35405, Tannerella forsythia ATCC 43037) that have been identified for the causation of periodontal diseases. Aspirin and diclofenac have been used as regular analgesics. Therefore, it is of interest to document the identification of aspirin and diclofenac binding proteins in the red complex pathogens using the STITCH v.5 pipeline. The virulence properties of these proteins were analyzed using VICMPred and VirulentPred software. Thus, we document 000 number of proteins having optimal binding features with the known analgesics.     

Altered Active Zones,Vesicle Pools,Nerve Terminal Conductivity,and Morphology during Experimental MuSK Myasthenia Gravis

Recent studies demonstrate reduced motor-nerve function during autoimmune muscle-specific tyrosine kinase (MuSK) myasthenia gravis (MG). To further understand the basis of motor-nerve dysfunction during MuSK-MG, we immunized female C57/B6 mice with purified rat MuSK ectodomain. Nerve-muscle preparations were dissected and neuromuscular junctions (NMJs) studied electrophysiologically, morphologically, and biochemically. While all mice produced antibodies to MuSK, only 40% developed respiratory muscle weakness. In vitro study of respiratory nerve-muscle preparations isolated from these affected mice revealed that 78% of NMJs produced endplate currents (EPCs) with significantly reduced quantal content, although potentiation and depression at 50 Hz remained qualitatively normal. EPC and mEPC amplitude variability indicated significantly reduced number of vesicle-release sites (active zones) and reduced probability of vesicle release. The readily releasable vesicle pool size and the frequency of large amplitude mEPCs also declined. The remaining NMJs had intermittent (4%) or complete (18%) failure of neurotransmitter release in response to 50 Hz nerve stimulation, presumably due to blocked action potential entry into the nerve terminal, which may arise from nerve terminal swelling and thinning. Since MuSK-MG-affected muscles do not express the AChR γ subunit, the observed prolongation of EPC decay time was not due to inactivity-induced expression of embryonic acetylcholine receptor, but rather to reduced catalytic activity of acetylcholinesterase. Muscle protein levels of MuSK did not change. These findings provide novel insight into the pathophysiology of autoimmune MuSK-MG.     

Wolbachia heat shock protein 60 induces pro-inflammatory cytokines and apoptosis in monocytes in vitro

Recombinant Wolbachia heat shock protein 60 (rWmhsp60) induces gene expression of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α in human monocytic cell line THP-1. In addition, it inhibits the phagocytic activity and does not alter the nitric oxide production by differentiated THP-1 macrophages, which corroborates with no significant change in inducible nitric oxide synthase gene expression in rWmhsp60 treated THP-1 monocytes. Further, 24 h stimulation of peripheral blood mononuclear cells from normal individuals by rWmhsp60 reveals that monocytes enter the late apoptotic stage, while lymphocytes do not show apoptosis. Thus these findings suggest that rWmhsp60 may contribute to inflammation mediated monocyte dysfunction in filarial pathogenesis.     

Parasite-Antigen Driven Expansion of IL-5? and IL-5+ Th2 Human Subpopulations in Lymphatic Filariasis and Their Differential Dependence on IL-10 and TGFβ

Background

Two different Th2 subsets have been defined recently on the basis of IL-5 expression – an IL-5⁺Th2 subset and an IL-5⁻Th2 subset in the setting of allergy. However, the role of these newly described CD4⁺ T cells subpopulations has not been explored in other contexts.

Methods

To study the role of the Th2 subpopulation in a chronic, tissue invasive parasitic infection (lymphatic filariasis), we examined the frequency of IL-5⁺IL-4⁺IL-13⁺ CD4⁺ T cells and IL-5⁻IL-4 IL-13⁺ CD4⁺ T cells in asymptomatic, infected individuals (INF) and compared them to frequencies (F_o) in filarial-uninfected (UN) individuals and to those with filarial lymphedema (CP).

Results

INF individuals exhibited a significant increase in the spontaneously expressed and antigen-induced F_o of both Th2 subpopulations compared to the UN and CP. Interestingly, there was a positive correlation between the F_o of IL-5⁺Th2 cells and the absolute eosinophil and neutrophil counts; in addition there was a positive correlation between the frequency of the CD4⁺IL-5⁻Th2 subpopulation and the levels of parasite antigen – specific IgE and IgG4 in INF individuals. Moreover, blockade of IL-10 and/or TGFβ demonstrated that each of these 2 regulatory cytokines exert opposite effects on the different Th2 subsets. Finally, in those INF individuals cured of infection by anti-filarial therapy, there was a significantly decreased F_o of both Th2 subsets.

Conclusions

Our findings suggest that both IL-5⁺ and IL-5⁻Th2 cells play an important role in the regulation of immune responses in filarial infection and that these two Th2 subpopulations may be regulated by different cytokine-receptor mediated processes.