Comparative characterization of mesenchymal bone marrow stromal cells at early and late stages of culturing

The mesenchymal stromal cell is a multipotent precursor of osteoblasts, adipocytes, and some other cell types. In this study, a comparative analysis of cultured mesenchymal stromal cells from the rat bone marrow at the early and late stages of subculturing has been performed using molecular genetic and cytological methods. The culture has undergone 11 passages during 140 days. Upon long-term culturing, the mesenchymal stromal cells have proved to lose their potential for adipogenic differentiation but preserve the potential for osteogenesis. Morphological characters typical of osteogenic differentiation can be observed at the earlier stages of culturing (passages 1–4) but disappear at later stages (passages 9–11), despite mineralization of the extracellular matrix and the expression of osteogenic differentiation markers. A comparative analysis of the proliferation potential of stromal cells has shown that differences in the period of cell population doubling at the early and later stages of culturing are insignificant. An almost complete arrest of cell growth has been observed in the middle of the culture period (passages 5 and 6).     

Developing pulmonary vasculopathy in systemic sclerosis, detected with non-invasive cardiopulmonary exercise testing

Background

Patients with systemic sclerosis (SSc) may develop exercise intolerance due to musculoskeletal involvement, restrictive lung disease, left ventricular dysfunction, or pulmonary vasculopathy (PV). The latter is particularly important since it may lead to lethal pulmonary arterial hypertension (PAH). We hypothesized that abnormalities during cardiopulmonary exercise testing (CPET) in patients with SSc can identify PV leading to overt PAH.

Methods

Thirty SSc patients from the Harbor-UCLA Rheumatology clinic, not clinically suspected of having significant pulmonary vascular disease, were referred for this prospective study. Resting pulmonary function and exercise gas exchange were assessed, including peakVO₂, anaerobic threshold (AT), heart rate- VO₂ relationship (O₂-pulse), exercise breathing reserve and parameters of ventilation-perfusion mismatching, as evidenced by elevated ventilatory equivalent for CO₂ (VE/VCO₂) and reduced end-tidal pCO₂ (P_ETCO₂) at the AT.

Results

Gas exchange patterns were abnormal in 16 pts with specific cardiopulmonary disease physiology: Eleven patients had findings consistent with PV, while five had findings consistent with left-ventricular dysfunction (LVD). Although both groups had low peak VO₂ and AT, a higher VE/VCO₂ at AT and decreasing P_ETCO₂ during early exercise distinguished PV from LVD.

Conclusions

Previously undiagnosed exercise impairments due to LVD or PV were common in our SSc patients. Cardiopulmonary exercise testing may help to differentiate and detect these disorders early in patients with SSc.     

The α isoform of topoisomerase II is required for hypercompaction of mitotic chromosomes in human cells

As proliferating cells transit from interphase into M-phase, chromatin undergoes extensive reorganization, and topoisomerase (topo) IIα, the major isoform of this enzyme present in cycling vertebrate cells, plays a key role in this process. In this study, a human cell line conditional null mutant for topo IIα and a derivative expressing an auxin-inducible degron (AID)-tagged version of the protein have been used to distinguish real mitotic chromosome functions of topo IIα from its more general role in DNA metabolism and to investigate whether topo IIβ makes any contribution to mitotic chromosome formation. We show that topo IIβ does contribute, with endogenous levels being sufficient for the initial stages of axial shortening. However, a significant effect of topo IIα depletion, seen with or without the co-depletion of topo IIβ, is the failure of chromosomes to hypercompact when delayed in M-phase. This requires much higher levels of topo II protein and is impaired by drugs or mutations that affect enzyme activity. A prolonged delay at the G2/M border results in hyperefficient axial shortening, a process that is topo IIα-dependent. Rapid depletion of topo IIα has allowed us to show that its function during late G2 and M-phase is truly required for shaping mitotic chromosomes.     

Development and optimization of a coupled cell-free system for the synthesis of the transmembrane domain of the receptor tyrosine kinase ErbB3

A coupled cell-free expression system (CECF) for the production of the transmembrane domain of the human receptor tyrosine kinase ErbB3 (residues from 632 to 675) has been developed based on the Escherichia coli S30 extract. The synthesis of the domain in the soluble form in the presence of various detergents and in the form of an insoluble precipitate of the reaction mixture has been examined. The conditions for the purification of the recombinant domain obtained using the two approaches have been determined. The final yield of the target protein under optimal conditions was 1.8–2.0 mg per 1 ml of the reaction mixture.     

A simplified method for purification of annexin V from human placenta

A simplified procedure for purification of annexin V from human placenta was developed. At first, the protein was separated from other proteins in membrane bound form in the presence of Ca2+, then was extracted with EDTA and purified by affinity chromatography on PAAG-immobilized phosphatidylserine. The purified protein gave a single band with a molecular weight of 35,000 in SDS-PAGE.