Cartilage-derived factor (CDF) : II. Somatomedin-like action on cultured chondrocytes

Previously, we showed that fetal bovine cartilage contains a polypeptide that stimulates the incorporation of [³⁵S]sulfate into proteoglycans synthesized by rat and rabbit costal chondrocytes in culture. In this paper, we report that the cartilage-derived factor (CDF) increases not only [³⁵S]sulfate incorporation but also [³H]thymidine incorporation into rabbit chondrocytes in monolayer culture. The dose-response curve of CDF stimulation of DNA synthesis was similar in profile to that of CDF stimulation of proteoglycan synthesis. In addition, CDF markedly enhanced [³H]uridine incorporation into rabbit chondrocytes and significantly enhanced [³H]serine incorporation into total protein. These findings indicate that fetal bovine cartilage contains a factor that shows somatomedin-like activity in monolayer cultures of rabbit chondrocytes.     

Electrophysiological study of coupling between cultured cells of the mouse mammary gland in five distinct physiological states.

Electrical coupling has been observed between cultured cells of the mouse mammary gland in five distinct physiological or pathological states. We have employed young primary cultures of cells dissociated from the following tissues: normal glands from young virgin or midpregnant females, hyperplastic alveolar nodules (believed to be precancerous) transplanted in gland-free mammary fat pads, and spontaneous mammary adenocarcinomas and their pulmonary metastases. All successfully impaled pairs of cells (a total of 97 pairs) were found to be ionically coupled. Furthermore, in normal and tumor cell cultures, electrical coupling was observed between dome-dome and dome-nondome cell pairs. This study correlates with electronmicroscopic studies of fresh normal, hyperplastic, and tumor samples, which show the presence of gap junctions in all three.     

Use of the 2-(2-Pyridyl)Ethyl Protecting Group in the Synthesis of DNA Fragments via Phosphoramidite Intermediates

Abstract

2-(2-Pyridyl)ethyl is a new protecting group for the internucleotidic linkages in the synthesis of oligodeoxyribo-nucleotides by the phosphoramidite method. This group is stable to alkali and acid, and can be removed by two step procedures under mild conditions. Furthermore, we have found that bis-(diiso-propylamino)chlorophosphine is much more effective for the preparation of bis-(diisopropylamino)alkoxyphosphine than various dichlorophosphines. The synthesis of oligodeoxyribonucleotides by using 2-(2-pyridyl)ethyl-deoxyribonucleoside-3′-0-N,N-diisopropyl-amidite units is also described.     

Anastrozole quantification in human plasma by high-performance liquid chromatography coupled to photospray tandem mass spectrometry applied to pharmacokinetic studies

A rapid, sensitive and specific method for quantifying the aromatase inhibitor (anastrozole) in human plasma using dexchlorpheniramine as the internal standard (I.S.) is described herein. The analyte and the I.S. were extracted from 200 microl of human plasma by liquid-liquid extraction using a mixture of diethyl ether:dichloromethane (70:30, v/v) solution. Extracts were removed and dried in the organic phase then reconstituted with 200 microl of acetonitrile:water (50:50; v/v). The extracts were analyzed by high performance liquid chromatography coupled with photospray tandem mass spectrometry (HPLC-MS-MS). Chromatography was performed isocratically on a Genesis, C18 4 microm analytical column (100 mm x 2.1mm i.d.). The method had a chromatographic run time of 2.5 min and a linear calibration curve ranging from 0.05-10 ng ml(-1). The limit of quantification (LOQ) was 0.05 ng ml(-1). This HPLC-MS-MS procedure was used to assess pharmacokinetic studies.     

GTP/GDP exchange by Sec12p enables COPII vesicle bud formation on synthetic liposomes

The generation of COPII vesicles from synthetic liposome membranes requires the minimum coat components Sar1p, Sec23/24p, Sec13/31p, and a nonhydrolyzable GTP analog such as GMP-PNP. However, in the presence of GTP and the full complement of coat subunits, nucleotide hydrolysis by Sar1p renders the coat insufficiently stable to sustain vesicle budding. In order to recapitulate a more authentic, GTP-dependent budding event, we introduced the Sar1p nucleotide exchange catalyst, Sec12p, and evaluated the dynamics of coat assembly and disassembly by light scattering and tryptophan fluorescence measurements. The catalytic, cytoplasmic domain of Sec12p (Sec12DeltaCp) activated Sar1p with a turnover 10-fold higher than the GAP activity of Sec23p stimulated by the full coat. COPII assembly was stabilized on liposomes incubated with Sec12DeltaCp and GTP. Numerous COPII budding profiles were visualized on membranes, whereas a parallel reaction conducted in the absence of Sec12DeltaCp produced no such profiles. We suggest that Sec12p participates actively in the growth of COPII vesicles by charging new Sar1p-GTP molecules that insert at the boundary between a bud and the surrounding endoplasmic reticulum membrane.     

A role of the Duffy antigen for the maintenance of plasma chemokine concentrations

We examined plasma chemokine concentrations and chemokine clearance rates in Duffy antigen knockout mice. The plasma concentrations of eotaxin and MCP-1 in Duffy antigen knockout mice were less than one-third of those in wild-type mice. When eotaxin or hMGSA was intravenously injected, the chemokine disappeared more rapidly from the plasma of Duffy antigen knockout mice than from the plasma of wild-type mice. The half-lives of hIP-10 and interferon-gamma, which do not have an affinity for the Duffy antigen, in plasma were indistinguishable between Duffy antigen knockout mice and wild-type mice. These results suggest that the Duffy antigen delays the disappearance of chemokines from the plasma, resulting in the maintenance of plasma chemokine concentrations.