Contaminant eluted from solid-phase plasmid affinity-purification protocol columns is not found using liquid-phase methods and can be prevented.

The preparation of high quality plasmid DNA is a necessary requirement for most molecular biology applications. We compared four different large plasmid preparation protocols, which were based on either a liquid-phase approach (Triton lysis) or purification of alkaline lysis bacterial extracts followed by supercoiled plasmid purification on affinity columns. Two host Escherichia coli strains, JM 109 and INValphaF', were used to grow the test plasmids for comparison of product plasmid DNA produced from the four different plasmid isolation methods. While the DNA grown in E. coli strain JM109, prepared by liquid-phase Triton lysis was appropriately restricted by 12 restriction enzymes, this was not the case for any of the JM109-grown DNA purified by any of the affinity column solid-phase approaches. In contrast to this, when the plasmid DNA was grown in E. coli strain INValphaF', most restriction enzymes cut DNA appropriately, irregardless of the plasmid preparation protocol used. It seems that an impurity commonly eluted with the DNA from all three of the solid-phase DNA columns had an equal effect on the above enzymes using the common host strain JM109, but not strain INValphaF'.     

Adjuvant Docetaxel and Cyclophosphamide (DC) with Prophylactic Granulocyte Colony-Stimulating Factor (G-CSF) on Days 8 &12 in Breast Cancer Patients: A Retrospective Analysis

Purpose

Four cycles of docetaxel/cyclophosphamide (DC) resulted in superior survival than doxorubicin/cyclophosphamide in the treatment of early breast cancer. The original study reported a 5% incidence of febrile neutropenia (FN) recommending prophylactic antibiotics with no granulocyte colony-stimulating factor (G-CSF) support. The worldwide adoption of this protocol yielded several reports on substantially higher rates of FN events. We explored the use of growth factor (GF) support on days 8 and 12 of the cycle with the original DC protocol.

Methods

Our study included all consecutive patients with stages I–II breast cancer who were treated with the DC protocol at the Institute of Oncology, Davidoff Center (Rabin Medical Center, Petah Tikva, Israel) from April, 2007 to March, 2012. Patient, tumor characteristics, and toxicity were reported. Results: In total, 123 patients received the DC regimen. Median age was 60 years, (range, 25–81 years). Thirty-three patients (26.8%) were aged 65 years and older. Most of the women (87%) adhered to the planned G-CSF protocol (days 8 &12). 96% of the patients completed the 4 planned cycles of chemotherapy. Six patients (5%) had dose reductions, 6 (5%) had treatment delays due to non-medical reasons. Thirteen patients (10.6%) experienced at least one event of FN (3 patients had 2 events), all requiring hospitalization. Eight patients (6.5%) required additional support with G-CSF after the first chemotherapy cycle, 7 because of FN and one due to neutropenia and diarrhea.

In Conclusion

Primary prophylactic G-CSF support on days 8 and 12 of the cycle provides a tolerable option to deliver the DC protocol. Our results are in line with other retrospective protocols using longer schedules of GF support.     

Distinct differentiation potential of blood monocyte subsets in the lung

Peripheral blood monocytes are a population of circulating mononuclear phagocytes that harbor potential to differentiate into macrophages and dendritic cells. As in humans, monocytes in the mouse comprise two phenotypically distinct subsets that are Gr1(high)CX(3)CR1(int) and Gr1(low)CX(3)CR1(high), respectively. The question remains whether these populations contribute differentially to the generation of peripheral mononuclear phagocytes. In this study, we track the fate of adoptively transferred, fractionated monocyte subsets in the lung of recipient mice. We show that under inflammatory and noninflammatory conditions, both monocyte subsets give rise to pulmonary dendritic cells. In contrast, under the conditions studied, only Gr1(low)CX(3)CR1(high) monocytes, but not Gr1(high)CX(3)CR1(int) cells, had the potential to differentiate into lung macrophages. However, Gr1(high)CX(3)CR1(int) monocytes could acquire this potential upon conversion into Gr1(low)CX(3)CR1(high) cells. Our results therefore indicate an intrinsic dichotomy in the differentiation potential of the two main blood monocyte subsets.     

Lung macrophages serve as obligatory intermediate between blood monocytes and alveolar macrophages

Alveolar macrophages are a unique type of mononuclear phagocytes that populate the external surface of the lung cavity. Early studies have suggested that alveolar macrophages originate from tissue-resident, local precursors, whereas others reported their derivation from blood-borne cells. However, the role of circulating monocytes as precursors of alveolar macrophages was never directly tested. In this study, we show through the combined use of conditional cell ablation and adoptive cell transfer that alveolar macrophages originate in vivo from blood monocytes. Interestingly, this process requires an obligate intermediate stage, the differentiation of blood monocytes into parenchymal lung macrophages, which subsequently migrate into the alveolar space. We also provide direct evidence for the ability of both lung and alveolar macrophages to proliferate.     

Mathematical model of excitation-contraction in a uterine smooth muscle cell

Uterine contractility is generated by contractions of myometrial smooth muscle cells (SMCs) that compose most of the myometrial layer of the uterine wall. Calcium ion (Ca²⁺) entry into the cell can be initiated by depolarization of the cell membrane. The increase in the free Ca²⁺ concentration within the cell initiates a chain of reactions, which lead to formation of cross bridges between actin and myosin filaments, and thereby the cell contracts. During contraction the SMC shortens while it exerts forces on neighboring cells. A mathematical model of myometrial SMC contraction has been developed to study this process of excitation and contraction. The model can be used to describe the intracellular Ca²⁺ concentration and stress produced by the cell in response to depolarization of the cell membrane. The model accounts for the operation of three Ca²⁺ control mechanisms: voltage-operated Ca²⁺ channels, Ca²⁺ pumps, and Na⁺/Ca²⁺ exchangers. The processes of myosin light chain (MLC) phosphorylation and stress production are accounted for using the cross-bridge model of Hai and Murphy (Am J Physiol Cell Physiol 254: C99–C106, 1988) and are coupled to the Ca²⁺ concentration through the rate constant of myosin phosphorylation. Measurements of Ca²⁺, MLC phosphorylation, and force in contracting cells were used to set the model parameters and test its ability to predict the cell response to stimulation. The model has been used to reproduce results of voltage-clamp experiments performed in myometrial cells of pregnant rats as well as the results of simultaneous measurements of MLC phosphorylation and force production in human nonpregnant myometrial cells. cellular calcium control mechanisms; myometrial contractions; myosin light chain phosphorylation     

25 hydroxy-vitamin D(3)-1alpha hydroxylase expression and activity in cultured human osteoblasts and their modulation by parathyroid hormone, estrogenic compounds and dihydrotestosterone

Human osteoblasts (hOB) produce and respond to 1,25(OH)(2)D(3) (1,25D), suggesting an autocrine/paracrine system. We therefore examined hormonal modulation of the expression and activity of 25 hydroxy-vitamin D(3)-1alpha hydroxylase (1-Ohase) in hOB. Cells from pre- and post-menopausal women or men, were treated with estrogenic compounds and 1-OHase expression and activity were measured. 1-OHase mRNA expression was highest in pre-menopausal women hOB and was increased by all hormones tested. In post-menopausal hOB all hormones except biochainin A (BA) and genistein (G) increased 1-OHase mRNA expressions to less extent. In male-derived hOB only dihydrotestosterone (DHT) and carboxy BA (cBA) increased 1-OHase mRNA expression. 1,25D production from 25(OH)D(3) had a K(m) of approximately 769-400 ng/ml (1.92-1.07 microM) and V(max) of 31.3-17.4 ng/ml (0.078-0.044 microM/60 min/5 x 10(6)cells) respectively, and was increased by all hormones except raloxifene (Ral) with higher stimulation in pre- than in post-menopausal cells. Only BA was almost five times more potent in pre- rather than post-menopausal hOBs. In male hOB only DHT and cBA increased 1,25D production whereas estradiol-17beta (E(2)) had no effect and BA decreased it. These results provide evidence for the expression of 1-OHase mRNA and production of 1,25D in hOBs, which are age and sex dependent and are hormonally modulated. The role of this local autocrine/paracrine 1,25D system in bone physiology deserves further investigation.     

Filamin A is a novel caveolin-1-dependent target in IGF-I-stimulated cancer cell migration

Caveolin-1 is an essential structural constituent of caveolae which is involved in regulation of mitogenic signaling and oncogenesis. Caveolin-1 has been implicated in cell migration but its exact role and mechanism of action in this process remained obscure. We have previously reported that expression of caveolin-1 in stably transfected MCF-7 human breast cancer (MCF-7/Cav1) cells up-regulates phosphorylation of a putative Akt substrate protein, designated pp340 [D. Ravid, S. Maor, H. Werner, M. Liscovitch, Caveolin-1 inhibits cell detachment-induced p53 activation and anoikis by upregulation of insulin-like growth factor-I receptors and signaling, Oncogene 24 (2005) 1338-1347.]. We now show, using differential detergent extraction, SDS-PAGE and mass spectrometry, that the major protein in the pp340 band is the actin filament cross-linking protein filamin A. The identity of pp340 as filamin A was confirmed by immunoprecipitation of pp340 with specific filamin A antibodies. RT-PCR, flow cytometry and Western blot analyses show that filamin A mRNA and protein levels are respectively 3.5- and 2.5-fold higher in MCF-7/Cav1 cells than in MCF-7 cells. Basal filamin A phosphorylation on Ser-2152, normalized to total filamin A levels, is 7.8-fold higher in MCF-7/Cav1 than in MCF-7 cells. Insulin-like growth factor-I (IGF-I) stimulates phosphorylation of filamin A on Ser-2152 in MCF-7 cells and further enhances Ser-2152 phosphorylation over its already high basal level in MCF-7/Cav1 cells. The effect of IGF-I is inhibited by the PI3K inhibitor wortmannin, indicating that IGF-I-stimulated phosphorylation of filamin A occurs via the PI3K/Akt pathway. Co-immunoprecipitation experiments have confirmed a previous report showing that filamin A and caveolin-1 co-exist in a complex and have revealed the presence of active phospho-Akt in this complex. Ser-2152 phosphorylation of filamin A has been implicated in cancer cell migration. Accordingly, caveolin-1 expression dramatically enhances IGF-I-dependent MCF-7 cell migration. These data indicate that caveolin-1 specifies filamin A as a novel target for Akt-mediated filamin A Ser-2152 phosphorylation thus mediating the effects of caveolin-1 on IGF-I-induced cancer cell migration.