Isolation of lactoperoxidase, lactoferrin, α-lactalbumin, β-lactoglobulin B and β-lactoglobulin A from bovine rennet whey using ion exchange chromatography

A mild and rapid method is described for isolating various milk proteins from bovine rennet whey. β-Lactoglobulin from bovine rennet whey was easily adsorbed on and desorbed from a weak anion exchanger, diethylaminoethyl-Toyopearl. However, α-lactalbumin could not be adsorbed onto the resin. α-Lactalbumin and β-lactoglobulin from rennet whey could also be adsorbed and separated using a strong anion exchanger, quaternary aminoethyl-Toyopearl. The rennet whey was passed through a strong cation exchanger, sulphopropyl-Toyopearl, to separate lactoperoxidase and lactoferrin. α-Lactalbumin and β-lactoglobulin were adsorbed onto quaternary aminoethyl-Toyopearl. α-Lactalbumin was eluted using a linear (0–0.15 M) concentration gradient of NaCl in 0.05 M Tris–HCl buffer (pH 8.5). Subsequently, β-lactoglobulin B and β-lactoglobulin A were eluted from the column with 0.05 M Tris–HCl (pH 6.8), using a linear (0.1–0.25 M) concentration gradient of NaCl. The yields were 1260 mg α-lactalbumin, 1290 mg β-lactoglobulin B and 2280 mg β-lactoglobulin A from 1 l rennet whey.     

Hypotonic swelling-induced Ca2+ release by an IP3-insensitive Ca2+ store

Hypotonicswelling increases the intracellular Ca²⁺ concentration([Ca²⁺]_i) in vascular smooth muscle cells(VSMC). The source of this Ca²⁺ is not clear. To study thesource of increase in [Ca²⁺]_i in response tohypotonic swelling, we measured [Ca²⁺]_i infura 2-loaded cultured VSMC (A7r5 cells). Hypotonic swelling produced a40.7-nM increase in [Ca²⁺]_i that was notinhibited by EGTA but was inhibited by 1 µM thapsigargin. Priordepletion of inositol 1,4,5-trisphosphate (IP₃)-sensitive Ca²⁺ stores with vasopressin did not inhibit the increasein [Ca²⁺]_i in response to hypotonic swelling.Exposure of ⁴⁵Ca²⁺-loaded intracellular storesto hypotonic swelling in permeabilized VSMC produced an increase in⁴⁵Ca²⁺ efflux, which was inhibited by 1 µMthapsigargin but not by 50 µg/ml heparin, 50 µM ruthenium red, or25 µM thio-NADP. Thus hypotonic swelling of VSMC causes a release ofCa²⁺ from the intracellular stores from a novel sitedistinct from the IP₃-, ryanodine-, and nicotinic acidadenine dinucleotide phosphate-sensitive stores.

     

The use of microcomputers for the quantitation of light intensity patterns using digitized video signals

We have developed an inexpensive yet versatile microcomputer-basedsystem for quantitating light intensity levels in autoradiographs.This system employs a standard video camera interfaced to ananalog-to-digital convertor. A program has been written forthis system which can measure intensities within a defined regionof an autoradiograph, permitting an easy and accurate quantitationof spots or bands of irregular shape. Received on June 18, 1985; accepted on September 3, 1985     

Selective pericentromeric heterochromatin dismantling caused by TP53 activation during senescence

Cellular senescence triggers various types of heterochromatin remodeling that contribute to aging. However, the age-related mechanisms that lead to these epigenetic alterations remain elusive. Here, we asked how two key aging hallmarks, telomere shortening and constitutive heterochromatin loss, are mechanistically connected during senescence. We show that, at the onset of senescence, pericentromeric heterochromatin is specifically dismantled consisting of chromatin decondensation, accumulation of DNA breakages, illegitimate recombination and loss of DNA. This process is caused by telomere shortening or genotoxic stress by a sequence of events starting from TP53-dependent downregulation of the telomere protective protein TRF2. The resulting loss of TRF2 at pericentromeres triggers DNA breaks activating ATM, which in turn leads to heterochromatin decondensation by releasing KAP1 and Lamin B1, recombination and satellite DNA excision found in the cytosol associated with cGAS. This TP53–TRF2 axis activates the interferon response and the formation of chromosome rearrangements when the cells escape the senescent growth arrest. Overall, these results reveal the role of TP53 as pericentromeric disassembler and define the basic principles of how a TP53-dependent senescence inducer hierarchically leads to selective pericentromeric dismantling through the downregulation of TRF2.     

Efficient mucosal vaccination mediated by the neonatal Fc receptor

Almost all infectious diseases are initiated at mucosal surfaces, yet intramuscular or subcutaneous vaccination usually provides only minimal protection at sites of infection owing to suboptimal activation of the mucosal immune system. The neonatal Fc receptor (FcRn) mediates the transport of IgG across polarized epithelial cells lining mucosal surfaces. We mimicked this process by fusing a model antigen, herpes simplex virus type-2 (HSV-2) glycoprotein gD, to an IgG Fc fragment. Intranasal immunization, together with the adjuvant CpG, completely protected wild-type, but not FcRn knockout, mice after intravaginal challenge with virulent HSV-2 186. This immunization strategy induced efficient mucosal and systemic antibody, B- and T-cell immune responses, with stable protection for at least 6 months after vaccination in most of the immunized animals. The FcRn-IgG transcellular transport pathway may provide a general delivery route for subunit vaccines against many mucosal pathogens.     

Sunitinib Reverse Multidrug Resistance in Gastric Cancer Cells by Modulating Stat3 and Inhibiting P-gp Function

Sunitinib, a small-molecule multi-targeted tyrosine kinase inhibitor, has been applied in phase II clinical trial as second-line treatment for advanced gastric cancer. In this study, we determined the effect of Sunitinib on the multidrug resistance in gastric cancer cells selected by vincristine. Our results showed that Sunitinib significantly enhanced the cytotoxicity of adriamycin, vincristine, etoposide, 5-Fluorouracil, and cisplatin in multidrug-resistant gastric cancer cells (SGC7901/VCR). Sunitinib significantly increased the intracellular accumulation and retention of rhodamine 123 in the SGC7901/VCR cells. However, Sunitinib, at a concentration that reverses MDR, had no significant effect on P-gp protein or mRNA expression levels. In addition, the present study revealed that Sunitinib inhibited Stat3 and down-regulated Bcl-2 in SGC7901/VCR cells, which might also contribute to the reversal of MDR. In conclusion, Sunitinib reverses multidrug resistance in gastric cancer cells by inhibiting P-gp transporter function and modulating Stat3 and Bcl-2. Further study with Sunitinib may be helpful for developing combination therapeutic strategy or circumventing gastric cancer MDR to other conventional anti-cancer drugs.