Determining the structure and mechanism of the human multidrug resistance P-glycoprotein using cysteine-scanning mutagenesis and thiol-modification techniques

The multidrug resistance P-glycoprotein is an ATP-dependent drug pump that extrudes a broad range of hydrophobic compounds out of cells. Its physiological role is likely to protect us from exogenous and endogenous toxins. The protein is important because it contributes to the phenomenon of multidrug resistance during AIDS and cancer chemotherapy. We have used cysteine-scanning mutagenesis and thiol-modification techniques to map the topology of the protein, show that both nucleotide-binding domains are essential for activity, examine packing of the transmembrane segments, map the drug-binding site, and show that there is cross-talk between the ATP-binding sites and the transmembrane segments.     

Functional and morphological correlates of connexin50 expressed in Xenopus laevis oocytes

Electrophysiological and morphological methods were used to study connexin50 (Cx50) expressed in Xenopus laevis oocytes. Oocytes expressing Cx50 exhibited a new population of intramembrane particles (9.0 nm in diameter) in the plasma membrane. The particles represented hemichannels (connexin hexamers) because (a) their cross-sectional area could accommodate 24 +/- 3 helices, (b) when their density reached 300-400/microm2, they formed complete channels (dodecamers) in single oocytes, and assembled into plaques, and (c) their appearance in the plasma membrane was associated with a whole-cell current, which was activated at low external Ca2+ concentration ([Ca2+]o), and was blocked by octanol and by intracellular acidification. The Cx50 hemichannel density was directly proportional to the magnitude of the Cx50 Ca2+-sensitive current. Measurements of hemichannel density and the Ca2+-sensitive current in the same oocytes suggested that at physiological [Ca2+]o (1-2 mM), hemichannels rarely open. In the cytoplasm, hemichannels were present in approximately 0.1-microm diameter "coated" and in larger 0.2-0.5-microm diameter vesicles. The smaller coated vesicles contained endogenous plasma membrane proteins of the oocyte intermingled with 5-40 Cx50 hemichannels, and were observed to fuse with the plasma membrane. The larger vesicles, which contained Cx50 hemichannels, gap junction channels, and endogenous membrane proteins, originated from invaginations of the plasma membrane, as their lumen was labeled with the extracellular marker peroxidase. The insertion rate of hemichannels into the plasma membrane (80, 000/s), suggested that an average of 4,000 small coated vesicles were inserted every second. However, insertion of hemichannels occurred at a constant plasma membrane area, indicating that insertion by vesicle exocytosis (60-500 microm2 membranes/s) was balanced by plasma membrane endocytosis. These exocytotic and endocytotic rates suggest that the entire plasma membrane of the oocyte is replaced in approximately 24 h.     

Estrogen receptor alpha mediates estrogen's immune protection in autoimmune disease

Estrogens are known to influence a variety of autoimmune diseases, but it is not known whether their actions are mediated through classic estrogen receptor alpha (ERalpha). The presence of a functional ER was demonstrated in secondary lymphoid tissues, then ERalpha expression was shown at both the RNA and protein levels in these tissues. Use of ERalpha knockout mice revealed that both the estrogen-induced disease protection and the estrogen-induced reduction in proinflammatory cytokines were dependent upon ERalpha in the prototypic Th1-mediated autoimmune disease experimental autoimmune encephalomyelitis. These findings are central to the design of selective ER modifiers which aim to target biologic responses in specific organ systems.     

A rat cell line derived from DMBA-induced mammary carcinoma

A new cell line, designated UHKBR-01, was successfully established from a 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat mammary tumour. DMBA was administered orally at a dose of 4 mg/ml per rat on the first day of the experiment and thereafter at weekly intervals of same dosage, until the rats have reached a weight of around 150-200 g. The tumours grew rapidly after the injection, and were transplanted into nude mice one the harvest size (2.5 x 2 x 1 mm(3)) was reached, it was transplanted onto nude mice. We have developed a cell line from a portion of the DMBA-induced carcinoma of the nude mice. The UHKBR-01 cell exhibited a slow increase in growth rate during the time of culture and was highly tumourigenic in nude mice. The cells have been grown in culture for over 40 passages. Characterization of the cell line was performed. This included morphology by light and transmission electron microscopy, karyotype, growth rate, tumour antigen expression and xenograft implantation into nude mice. These cells exhibit ultrastructural and immunohistochemical features of epithelial cells of mammary origin. The above analyses also demonstrated that UHKBR-01 cells were oestrogen- and progesterone-receptor positive, in likeness to other established breast cancer cell lines such as MDA-MB-231 and MCF-7. The cell line grows as monolayers of oval-shaped cells with large folded nuclei accompanied by a rich supply of mitochondria. This report describes the first in vitro cell line from transplantable DMBA-induced mammary carcinoma of nude mice, which presents unique characteristics that may prove to be a good experimental model for investigating breast cancer biology.     

A quantitative diffraction-based sandwich immunoassay

It is shown that diffraction-based sensing can be enhanced for diagnostic purposes through the use of a secondary label. The limit of detection for anti-rabbit IgG was reduced more than 40-fold by using a gold-conjugated secondary antibody. The response to secondary antibody binding was linear for concentrations from 25 to 500 ng/ml of anti-rabbit IgG, suggesting that quantitative determinations can be readily done. Moreover, the binding of the secondary antibody was observed as soon as 1 min after its introduction to the surface-bound primary complex.     

Phosphate absorption in Aplysia californica gut: glucocorticoid inhibition

Apical membranes of Aplysia californica foregut epithelia contain a sodium-phosphate symporter. Dexamethasone inhibited the absorptive activity of the sodium-phosphate symporter, whereas amiloride had no effect on the sodium-phosphate symporter. It appears that glucocorticoids or their molluscan equivalent play a role in the overall regulation of phosphate homeostasis by the Aplysia californica gut.     

Restriction fragment alleles of the rabbit IGHG genes with reference to the rabbit IGHGCH2 or e locus polymorphism

Among domesticated mammals, rabbit (Oryctolagus cuniculus) is the only species possessing not more than one subclass of immunoglobulin (IgG) antibodies. The rabbit IGHGCH2 or e locus presents two serologically defined alleles, the e14 and e15 allotypes, which are correlated with amino acid variation at the IgG CH2-CH3 interface. Genetic studies, while revealing the adaptive value of this polymorphism, have relied so far entirely upon allo-antisera. Here we show how these alleles can be distinguished by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods. The proposed PCR-RFLP approach allows the monitoring of IGHG locus diversity in rabbit.     

Caspases are not localized in mitochondria during life or death

Caspases are crucial for the initiation, propagation and execution of apoptosis. They normally exist as proenzymes, which can be activated through recruitment into activating complexes and by proteolytic cleavage by other caspases or proteases. Perturbation of organelles such as nuclei, endoplasmatic reticulum and mitochondria results in the activation of caspases. A number of caspases (-2, -3, -8 and -9) were published as being localized in the intermembrane space of mitochondria. However, in three different models of apoptosis (anti-Fas-induced cell death in murine hepatocytes, Fas ligand-induced apoptosis in Jurkat cells and apoptosis induced by growth factor withdrawal in Ba/F3 cells) we could not identify a mitochondrial location of caspases, neither under control nor under apoptotic conditions. In all three apoptotic models caspases were found in the cytosolic (caspases-2, -3, -6, -7, -8, -9) and nuclear subcellular fractions (caspases-2, -3). In another approach we treated isolated liver mitochondria with truncated Bid. Although tBid-dependent release of Cytochrome c, AIF, adenylate kinase, Smac/DIABLO and Omi/HtrA2 could be demonstrated, none of the caspases were detectable both in the supernatant and the mitochondrial fraction after treatment. Our results demonstrate that, in contrast to previous studies, no caspases-2, -3, -8 and -9 are associated with the mitochondrial fraction. These findings support the concept of a separate compartmentalization between proapoptotic cofactors in the mitochondria and silent precursor caspases in the cytosol.     

A Bcl-2 transgene expressed in hepatocytes does not protect mice from fulminant liver destruction induced by Fas ligand

We compared the biological mechanism of cell death during hepatotoxicity induced by ligation of the Fas receptor in wild-type and liver-specific bcl-2 transgenic mice. Transgenic overexpression of Bcl-2 in mouse hepatocytes can prevent lethal hepatitis induced by agonistic anti-Fas antibodies. In contrast, Fas ligand (FasL)-induced death cannot be overcome in bcl-2 transgenic mice, indicating that anti-Fas antibodies do not reliably mimic the more physiological ligand. Different apoptotic parameters, viz. caspase activation, cytochrome c release and nuclear DNA degradation were analysed. No differences, however, could be observed between wild-type and bcl-2 transgenic mice after injection with a lethal dose of soluble FasL, indicating that apoptosis by FasL-dependent ligation is not modulated by Bcl-2 in vivo. These results demonstrate that the stimulus determines the outcome between type I mitochondria-independent apoptosis, in the case of FasL, or type II mitochondria-dependent and Bcl-2-inhibitable apoptosis, in the case of anti-Fas antibodies.