Visualization of caveolin-1, a caveolar marker protein, in living cells using green fluorescent protein (GFP) chimeras. The subcellular distribution of caveolin-1 is modulated by cell-cell contact.
(9/999)
Caveolin-1, a suspected tumor suppressor, is a principal protein component of caveolae in vivo. Recently, we have shown that NIH 3T3 cells harboring anti-sense caveolin-1 exhibit a loss of contact inhibition and anchorage-independent growth. These observations may be related to the ability of caveolin-1 expression to positively regulate contact inhibition. In order to understand the postulated role of caveolin-1 in contact inhibition, it will be necessary to follow the distribution of caveolins in living cells in response to a variety of stimuli, such as cell density. Here, we visualize the distribution of caveolin-1 in living normal NIH 3T3 cells by creating GFP-fusion proteins. In many respects, the behavior of these GFP-caveolin-1 fusion proteins is indistinguishable from endogenous caveolin-1. These GFP-caveolin-1 fusion proteins co-fractionated with endogenous caveolin-1 using an established protocol that separates caveolae-derived membranes from the bulk of cellular membranes and cytosolic proteins, and co-localized with endogenous caveolin-2 in vivo as seen by immunofluorescence microscopy. We show here that as NIH 3T3 cells become confluent, the distribution of GFP-caveolin-1 and endogenous caveolin-1 shifts to areas of cell-cell contact, coincident with contact inhibition. However, unlike endogenous caveolin-1, the levels of GFP-caveolin-1 expression are unaffected by changes in cell density, serum starvation, or growth factor stimulation. These results are consistent with the idea that the levels of endogenous caveolin-1 are modulated by either transcriptional or translational control, and that this modulation is separable from density-dependent regulation of the distribution of caveolin-1. These studies provide a new living-model system for elucidating the dynamic mechanisms underlying the density-dependent regulation of the distribution of caveolin-1 and how this relates to contact inhibition. (+info)
Glutathione and ascorbate are negatively correlated with oxidative DNA damage in human lymphocytes.
(10/999)
Intracellular antioxidants, glutathione and ascorbate, and two molecular markers of oxidative DNA damage, 5-hydroxy-2'-deoxycytidine (5-OH-dCyd) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo), were measured in lymphocytes from 105 healthy volunteers. The analysis of 5-OH-dCyd and 8-oxo-dGuo was carried out by HPLC with electrochemical detection such that both compounds were detected on the same chromatography run. There was no significant difference in oxidative DNA damage when the extraction of DNA from cells using phenol was carried out under anaerobic conditions or in the presence of metal ion chelators. This indicates that auto-oxidation of DNA during sample preparation was minimal. Using the above methods, the average level of oxidative DNA damage in lymphocytes was 2.9 +/- 1.4 for 5-OH-dCyd and 4.5 +/- 1.8 for 8-oxo-dGuo lesions per 10(6) dGuo (n = 105). It is unlikely that artifactual oxidation contributed to the observed damage because the level of 5-OH-dCyd was comparable with that of 8-oxo-dGuo in lymphocyte DNA, whereas 8-oxo-dGuo outnumbers 5-OH-dCyd by a ratio of >5:1 when DNA is exposed to various oxidants, including ionizing radiation or Fenton reagents. Rather, the nearly equal levels of 5-OH-dCyd and 8-oxo-dGuo in cellular DNA implies that 8-oxo-dGuo may be more efficiently removed by DNA repair. Finally, and most importantly, the correlation of our endpoints revealed that the naturally occurring level of intracellular antioxidants was negatively correlated to the level of oxidative DNA damage with the strongest correlation observed for glutathione and 8-oxo-dGuo (r = -0.36; P < 0.001). These results strongly suggest that intracellular glutathione and ascorbate protect human lymphocytes against oxidative DNA damage. (+info)
Production of beta-defensin antimicrobial peptides by the oral mucosa and salivary glands.
(11/999)
beta-Defensins are cationic peptides with broad-spectrum antimicrobial activity that are produced by epithelia at mucosal surfaces. Two human beta-defensins, HBD-1 and HBD-2, were discovered in 1995 and 1997, respectively. However, little is known about the expression of HBD-1 or HBD-2 in tissues of the oral cavity and whether these proteins are secreted. In this study, we characterized the expression of HBD-1 and HBD-2 mRNAs within the major salivary glands, tongue, gingiva, and buccal mucosa and detected beta-defensin peptides in salivary secretions. Defensin mRNA expression was quantitated by RNase protection assays. HBD-1 mRNA expression was detected in the gingiva, parotid gland, buccal mucosa, and tongue. Expression of HBD-2 mRNA was detected only in the gingival mucosa and was most abundant in tissues with associated inflammation. To test whether beta-defensin expression was inducible, gingival keratinocyte cell cultures were treated with interleukin-1beta (IL-1beta) or bacterial lipopolysaccharide (LPS) for 24 h. HBD-2 expression increased approximately 16-fold with IL-1beta treatment and approximately 5-fold in the presence of LPS. Western immunoblotting, liquid chromatography, and mass spectrometry were used to identify the HBD-1 and HBD-2 peptides in human saliva. Human beta-defensins are expressed in oral tissues, and the proteins are secreted in saliva; HBD-1 expression was constitutive, while HBD-2 expression was induced by IL-1beta and LPS. Human beta-defensins may play an important role in the innate defenses against oral microorganisms. (+info)
32P-postlabeling high-performance liquid chromatography (32P-HPLC) adapted for analysis of 8-hydroxy-2'-deoxyguanosine.
(12/999)
8-Hydroxy-2'-deoxyguanosine (8-OH-dG) is a promutagenic lesion in DNA caused by reactive oxygen species. It normally exists at a level of 0.1-1 per 10(5) 2'-deoxyguanosines (dG). To analyze the lesion in easily obtainable biological samples, a very sensitive analytical method is required. The method should also handle the problem with potential oxidation of dG to 8-OH-dG during workup and analysis. 32P-postlabeling high-performance liquid chromatography (32P-HPLC) is an analytical method previously used to analyze lipophilic DNA adducts at levels as low as 1 per 10(9) normal nucleotides when analyzing microgram amounts of DNA. This method was adapted for analysis of 8-OH-dG. The aim was to develop an analytical method that provided a high sensitivity and good reproducibility, prevented oxidation of dG present in samples to 8-OH-dG, was capable of analyzing DNA from very small samples and still offered high sample throughput and ease of use. In analysis of calf thymus DNA, the method had a detection limit of 0.1 8-OH-dG per 10(5) dG when 1 microgram of DNA was used. The standard deviation of repeated analyses of the same sample was +/-10% and the result corresponded well with the established analytical method using HPLC with electrochemical detection. 32P-HPLC is sensitive enough to enable analysis of low levels of 8-OH-dG in biological samples such as small volumes of blood, needle biopsies and tissue swabs. It also substantially reduces oxidation of dG to 8-OH-dG during sample workup and analysis. (+info)
Saturable stimulation of fatty acid transport through model cytoplasm by soluble binding protein.
(13/999)
To better define the role of soluble binding proteins in the cytoplasmic transport of amphipathic molecules, we measured the diffusional mobility of a fluorescent long-chain fatty acid, 12-N-methyl-(7-nitrobenz-2-oxa-1,3-diazol)aminostearate (NBD-stearate), through model cytoplasm as a function of soluble binding protein concentration. Diffusional mobilities were correlated with the partition of the fatty acid between membrane and protein binding sites. Cytoplasm was modeled as a dense suspension of liposomes, and albumin was used as a model binding protein. Albumin saturably increased NBD-stearate mobility through the membrane suspension approximately eightfold. Fatty acid mobility in the absence of albumin was identical to the mobility of the membrane vesicles (1.99 +/- 0.33 x 10(-8) cm(2)/s), whereas the mobility at saturating concentrations was identical to the mobility of albumin (1.65 +/- 0.12 x 10(-7) cm(2)/s). The protein concentration producing half-maximal stimulation of NBD-stearate diffusion (42.8 +/- 0.3 microM) was unexpectedly greater than that required to solubilize half of the NBD-stearate (17.9 +/- 3.0 microM). These results support a proposed mechanism for cytoplasmic transport of small amphipathic molecules in which aqueous diffusion of the protein-bound form of the molecule largely determines the transport rate. However, slow interchange of fatty acid between the binding protein and membranes also appears to influence the transport rate in this model system. (+info)
Osmolarity effects on red blood cell elution in sedimentation field-flow fractionation.
(14/999)
Field-flow fractionation (FFF) is an analytical technique particularly suitable for the separation, isolation, and characterization of macromolecules and micrometer- or submicrometer-sized particles. This chromatographic-like methodology can modulate the retention of micron-sized species according to an elution mode described to date as "steric hyperlayer". In such a model, differences in sample species size, density, or other physical parameters make particle selective elution possible depending on the configuration and the operating conditions of the FFF system. Elution characteristics of micron-sized particles of biological origin, such as cells, can be modified using media and carrier phases of different osmolarities. In these media, a cells average size, density, and shape are modified. Therefore, systematic studies of a single reference cell population, red blood cells (RBCs), are performed with 2 sedimentation FFF systems using either gravity (GrFFF) or a centrifugational field (SdFFF). However, in all cases, normal erythrocyte in isotonic suspension elutes as a single peak when fractionated in these systems. With carrier phases of different osmolarities, FFF elution characteristics of RBCs are modified. Retention modifications are qualitatively consistent with the "steric-hyperlayer" model. Such systematic studies confirm the key role of size, density, and shape in the elution mode of RBCs in sedimentation FFF for living, micronsized biological species. Using polymers as an analogy, the RBC population is described as highly "polydisperse". However, this definition must be reconsidered depending on the parameters under concern, leading to a matricial concept: multipolydispersity. It is observed that multipolydispersity modifications of a given RBC population are qualitatively correlated to the eluted sample band width. (+info)
Immunological determination and size characterization of poly(ADP-ribose) synthesized in vitro and in vivo.
(15/999)
Poly(ADP-ribose) polymerase is a DNA break detecting enzyme playing a role in the surveillance of genome integrity. Poly(ADP-ribose) is synthesized rapidly and transiently from beta-NAD in response to DNA damaging agents. In order to study the physiological significance of poly(ADP-ribose) metabolism, we have developed immunological methods which enable us to study endogenous poly(ADP-ribose) without interfering with cell metabolism and integrity. For this purpose, we produced a highly specific polyclonal anti-poly(ADP-ribose) antibody which immunoreacts with polymers and oligomers. In addition to the immunodot blot method recently described by us (Affar et al., Anal. Biochem. 259 (1998) 280-283), other applications were investigated in cells: (i) detection of poly(ADP-ribose) by ELISA; (ii) characterization of poly(ADP-ribose) size using high resolution gel electrophoresis of polymers, followed by its transfer onto a positively charged membrane and detection with anti-poly(ADP-ribose) antibody; (iii) immunocytochemistry and flow cytometry analyses allowing poly(ADP-ribose) study at the level of individual cells. (+info)
Roles for the E4 orf6, orf3, and E1B 55-kilodalton proteins in cell cycle-independent adenovirus replication.
(16/999)
Adenoviruses bearing lesions in the E1B 55-kDa protein (E1B 55-kDa) gene are restricted by the cell cycle such that mutant virus growth is most impaired in cells infected during G(1) and least restricted in cells infected during S phase (F. D. Goodrum and D. A. Ornelles, J. Virol. 71:548-561, 1997). A similar defect is reported here for E4 orf6-mutant viruses. An E4 orf3-mutant virus was not restricted for growth by the cell cycle. However, orf3 was required for enhanced growth of an E4 orf6-mutant virus in cells infected during S phase. The cell cycle restriction may be linked to virus-mediated mRNA transport because both E1B 55-kDa- and E4 orf6-mutant viruses are defective at regulating mRNA transport at late times of infection. Accordingly, the cytoplasmic-to-nuclear ratio of late viral mRNA was reduced in G(1) cells infected with the mutant viruses compared to that in G(1) cells infected with the wild-type virus. By contrast, this ratio was equivalent among cells infected during S phase with the wild-type or mutant viruses. Furthermore, cells infected during S phase with the E1B 55-kDa- or E4 orf6-mutant viruses synthesized more late viral protein than did cells infected during G(1). However, the total amount of cytoplasmic late viral mRNA was greater in cells infected during G(1) than in cells infected during S phase with either the wild-type or mutant viruses, indicating that enhanced transport of viral mRNA in cells infected during S phase cannot account for the difference in yields in cells infected during S phase and in cells infected during G(1). Thus, additional factors affect the cell cycle restriction. These results indicate that the E4 orf6 and orf3 proteins, in addition to the E1B 55-kDa protein, may cooperate to promote cell cycle-independent adenovirus growth. (+info)