We have investigated properties relevant to quantitative imaging in living cells of five green fluorescent protein (GFP) variants that have been used extensively or are potentially useful. We measured the extinction coefficients, quantum yields, pH effects, photobleaching effects, and temperature-dependent chromophore formation of wtGFP, alphaGFP (F99S/M153T/V163A), S65T, EGFP (F64L/S65T), and a blue-shifted variant, EBFP (F64L/S65T/Y66H/Y145F). Absorbance and fluorescence spectroscopy showed little difference between the extinction coefficients and quantum yields of wtGFP and alphaGFP. In contrast, S65T and EGFP extinction coefficients made them both approximately 6-fold brighter than wtGFP when excited at 488 nm, and EBFP absorbed more strongly than the wtGFP when excited in the near-UV wavelength region, although it had a much lower quantum efficiency. When excited at 488 nm, the GFPs were all more resistant to photobleaching than fluorescein. However, the wtGFP and alphaGFP photobleaching patterns showed initial increases in fluorescence emission caused by photoconversion of the protein chromophore. The wtGFP fluorescence decreased more quickly when excited at 395 nm than 488 nm, but it was still more photostable than the EBFP when excited at this wavelength. The wtGFP and alphaGFP were quite stable over a broad pH range, but fluorescence of the other variants decreased rapidly below pH 7. When expressed in bacteria, chromophore formation in wtGFP and S65T was found to be less efficient at 37 degrees C than at 28 degrees C, but the other three variants showed little differences between 37 degrees C and 28 degrees C. In conclusion, no single GFP variant is ideal for every application, but each one offers advantages and disadvantages for quantitative imaging in living cells.

Complete lack of transcription factor PDX-1 leads to pancreatic agenesis, whereas heterozygosity for PDX-1 mutations has been recently noted in some individuals with maturity-onset diabetes of the young (MODY) and in some individuals with type 2 diabetes. To determine how alterations in PDX-1 affect islet function, we examined insulin secretion and islet physiology in mice with one PDX-1 allele inactivated. PDX-1 ؉/؊ mice had a normal fasting blood glucose and pancreatic insulin content but had impaired glucose tolerance and secreted less insulin during glucose tolerance testing. The expression of PDX-1 and glucose transporter 2 in islets from PDX-1 ؉/؊ mice was reduced to 68 and 55%, respectively, whereas glucokinase expression was not significantly altered. NAD(P)H generation in response to glucose was reduced by 30% in PDX-1 ؉/؊ mice. The in situ perfused pancreas of PDX-1 ؉/؊ mice secreted about 45% less insulin when stimulated with 16.7 mM glucose. The K m for insulin release was similar in wild type and PDX-1 ؉/؊ mice. Insulin secretion in response to 20 mM arginine was unchanged; the response to 10 nM glucagonlike peptide-1 was slightly increased. However, insulin secretory responses to 10 mM 2-ketoisocaproate and 20 mM KCl were significantly reduced (by 61 and 66%, respectively). These results indicate that a modest reduction in PDX-1 impairs several events in glucose-stimulated insulin secretion (such as NAD(P)H generation, mitochondrial function, and/or mobilization of intracellular Ca 2؉ ) and that PDX-1 is important for normal function of adult pancreatic islets.The islet transcription factor PDX-1 (also known as IPF-1, IDX-1, and STF-1) was originally discovered as an activator of the insulin and somatostatin genes and from developmental work in the frog (1-9). Moreover, several laboratories have demonstrated that PDX-1 plays a key role in pancreatic development and that animals and humans lacking PDX-1 have pancreatic agenesis (1-3, 6 -10). PDX-1 also regulates transcription of other islet genes such as GLUT2, glucokinase (GK), 1 and islet amyloid polypeptide (IAPP) (1, 2, 9, 11-14). How PDX-1 functions in concert with other islet transcription factors and leads to normal pancreatic exocrine and islet development is unknown.Mutations in PDX-1 lead to abnormalities in islet function and diabetes in humans and mice (1,9,(15)(16)(17)(18)(19). Individuals who are heterozygous for the Pro63fsdelC mutation develop maturity-onset diabetes of the young (MODY4) and have impaired insulin secretion in response to glucose (19). This mutation is thought to reduce PDX-1 activity by creating an alternate internal translation start site that produces a dominant negative isoform of PDX-1 (20). Some recent reports indicate that mutations in PDX-1 may predispose individuals to late onset type 2 diabetes mellitus (15)(16)(17)21). Similarly in murine models both conventional and postnatal, cell-specific inactivation of one PDX-1 allele leads to impaired glucose tolerance (2,6,8,18). In addition, Edlund and co-workers (...

Two-photon excitation microscopy was used to image and quantify NAD(P)H autofluorescence from intact pancreatic islets under glucose stimulation. At maximal glucose stimulation, the rise in whole-cell NAD(P)H levels was estimated to be Ϸ30 M. However, because glucose-stimulated insulin secretion involves both glycolytic and Kreb's cycle metabolism, islets were cultured on extracellular matrix that promotes cell spreading and allows spatial resolution of the NAD(P)H signals from the cytoplasm and mitochondria. The metabolic responses in these two compartments are shown to be differentially stimulated by various nutrient applications. The glucose-stimulated increase of NAD(P)H fluorescence within the cytoplasmic domain is estimated to be Ϸ7 M. Likewise, the NAD(P)H increase of the mitochondrial domain is Ϸ60 M and is delayed with respect to the change in cytoplasmic NAD(P)H by Ϸ20 sec. The large mitochondrial change in glucose-stimulated NAD(P)H thus dominates the total signal but may depend on the smaller but more rapid cytoplasmic increase.G lucose-induced insulin secretion is coupled to the metabolic state of the ␤ cell. After transport into the cell, glucose is phosphorylated and shunted into glycolysis, which increases metabolic flux. This altered metabolic state, which can be monitored by NAD(P)H autofluorescence increase, leads to an increase in the ATP͞ADP ratio that closes the plasma membrane-associated ATP-sensitive potassium (K ATP ) channel. Closure of this channel depolarizes the membrane, leading to the activation of voltage-sensitive calcium (Ca 2ϩ ) channels, Ca 2ϩ influx, and insulin secretion (1).Glucose usage in stimulated pancreatic ␤ cells is principally glycolytic, with the polyol pathway, glycogen synthesis, and pentose phosphate pathway accounting for Ͻ10% of total usage (2). Thus, the glucose metabolic signal is derived from glycolysis in the cytoplasm and pyruvate metabolism in the mitochondria. The absence of stimulated insulin secretion with nonmetabolizable glucose derivatives (1) and the abolition of glucosestimulated insulin secretion in a pancreatic ␤ cell line lacking mitochondrial DNA (3) suggest roles for both cytoplasmic and mitochondrial metabolism in normal secretion. Additionally, both glycolytic intermediates, such as glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (2), and mitochondrial substrates, such as leucine (4) or methyl pyruvate (5), stimulate insulin secretion, which further supports a role for metabolism in both compartments in glucose signaling.Attempts to resolve the glycolytic and mitochondrial contributions to glucose-stimulated insulin secretion have relied on nutrient secretagogues that couple at various points into glycolysis or Kreb's cycle or on pharmacological inhibition at various points along each pathway (1, 6). Although various nutrient supplements indicate that couplings of the pathway can lead to insulin secretion, they seldom mimic the effects of glucose. For example, pyruvate potentiates glucose-stimulated secretion but does not cause s...

BackgroundCongenital human cytomegalovirus (HCMV) infections can result in CNS abnormalities in newborn babies including vision loss, mental retardation, motor deficits, seizures, and hearing loss. Brain pericytes play an essential role in the development and function of the blood–brain barrier yet their unique role in HCMV dissemination and neuropathlogy has not been reported.MethodsPrimary human brain vascular pericytes were exposed to a primary clinical isolate of HCMV designated ‘SBCMV’. Infectivity was analyzed by microscopy, immunofluorescence, Western blot, and qRT-PCR. Microarrays were performed to identify proinflammatory cytokines upregulated after SBCMV exposure, and the results validated by real-time quantitative polymerase chain reaction (qPCR) methodology. In situ cytokine expression of pericytes after exposure to HCMV was examined by ELISA and in vivo evidence of HCMV infection of brain pericytes was shown by dual-labeled immunohistochemistry.ResultsHCMV-infected human brain vascular pericytes as evidenced by several markers. Using a clinical isolate of HCMV (SBCMV), microscopy of infected pericytes showed virion production and typical cytomegalic cytopathology. This finding was confirmed by the expression of major immediate early and late virion proteins and by the presence of HCMV mRNA. Brain pericytes were fully permissive for CMV lytic replication after 72 to 96 hours in culture compared to human astrocytes or human brain microvascular endothelial cells (BMVEC). However, temporal transcriptional expression of pp65 virion protein after SBCMV infection was lower than that seen with the HCMV Towne laboratory strain. Using RT-PCR and dual-labeled immunofluorescence, proinflammatory cytokines CXCL8/IL-8, CXCL11/ITAC, and CCL5/Rantes were upregulated in SBCMV-infected cells, as were tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1beta), and interleukin-6 (IL-6). Pericytes exposed to SBCMV elicited higher levels of IL-6 compared to both mock-infected as well as heat-killed virus controls. A 6.6-fold induction of IL-6 and no induction TNF-alpha was observed in SBCMV-infected cell supernatants at 24 hours postinfection. Using archival brain tissue from a patient coinfected with HCMV and HIV, we also found evidence of HCMV infection of pericytes using dual-label immunohistochemistry, as monitored by NG2 proteoglycan staining.ConclusionHCMV lytic infection of primary human brain pericytes suggests that pericytes contribute to both virus dissemination in the CNS as well as neuroinflammation.

Long-chain fatty acids (FA) have been shown to regulate expression of the gene for the adipocyte FA-binding protein aP2. We examined whether this effect was exerted by FA themselves or by a FA metabolite. The a-bromo derivative of palmitate, an inhibitor of FA oxidation, was synthesized in the radioactive form, and its metabolism was investigated and correlated with its ability to induce aP2 in Ob1771 preadipocytes. a-Bromopalmitate was not utilized by preadipocytes. It was not cleared from the medium over a 24-hr period and was not incorporated into cellular lipids. Short incubations indicated that a-bromopalmitate exchanged across the preadipocyte membrane but remained in the free form inside the cell. In line with this, preadipocyte homogenates did not activate a-bromopalmitate to the acyl form. However, although it was not metabolized, bromopalmitate was much more potent than native FA in inducing aP2 gene expression. Induction exhibited the characteristics previously described for native FA, indicating that a similar if not identical mechanism was involved. The data indicated that induction of aP2 was exerted by unprocessed FA. Finally, in contrast to preadipocytes, adipocytes metabolized bromopalmitate. This reflected increased activity with cell differentiation of a palmitoyl-CoA synthase that could activate palmitate and bromopalmitate at about one-fifth the rate for palmitate. In preadipocytes, the predominant fattyacyl-CoA synthase, arachidonyl-CoA synthase, had very low affinity for both FA. Increased activity of the palmitoyl-CoA synthase, which has a wider substrate range, is likely to be important for initiation of lipid deposition.Fatty acids (FA) (10)(11)(12). In this report, we investigated whether the effect of long-chain FA to induce aP2 in Ob1771 was exerted by FA themselves or by FA metabolism. This information would be essential to elucidate the molecular mechanism of the FA effect. Furthermore, a similar regulatory effect of FA might apply to other genes induced during adipose differentiation.For example, in Ob1771 preadipocytes, FA also induced the gene for fatty-acyl-CoA synthase (EC 2.3.1.86) (8). In BFC-1 preadipocytes, an increase in membrane transport of FA was an early event during cell differentiation, which would be consistent with FA playing a role in modulating subsequent differentiation steps (9).Our results, using a-bromopalmitate, indicate that induction of aP2 in preadipocytes is exerted by unprocessed FA. The data also demonstrate increased expression, with cell differentiation, of a palmitoyl-CoA synthase that has a wide range of FA substrates (which include a-bromopalmitate) and that is likely to be important for the initiation of adipogenesis. MATERIALS AND METHODSMaterials. Guanidinium isothiocyanate and films for autoradiography (X-Omat, AR) were from Kodak. Cesium chloride was from Fisher Scientific. The multiprime labeling kit for cDNA, the [32P]dCTP, and the nitrocellulose filters were from Amersham. The cDNA for aP2 (13) was a gift from H. Green (Harvard...