Density-enhanced phosphatase 1 regulates phosphorylation of tight junction proteins and enhances barrier function of epithelial cells.

Cell-cell adhesion is a dynamic process that can activate multiple signaling pathways. These signaling pathways can be regulated through reversible tyrosine phosphorylation events. The level of tyrosine phosphorylation of junctional proteins reflects the balance between protein-tyrosine kinase and protein-tyrosine phosphatase activity.

VHL gene mutations in renal cell carcinoma: role as a biomarker of disease outcome and drug efficacy.

The therapeutic landscape for renal cell carcinoma (RCC) has changed drastically over the past several years with the emergence of molecularly targeted therapies. With previous prognostic and predictive tools based on studies of patients treated with cytokine therapies, confirmation of these prior methods and discovery of new markers in this new era of targeted therapy are of great importance. Alteration of the von Hippel-Lindau gene (VHL) by mutation, loss of heterozygosity, and promoter methylation has been found to be important to RCC pathogenesis.

Qualitative human immunodeficiency virus RNA analysis of dried blood spots for diagnosis of infections in infants.

The Gen-Probe Aptima human immunodeficiency virus type 1 (HIV-1) RNA assay was adapted for the diagnosis of HIV infection in infants by using dried blood spots. The assay was 99% sensitive (128/129) and 100% specific (162/162). This may prove useful in resource-limited settings, since it precludes the need for a phlebotomist and maintenance of a cold chain.

Inactivation of gadd45a sensitizes epithelial cancer cells to ionizing radiation in vivo resulting in prolonged survival.

Ionizing radiation (IR) therapy is one of the most commonly used treatments for cancer patients. The responses of tumor cells to IR are often tissue specific and depend on pathway aberrations present in the tumor. Identifying molecules and mechanisms that sensitize tumor cells to IR provides new potential therapeutic strategies for cancer treatment.

Digital differential interference contrast autofocus for high-resolution oil-immersion microscopy.

Continued advances in cellular fluorescent biosensors enable studying intracellular protein dynamics in individual, living cells. Autofocus is valuable in such studies to compensate for temperature drift, uneven substrate over multiple fields of view, and cell growth during long-term high-resolution time-lapse studies of hours to days. Observing cellular dynamics with the highest possible resolution and sensitivity motivates the use of high numerical aperture (NA) oil-immersion objectives, and control of fluorescence exposure to minimize phototoxicity.

Upregulation of the TLR3 pathway by Kaposi's sarcoma-associated herpesvirus during primary infection.

Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with several different human malignancies, including Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. KSHV establishes lifelong latency in the host and modulates the host immune response. Innate immunity is critical for controlling de novo viral infection.

Design and optimization of genetically encoded fluorescent biosensors: GTPase biosensors.

This chapter details the design and optimization of biosensors based on a design used successfully to study nucleotide loading of small GTPase proteins in living cells. This design can be generalized to study many other protein activities, using a single, genetically encoded chain incorporating the protein to be studied, an "affinity reagent" which binds only to the activated form of the targeted protein, and mutants of the green fluorescent protein (GFP) that undergo fluorescence resonance energy transfer (FRET). Specific topics include procedures and caveats in the design and cloning of single-chain FRET sensors, in vitro and in vivo validation, expression in living cell systems for biological studies, and some general considerations in quantitative fluorescence imaging.

Development of a fluorescence-based assay to screen antiviral drugs against Kaposi's sarcoma associated herpesvirus.

Tumors associated with Kaposi's sarcoma-associated herpesvirus infection include Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. Virtually all of the tumor cells in these cancers are latently infected and dependent on the virus for survival. Latent viral proteins maintain the viral genome and are required for tumorigenesis.

Grading of follicular lymphoma: comparison of routine histology with immunohistochemistry.

Context: Follicular lymphoma (FL) grading is based on the average number of large transformed cells in 10 neoplastic follicles at x40 high-power field (x10-40 high-power field) examination (grade 1, 0-5 centroblasts per high-power field; grade 2, 6-15 centroblasts per high-power field; grade 3, >15 centroblasts per high-power field).

Objective: Since there may be significant interobserver variability, we analyzed the usefulness of immunohistochemical stains in grading FLs more reliably.

Design: Forty-three FLs initially graded by World Health Organization criteria (grade 1, 12; grade 2, 18; grade 3, 13) were reviewed and stained with CD3, CD20, Ki-67, CD30, CD68, PAX-5, and BCL-6.

Merocyanine dyes with improved photostability.

Merocyanine dyes have proven valuable for live cell fluorescence imaging applications, but many structures have been limited by rapid photobleaching. We show that photostability is substantially enhanced for merocyanines having a cyano group at a specific position in the central polymethine chain. Evidence is presented that this is due to reduction in reactivity of the dyes with singlet oxygen.

Simple one-pot preparation of water-soluble, cysteine-reactive cyanine and merocyanine dyes for biological imaging.

A simple one-pot-procedure for preparation of protein-reactive, water-soluble merocyanine and cyanine dyes has been developed. The 1-(3-ammoniopropyl)-2,3,3-trimethyl-3H-indolium-5-sulfonate bromide (1) was used as a common starting intermediate. The method allows easy preparation of dyes with chloro- and iodoacetamide side chains for covalent attachment to cysteine.

Independent evolution of human immunodeficiency virus type 1 env V1/V2 and V4/V5 hypervariable regions during chronic infection.

Using DNA heteroduplex tracking assays, we characterized human immunodeficiency virus type 1 env V4/V5 genetic populations in multiple blood plasma samples collected over an average of 7 months from 24 chronically infected human subjects. We observed complex and dynamic V4/V5 genetic populations in most subjects. Comparisons of V4/V5 and V1/V2 population changes over the course of the study showed that major shifts in genetic populations frequently occurred in one region but not the other, and these observations were independently confirmed in one subject by single-genome sequencing.

Spatiotemporal dynamics of RhoA activity in migrating cells.

Rho family GTPases regulate the actin and adhesion dynamics that control cell migration. Current models postulate that Rac promotes membrane protrusion at the leading edge and that RhoA regulates contractility in the cell body. However, there is evidence that RhoA also regulates membrane protrusion.

Regulation of cell adhesion by protein-tyrosine phosphatases. I. Cell-matrix adhesion.

Protein-tyrosine phosphatases are key regulators of protein tyrosine phosphorylation. More than merely terminating the pathways initiated by protein-tyrosine kinases, phosphatases are active participants in many signaling pathways. Signals involving tyrosine phosphorylation are frequently generated in response to cell-matrix adhesion.

Regulation of cell adhesion by protein-tyrosine phosphatases: II. Cell-cell adhesion.

Cell-cell adhesion is critical to the development and maintenance of multicellular organisms. The stability of many adhesions is regulated by protein tyrosine phosphorylation of cell adhesion molecules and their associated components, with high levels of phosphorylation promoting disassembly. The level of tyrosine phosphorylation reflects the balance between protein-tyrosine kinase and protein-tyrosine phosphatase activity.

Compartmentalized human immunodeficiency virus type 1 present in cerebrospinal fluid is produced by short-lived cells.

Human immunodeficiency virus type 1 (HIV-1) invades the central nervous system (CNS) during primary infection and persists in this compartment by unknown mechanisms over the course of infection. In this study, we examined viral population dynamics in four asymptomatic subjects commencing antiretroviral therapy to characterize cellular sources of HIV-1 in the CNS. The inability to monitor viruses directly in the brain poses a major challenge in studying HIV-1 dynamics in the CNS.

Rap1 GTPase inhibits leukocyte transmigration by promoting endothelial barrier function.

The passage of leukocytes out of the blood circulation and into tissues is necessary for the normal inflammatory response, but it also occurs inappropriately in many pathological situations. This process is limited by the barrier presented by the junctions between adjacent endothelial cells that line blood vessels. Here we show that activation of the Rap1 GTPase in endothelial cells accelerated de novo assembly of endothelial cell-cell junctions and increased the barrier function of endothelial monolayers.

Trading spaces: Rap, Rac, and Rho as architects of transendothelial migration.

Purpose Of Review: This review focuses on recent developments in understanding regulation of leukocyte transendothelial migration by small GTPase signaling.

Recent Findings: New studies are refining the model for GTPase regulation of leukocyte-endothelial cell interactions that occur during leukocyte transmigration. An emerging theme is that the endothelial cell is an active participant in this process; an example of this is the identification of a novel leukocyte docking structure.

The differential impact of p16(INK4a) or p19(ARF) deficiency on cell growth and tumorigenesis.

Mounting genetic evidence suggests that each product of the Ink4a/Arf locus, p16(INK4a) and p19(ARF), possesses tumor-suppressor activity (Kamijo et al., 1997; Krimpenfort et al., 2001; Sharpless et al.

A genetic approach to inactivating chemokine receptors using a modified viral protein.

We have developed a genetic system, called degrakine, that specifically and stably inactivates chemokine receptors (CKR) by redirecting the ability of the HIV-1 protein, Vpu, to degrade CD4 in the endoplasmic reticulum (ER) via the host proteasome machinery. To harness Vpu's proteolytic targeting capability to degrade new receptors, we fused a chemokine with the C terminal region of Vpu. The fusion protein, or degrakine, accumulates in the ER, trapping and functionally inactivating its target CKR.

Information perspectives of the Haseman-Elston method.

The Haseman-Elston regression approach can be viewed as a special case in a larger framework in which independent sibling pairs are ascertained, perhaps based on their phenotypes. Using likelihood arguments in an idealized setting, expressions are obtained for the inherent linkage information contained in the phenotype distributions. The results provide insight and analytic results that apply to general phenotype distributions and can be used to judge the effects of various data transformations and selective sampling of extreme phenotypes.

The persistence of senescence.

Senescence is a potent anticancer mechanism, representing a barrier that most, if not all, would-be tumor cells must traverse on their path to malignant transformation. In this Perspective, I discuss two recent publications (1, 2) that deal with the durability of senescence. These findings are of interest not only to those who study aging, but to those who study cancer as well.