Visual acuity and signal color pattern in an Anolis lizard.

Anolis lizards communicate with colorful dewlaps that often include detailed patterns. We measured the visual acuity of Anolis sagrei. Lizards viewed a checkerboard pattern of red and yellow-green squares that were too small to resolve, and thus appeared uniform in color. We quickly replaced the center portion of the display with a pattern of larger squares. If the new pattern could be resolved, the lizards perceived a change in color and reflexively shifted their gaze toward the target. The acuity threshold was 1.21 cycles deg-1 We also calculated acuity based on published anatomical data for Anolis carolinensis It was similar to that of A.sagrei for the visual periphery. Foveal acuity was 10 times greater. We approximated the effects of viewing conditions on the visibility of fine details of a conspecific's dewlap. For peripheral vision, no detailed patterns were visible at ≥0.5 m. For foveal vision, color-pattern details were visible at 1.0 m.

Circulating cytokines and monocyte subpopulations as biomarkers of outcome and biological activity in sunitinib-treated patients with advanced neuroendocrine tumours.

BACKGROUND: Sunitinib is approved worldwide for treatment of advanced pancreatic neuroendocrine tumours (pNET), but no validated markers exist to predict response. This analysis explored biomarkers associated with sunitinib activity and clinical benefit in patients with pNET and carcinoid tumours in a phase II study. METHODS: Plasma was assessed for vascular endothelial growth factor (VEGF)-A, soluble VEGF receptor (sVEGFR)-2, sVEGFR-3, interleukin (IL)-8 (n=105), and stromal cell-derived factor (SDF)-1α (n=28). Pre-treatment levels were compared between tumour types and correlated with response, progression-free (PFS), and overall survival (OS). Changes in circulating myelomonocytic and endothelial cells were also analysed. RESULTS: Stromal cell-derived factor-1α and sVEGFR-2 levels were higher in pNET than in carcinoid (P=0.003 and 0.041, respectively). High (above-median) baseline SDF-1α was associated with worse PFS, OS, and response in pNET, and high sVEGFR-2 with longer OS (P⩽0.05). For carcinoid, high IL-8, sVEGFR-3, and SDF-1α were associated with shorter PFS and OS, and high IL-8 and SDF-1α with worse response (P⩽0.05). Among circulating cell types, monocytes showed the largest on-treatment decrease, particularly CD14+ monocytes co-expressing VEGFR-1 or CXCR4. CONCLUSIONS: Interleukin-8, sVEGFR-3, and SDF-1α were identified as predictors of sunitinib clinical outcome. Putative pro-tumorigenic CXCR4+ and VEGFR-1+ monocytes represent novel candidate markers and biologically relevant targets explaining the activity of sunitinib.

F-actin aggregates in transformed cells.

Polymerized actin has been found aggregated into distinctive patches inside transformed cells in culture. The F-actin-specific fluorescent probe, nitrobenzoxadiazole-phallacidin, labels these F-actin aggregates near the ventral cell surface of cells transformed by RNA or DNA tumor viruses, or by chemical mutagens, or spontaneously. Their appearance in all eight transformed cell types studied suggests their ubiquity and involvement in transformation morphology. Actin patches developed in normal rat kidney (NRK) cells transformed by a temperature-sensitive mutant of Rous sarcoma virus (LA23-NRK) within 30 min after a shift from the nonpermissive (39 degrees C) to the permissive temperature (32 degrees C). Patch appearance paralleling viral src gene expression tends to implicate pp60src kinase activity in destabilizing the cytoskeleton. However, appearance of the actin aggregates in cells not transformed by retrovirus calls for alternative mechanisms, perhaps involving an endogenous kinase, for this apparently common trait.

Endothelial plasmalemmal vesicles have a characteristic striped bipolar surface structure.

Capillary endothelial cells have a large population of small (65-80 nm diameter in transmission electron microscopy) vesicles of which a large fraction is associated with the plasmalemma of the luminal and abluminal side. We studied the fine structure and distribution of these plasmalemmal vesicles by high resolution scanning electron microscopy in cultured endothelial cells obtained from bovine adrenal cortical capillaries. Cell monolayers were covered with polylysine-coated silicon chips, split in high potassium buffer, fixed in aldehyde mixtures, and then treated with OsO4 and thiocarbohydrazide. After critical point drying, the specimens were coated with a thin (less than 2 nm) continuous film of chromium. On the cytoplasmic aspect of the dorsal plasmalemmal fragments seen in such specimens, plasmalemmal vesicles appear as uniform vesicular protrusions approximately 70-90 nm in diameter, preferentially concentrated in distinct large fields in which they occur primarily as single units. Individual plasmalemmal vesicles exhibit a striped surface fine structure which consists of ridges approximately 10 nm in diameter, separated by furrows and oriented as meridians, often ending at two poles on opposite sides of the vesicles in a plane parallel to the plasmalemma. This striped surface structure is clearly distinct from the cage structure of coated pits found, at low surface density, on the same specimens. The cytoplasmic aspect of the plasmalemma proper is covered by a fibrillar infrastructure which does not extend over plasmalemmal vesicles but on which the latter appear to be anchored by fine filaments.

ATP-dependent phosphate transport in sarcoplasmic reticulum and reconstituted proteoliposomes.

During uptake of Ca2+ by rabbit sarcoplasmic reticulum, about 1 mumol of 32Pi was taken up per mumol 45Ca2+ transported. The uptake of Pi was dependent on external Ca2+, Mg2+ and ATP. Intravesicular Ca2+ did not substitute for external Ca2+. In contrast to the accumulation of Ca2+ which was abolished by the ionophore A23187, the uptake of Pi continued to take place provided sufficient Ca2+ was present in the medium. Thus, a Ca2+ gradient did not seem to be required. Similar observations were made with proteoliposomes reconstituted with membrane preparations of sarcoplasmic reticulum and soybean phospholipids. However, when purified Ca2+ -ATPase was used for reconstitution, there was ATP-dependent Ca2+ uptake but no ATP-dependent Pi transport was observed. These data show that the mechanism of Pi transport cannot be a passive movement in response to a Ca2+ gradient but appears to be catalyzed by a specific protein, which is inactivated during purification of the Ca2+ -ATPase. A protein that catalyzes Pi transport in reconstituted vesicles has been solubilized by extraction of sarcoplasmic reticulum with sodium cholate.

F-actin aggregates in transformed cells contain alpha-actinin and fimbrin but apparently lack tropomyosin.

Transformation-specific F-actin structures are examined in tumor cells after in vitro tumor cell growth alone or on an untransformed cell monolayer. In transformed cells F-actin aggregates near the ventral plasma membrane in close substrate adhesion areas contain the cytoskeletal proteins alpha-actinin and fimbrin but, unlike microfilament bundles, are not labeled with antibody against tropomyosin. By electron microscopy the dense ventral aggregates in transformed cells resemble stress fiber termini found at the membrane in normal cells. These transformed-cell cytoskeletal structures are not limited solely to substrate adhesion areas; they are also expressed at cell-cell contacts about 48 h after transformed cells are plated on untransformed cells. These specialized F-actin aggregates appear to be implicated in the processes of penetration of these transformed cells between adjoining untransformed cells in vitro.

Fluorescent erythrosin B is preferable to trypan blue as a vital exclusion dye for mammalian cells in monolayer culture.

Erythrosin B and trypan blue are tested and compared for their effectiveness as vital exclusion stains for mammalian cells in monolayer culture. Both stains are supposed to mark cells that have lost membrane integrity. Fluorescein diacetate (FDA), an efficient vital inclusion stain, is used as a control, as it marks cells retaining membrane integrity. Erythrosin B and FDA are used as fluorescent dyes, whereas trypan blue colors via light absorption. The effectiveness of both vital exclusion stains is assayed by their ability to stain a high percentage of monolayer cells exposed to treatments lethal to an entire cell population. Two types of lethal treatment, severe heat and metabolic poison, are employed. Erythrosin B stains all monolayer cells immediately after complete lethal treatment. Trypan blue optimally stains only about 60% of monolayer cells. Cell staining by erythrosin B and by FDA are found to be mutually exclusive. This result demonstrates the coincidence of viability indications by erythrosin B and FDA and thus confirms the reliability of both viability stains as they probe membrane permeability via independent mechanisms. This study shows that erythrosin B is an effective, nontoxic, and convenient fluorescent vital exclusion dye for three mammalian cell lines in monolayer culture, but tends to disqualify trypan blue for this application.

Fluorescence light microscopy of F-actin in retinal rods and glial cells.

The actin cytoskeleton of rod photoreceptors and glial cells in toad retina has been directly viewed using fluorescence microscopy of cells labeled with a potent phallotoxin that specifically binds to F-actin. The three-dimensional organization of this cytoskeletal protein consists of actin filaments, which course through the inner segment and end at the tips of the calycal processes surrounding the base of the outer segment. A transverse layer of actin staining is also observed at the base of rod outer segments in the region where new discs are formed. At the level of the external limiting membrane, evidence has been found for rings of actin within the glial cells that surround the photoreceptors. These actin rings form a structural meshwork in which photoreceptor cells are embedded.

In situ iodination of angiotensin-converting enzyme and other pulmonary endothelial membrane proteins.

Biochemical responses of endothelial cells in culture to pharmacological or physiological stimuli are often extrapolated to define the behavior of the vascular endothelium in vivo. However, culture conditions cannot recreate the environment of endothelial cells in vivo. To compare cell functions in vivo and in vitro, we iodinated endothelial membrane proteins of both the perfused rabbit lung and cultured rabbit lung endothelial cells. Endothelial cell protein 125I-labeling in the perfused intact lung was catalyzed by lactoperoxidase and glucose oxidase immobilized on 3-10 microns polyacrylamide beads (Enzymobeads, Bio-Rad). Changes in 5-hydroxytryptamine uptake, angiotensin converting enzyme activity and perfusion pressure made before, during and/or after iodination were small, suggesting that the procedure does not grossly injury the lung. As confirmed by tissue autoradiography, iodination was confined to the vascular space. A subcellular "membrane" fraction of the whole homogenate was enriched for several iodinated proteins. Lectin binding further purified a library of putative iodinated endothelial membranes proteins, one of which was angiotensin-converting enzyme as shown by immunoprecipitation with goat anti-rabbit antibody to angiotensin-converting enzyme. Iodinated proteins of similar molecular weights were also isolated from cultured rabbit lung endothelium iodinated under the same conditions, thus confirming the endothelial lineage of proteins iodinated in the intact lung. We conclude that this technique labels endothelial surface proteins in the intact lung without causing observable tissue injury and thus should be valuable in the study of the physiology and pathophysiology of the vascular lining in vivo.

Scatter factor stimulates migration of vascular endothelium and capillary-like tube formation.

Scatter factors (SFs) are heat- and trypsin-sensitive cytokines secreted by fibroblastic and vascular smooth muscle cell lines which stimulate motility of normal epithelium, carcinoma cells, and vascular endothelium. Human and mouse SFs have been purified and identified as 90 kD heterodimeric proteins consisting of heavy (58 kD) and light (31 kD) disulfide-bonded subunits. Partial amino acid sequence data from SF-derived tryptic peptides indicate marked sequence homology with hepatocyte growth factors, suggesting a common multigene family. In this chapter we describe the regulation by SF of vascular endothelial cell chemotaxis and chemokinesis; migration from microcarrier beads to flat surfaces; invasion through porous filters coated with reconstituted basement membrane; secretion of plasminogen activator; and in vitro capillary-like tube formation on a basement membrane surface.

Non-rotational teaching of obstetrics in a family practice residency.

The E. W. Sparrow Family Practice Residency Program has developed a unique system of training family practice residents in obstetrics. A continuous obstetrical training experience is provided on a non-rotational basis over the three-year residency training period. This experience has been arranged through the creation of the family practice obstetrical population, the use of family practice faculty as primary teachers, and the use of residents and faculty in obstetrics-glynecology as consultants. Extensive documentation and evaluation is used to allow residents to progress through varied levels of privileges in preparation for private practice.

Attachment of plasma membranes of cultured cells to silicon chips for high magnification imaging in scanning electron microscopy.

In the membrane preparation method described in this paper, a polylysine-coated silicon chip is adsorbed to the exposed apical surface of a cell monolayer. Upon removal, the adsorbed chip separates the plasmalemma from the residual bodies of the cultured cells. This sandwich-membrane separation approach simplifies access to the cytoplasmic aspects of both the apical and the basal plasmalemma which remains on the culture substrate and is covered to a varied extent by cytoplasmic infrastructures. To stabilize the attached membranes, small crosslinking agents are used in a controlled osmium impregnation. Large crosslinkers are avoided since they induce thickening of fine structures. Optimal conditions for attachment of plasmalemma of cultured adrenal endothelial cells to the cationic chip are defined. Effective cleaning procedures of the chips and useful molecular weights of polylysine are determined by quantitating colloidal gold adherence to the surface of the chips. The specimens are examined by high resolution scanning electron microscopy.

F-actin aggregates may activate transformed cell surfaces.

Observations on the role of transformation-specific F-actin aggregates [Carley et al, 1981] in altering morphology, adhesion and intercellular interaction in transformed cells are reported here. The appearance and disappearance of membrane- and substrate-associated F-actin aggregates (MAG and SAG, respectively) are followed in a cell line temperature-sensitive for transformation. Since MAG structures also appear near the membrane in suspension cultures of transformed cells and in transformed cells in coculture with untransformed cells, they appear to function at cell-cell contacts. Unlike microfilament bundles in untransformed cells, MAG and SAG do not contain the F-actin regulatory protein tropomyosin. The lack of tropomyosin in these structures near the membrane is reminiscent of areas of an exceptionally active actin cytoskeleton usually associated with motile processes of the normal cell membrane. Such areas of membrane-cytoskeletal interaction may be involved in the aberrant cell-cell communication as well as the aggressive behavior often seen in transformed cells.

Isolation, cultivation, and partial characterization of microvascular endothelium derived from human lung.

Primary cultures of peripheral lung lobes were grown in a highly supplemented medium. Human lung endothelial cells (HLE) were isolated from the mixed population by FACS. The cells proliferated rapidly and were serially cultivated for at least 16 passages. Both early and late passage cells were positive for the standard endothelial markers. Factor VIII related-antigen (Factor VIII R-Ag), angiotensin-converting enzyme, acetylated low-density lipoprotein labeled with 1,1'-dioctadecyl-1,3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI-Ac-LDL) uptake, and bound the lectin Ulex europaeus agglutinin (UEA). Prostaglandin E2 was the major cyclooxygenase product of HLE, in contrast to human umbilical vein endothelial cells (HUVE), which synthesized PGI2 in excess of PGE2. Factor VIII R-Ag exhibited a diffuse cytoplasmic as well as an extracellular fibrillar distribution in HLE, in contrast to a vesicular (Weibel-Palade body) cytoplasmic distribution in HUVE. The HUVE did demonstrate some extracellular fibrillar Factor VIII R-Ag as well. Urokinase was the predominant plasminogen activator (PA) secreted by HLE, whereas tissue PA was predominant in HUVE cultures. HLE formed tube-like structures within 2 h of plating on a Matrigel matrix whereas HUVE formed larger tube-like structures only after 1 or more days. The properties described here indicate that human lung microvessel endothelium can be isolated and continuously grown from small tissue segments and express a number of properties that differ from those of HUVE. These studies provide further support for the concept that endothelial cells from different sources can exhibit considerable heterogeneity relating to their phenotypic and biochemical properties.

Endotoxin-induced exposure of pulmonary glycoproteins in an intact animal.

A method has been developed to radioiodinate luminally disposed endothelial proteins in an in situ perfused lung system without causing obvious vascular changes. The spectrum of endothelial cell proteins labeled in control animals and those treated with endotoxin for 45 min were compared. No changes in gross tissue morphology or in the distribution of radiolabel (125I-s-SHPP) were detected in control or treated lungs. Lectin affinity purification was applied to a lung membrane fraction to isolate labeled proteins, which were in turn resolved by gel electrophoresis and autoradiography. Comparisons of gel autoradiographs from control and treated lungs identified eight glycoproteins, the labeling of which was enhanced in endotoxin-treated animals. A similar lectin affinity analysis of radiolabeled effluent blood cells from the lungs identified only two proteins, neither of which were consistently changed by endotoxin pretreatment. A glycoprotein response can, therefore, be measured at the pulmonary endothelial surface on endotoxin administration to the whole animal without causing obvious lung injury.

Specific albumin binding to microvascular endothelium in culture.

The specific binding of rat serum albumin (RSA) to confluent microvascular endothelial cells in culture derived from the vasculature of the rat epididymal fat pad was studied at 4 degrees C by radioassay and immunocytochemistry. Radioiodinated RSA (125I-RSA) binding to the cells reached equilibrium at approximately 20 min incubation. Albumin binding was a slowly saturating function over concentrations ranging from 0.01 to 50 mg/ml. Specific RSA binding with a moderate apparent affinity constant of 1.0 mg/ml and with a maximum binding concentration of 90 ng/cm2 was immunolocalized with anti-RSA antibody to the outer (free) side of the endothelium. Scatchard analysis of the binding yielded a nonlinear binding curve with a concave-upward shape. Dissociation rate analysis supports negative cooperativity of albumin binding, but multiple binding sites may also be present. Albumin binding fulfilled many requirements for ligand specificity including saturability, reversibility, competibility, and dependence on both cell type and cell number. The results are discussed in terms of past in situ investigations on the localization of albumin binding to vascular endothelium and its effect on transendothelial molecular transport.

Albumin interacts specifically with a 60-kDa microvascular endothelial glycoprotein.

Confluent monolayers of microvascular endothelial cells, derived from the rat epididymal fat pad and grown in culture, were radioiodinated by using the lactoper-oxidase method. Their radioiodinated surface polypeptides were detected by NaDodSO4/PAGE (followed by autoradiography) and were characterized by both lectin affinity chromatography and protease digestion to identify the proteins involved in albumin binding. All detected polypeptides were sensitive to Pronase digestion, whereas several polypeptides were resistant to trypsin. Pronase treatment of the cell monolayer significantly reduced the specific binding of radioiodinated rat serum albumin, but trypsin digestion did not. Limax flavus, Ricinus communis, and Triticum vulgaris agglutinins competed significantly with radioiodinated rat serum albumin binding, whereas other lectins did not. A single 60-kDa glyco-protein was precipitated in common by these three lectins and was trypsin-resistant and Pronase-sensitive. Rat serum albumin affinity chromatography columns weakly but specifically bound a 60-kDa polypeptide from cell lysates derived from radioiodinated cell monolayers. These findings indicate that the 60-kDa glycoprotein is directly involved in a specific interaction of albumin with the cultured microvascular endothelial cells used in these experiments.

Extracellular matrix specificity for the differentiation of capillary endothelial cells.

Capillary endothelial cells in a fenestrated vasculature contain openings in attenuated areas of the cytoplasm which are often covered with one or two diaphragms. However, due to endothelial cell dedifferentiation upon culturing, such indicative membrane structure are often not expressed. The expression of a more differentiated phenotype can be regulated by the extracellular matrix to which the cells attach. In addition, during angiogenesis endothelial cell migration enables their interaction with other cell types and matrices which may directly affect endothelial cell growth and function. We report the ability to modify the expression of diaphragmed fenestrations and transcapillary channels in capillary endothelial cells by specific extracellular matrices. When the number of membrane openings is measured, growth of the cells on Madin--Darby canine kidney cell matrix results in the greatest number while other matrices or isolated matrix components are only partially active. Production of such a biologically active matrix not only allows an avenue to identify fenestrae-associated proteins but also provides an easy culture method to more closely mimic the in vivo phenotype of endothelial cells.