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Biomedical subjects

R H Kramer

Publications and source records attributed to R H Kramer.

At least 109 records · Page 6Linked to original sources
PubMed1985

Basement membrane and connective tissue proteins in early lesions of Kaposi's sarcoma associated with AIDS.

Nearly one-third of all young homosexual men diagnosed as having acquired immune-deficiency syndrome (AIDS) develop a disseminated form of dermal Kaposi's sarcoma (KS). Although the histogenesis of KS cells is unclear, certain evidence suggests that the aberrant cells are of endothelial derivation. We have examined the presence and distribution of connective tissue-specific and basement membrane-specific macromolecules by indirect immunofluorescence and immunoperoxidase staining of frozen sections in early cutaneous lesions of KS from individuals with AIDS. The KS cells typically line the spaces between collagen bundles of the reticular dermis. When stained for the connective tissue-specific glycoprotein fibronectin, all Kaposi's sarcoma lesions showed an intense staining pattern, revealing a complex array of linear deposits of antigen that outlined the exterior surface of the collagen bundles. Antibodies to laminin and type IV collagen, both basement membrane-specific macromolecules, produced an intense staining pattern similar to that found with the anti-fibronectin antiserum, indicating that all 3 antigens are closely codistributed. In contrast, antibodies to type I collagen, the major collagen of the dermis, uniformly stained the collagen bundles in the KS lesions and in the normal control skin. Antiserum to factor VIII-associated antigen, an antigen specific to blood vascular endothelium, frequently stained the KS lesions but the staining pattern was diffuse and of variable intensity. The results suggest that KS cells are derived from the endothelium of the blood microvasculature and maintain their secretory phenotype of secreting basement membrane-specific macromolecules.

Acquired Immunodeficiency Syndrome↗
PubMed1985

Distribution of fibronectin and laminin in oral leukoplakia and carcinoma.

We determined the distribution of 2 components of the extracellular matrix, fibronectin and laminin, in normal, premalignant, and malignant oral mucosa. Both indirect immunofluorescence and avidin-biotin complex immunoperoxidase techniques were used with antisera to each glycoprotein. Fibronectin was consistently present in the basement membrane and connective tissue of normal, leukoplakic and malignant oral mucosa regardless of anatomic site. A continuous laminin-reactive basement membrane structure separated tumor parenchyma from the associated stroma except in areas of invasion in which no laminin staining was evident.

Carcinoma, Squamous Cell↗
PubMed1985

Calcium-dependent inward current in Aplysia bursting pace-maker neurones.

Depolarizing voltage-clamp pulses elicit a triphasic series of tail currents (phase I, II and III) in Aplysia burst-firing neurones L2-L6. The sequence and time course of the tail currents resemble slow changes in membrane potential which follow bursts in the unclamped cell. The phase II tail current is an inward current with a time course similar to that of the depolarizing after-potential (d.a.p.) which follows bursts in the unclamped cell. The phase II tail current is suppressed by depolarizing pulses which approach ECa, is blocked by Ca2+ current antagonists (Co2+ and Mn2+), and is blocked by intracellular injection of EGTA. The phase II tail current is not blocked by agents which block Na+-dependent action potentials, the Na+-Ca2+ exchange pump, or the Na+-K+ exchange pump. The phase II tail current is not blocked by the elimination of large outward K+ currents which can lead to extracellular K+ accumulation. Thus, the phase II tail current is not generated by any of these processes. The phase II tail current is reduced by about 60% following substitution of tetramethylammonium (TMA+) for external Na+, but is unaffected by reducing external Cl-. The phase II tail current is distinct from a persistent inward Ca2+ current which underlies the negative resistance region of the steady-state current--voltage relation of bursting cells. The persistent inward current is only slightly reduced by TMA+ substitution for Na+, and is enhanced by EGTA injection. Injection of Ca2+ into Aplysia bursting cells elicits a biphasic (inward-outward) current. The inward current can be observed in isolation after blocking the outward component (Ca2+-activated K+ current) with 50 mM-external tetraethylammonium. The Ca2+-elicited inward current has a reversal potential near -22 mV, and is non-selective for Na+, K+ and Ca2+. The reversal potential is unaffected by changes in Cl- and pH. The Ca2+- activated conductance is apparently voltage independent. We propose that the phase II tail current, and hence the d.a.p., is due to the Ca2+-dependent activation of a voltage-independent non-specific cationic conductance. This conductance participates in generating the depolarizing phase of bursting pace-maker activity.

Action Potentials↗
PubMed1985

Calcium-induced inactivation of calcium current causes the inter-burst hyperpolarization of Aplysia bursting neurones.

A triphasic series of tail currents which follow depolarizing voltage-clamp pulses in Aplysia neurones L2-L6 was described in the preceding paper (Kramer & Zucker, 1985). In this paper, we examine the nature of the late outward component of the tail current (phase III) which generates the inter-burst hyperpolarization in unclamped cells. The phase III tail current does not reverse between -30 and -90 mV, and is relatively insensitive to the external K+ concentration. In contrast, Ca2+-dependent K+ current (IK(Ca)), elicited by intracellular Ca2+ injection, reverses near -65 mV, and the reversal potential is sensitive to the external K+ concentration. Addition of 50 mM-tetraethylammonium (TEA) to the bathing medium causes a small increase in the phase III tail current. In contrast, IK(Ca) is completely blocked by addition of 50 mM-TEA. The phase III tail current is suppressed by depolarizing pulses which approach ECa, is blocked by Ca2+ current antagonists (Co2+ and Mn2+), and is blocked by intracellular injection of EGTA. The phase III tail current is reduced by less than 10% after complete removal of extracellular Na+. These bursting neurones have a voltage-dependent Ca2+ conductance which exhibits steady-state activation at a membrane potential similar to the average resting potential of the unclamped cell (i.e. -40 mV). The steady-state Ca2+ conductance can be inactivated by Ca2+ injection, or by depolarizing pre-pulses which generate a large influx of Ca2+. The steady-state Ca2+ conductance has a voltage dependence similar to that of the phase III tail current. The Ca2+-dependent inactivation of the steady-state Ca2+ conductance occurs in parallel with the phase III tail current; both have a similar sensitivity to Ca2+ influx, and both processes decay with similar rates after a depolarizing pulse. Hence, we propose that the phase III tail current is due to the Ca2+- dependent inactivation of a steady-state Ca2+ conductance. The decay of IK(Ca) following simulated spikes or bursts of spikes is rapid (less than 1 s) compared to the time course of the phase III tail current and the inter-burst hyperpolarization (tens of seconds). Thus, we conclude that IK(Ca) does not have a major role in terminating bursts or generating the inter-burst hyperpolarization in these cells. We present a qualitative model of the ionic basis of the bursting pace-maker cycle. The central features of the model are the voltage-dependent activation and the Ca2+-dependent inactivation of a Ca2+ current.

Action Potentials↗
PubMed1985

Extracellular matrix interactions with the apical surface of vascular endothelial cells.

Cultured aortic endothelial cells, like their in vivo counterparts, form highly organized, confluent monolayers of polarized epithelioid cells that secrete, exclusively at their basal surface, an extracellular matrix to which they then attach. The influence of isolated subendothelial matrix preparations on cell polarity and monolayer organization was studied by presenting fragments of the matrix to confluent bovine aortic endothelial cell cultures. The matrix particles were immediately bound to the apical aspect of the cell monolayer and induced rapid reorganization of the monolayer into cells with a fibroblastoid morphology. To determine if fibronectin, the major glycoprotein of the subendothelial matrix, could be involved in the observed apical cell surface-matrix interactions, latex beads or small discs of Nucleopore filters were coated with the glycoprotein and presented to confluent monolayers. In a fashion similar to that observed with matrix fragments, materials coated with fibronectin caused focal reorganization of the cell layer. After contact with the coated beads, the underlying endothelial cells flowed upward and spread over the entire bead, forming a canopy of confluent cells that draped the particle. Contact of confluent monolayers with the coated filters induced similar behaviour, except that monolayer reorganization into the fibroblastoid phenotype was followed by emigration of the majority of underlying cells through the pores to the upper filter surface, where they formed a new organized cell monolayer with the typical endothelial cell morphology. Thus contact of the apical surface of endothelial cells with structures to which they adhere initiates a rapid disruption of the organized cell monolayer, followed immediately by a concerted effort of the local population to re-establish both cell polarity and monolayer contiguity. The expression of this behaviour may be important during tissue remodeling that occurs in neovascularization and during interactions with thromboemboli.

Animals↗
PubMed1984

Basal lamina formation by cultured microvascular endothelial cells.

The production of a basal lamina by microvascular endothelial cells (MEC) cultured on various substrata was examined. MEC were isolated from human dermis and plated on plastic dishes coated with fibronectin, or cell-free extracellular matrices elaborated by fibroblasts, smooth muscle cells, corneal endothelial cells, or PF HR9 endodermal cells. Examination of cultures by electron microscopy at selected intervals after plating revealed that on most substrates the MEC produced an extracellular matrix at the basal surface that was discontinuous, multilayered, and polymorphous. Immunocytochemical studies demonstrated that the MEC synthesize and deposit both type IV collagen and laminin into the subendothelial matrix. When cultured on matrices produced by the PF HR9 endodermal cells MEC deposit a subendothelial matrix that was present as a uniform sheet which usually exhibited lamina rara- and lamina densa-like regions. The results indicate that under the appropriate conditions, human MEC elaborate a basal lamina-like matrix that is ultrastructurally similar to basal lamina formed in vivo, which suggests that this experimental system may be a useful model for studies of basal lamina formation and metabolism.

Animals↗
PubMed1984

Selective degradation of basement membrane macromolecules by metastatic melanoma cells.

The extracellular matrix deposited in culture by the mouse endodermal cell line (PF HR9) was used as an experimental model to study the interactions between basement membranes and several tumorigenic and nontumorigenic cell lines, including the metastatic B16 melanoma cell sublines. Analysis by biochemical and immunologic methods indicated that the isolated HR9 extracellular matrix was composed of laminin, type IV collagen, entactin, and heparan sulfate proteoglycans--all basement membrane-specific macromolecules. Ultrastructurally, the extracellular matrix elaborated by the HR9 cells appears as a meshwork of finely fibrillar material. B16 melanoma cells were found to adhere rapidly to the isolated HR9 matrix, but significant penetration into the structure was not observed. However, the melanoma cells did induce an extensive solubilization of 35S-sulfate-labeled heparan sulfate to fragments that were approximately one-third the size of the intact heparan sulfate chains. Only the B16 melanoma sublines exhibited this unique pattern of proteoglycan degradation, which suggests that they possess an endoglycosidase specific for heparan sulfate chains. B16 melanoma cells seeded onto HR9 matrices labeled with [1H]leucine or [3H]proline caused no detectable increase in matrix protein solubilization compared to controls. The results indicate that the metastatic tumor cells degrade the proteoglycans in the HR9 matrix more readily than the other matrix proteins.

Basement Membrane↗
PubMed1980

Metastatic tumor cells adhere preferentially to the extracellular matrix underlying vascular endothelial cells.

Two metastatic cell lines, mouse B16-Fl melanoma and human Hs939 melanoma, were examined for their abilities to adhere to confluent vascular endothelial cell monolayers and to the underlying endothelial extracellular matrix. Tumor cells attacked slowly to the endothelial cell monolayers while they adhered rapidly to isolated extracellular matrix. When analyzed by polyacrylamide gel electrophoresis in sodium dodecylsulfate solutions, the extracellular matrix was shown to be primarily composed of a protein of identical migration and molecular weight to fibronectin. Tumor-cell adhesion to fibronectin-coated polyvinyl surfaces mimicked the rapid rate of attachment of tumor cells to extracellular matrix, and tumor cells adherent to either extracellular matrix or fibronectin-coated polyvinyl dishes adopted an unusual, highly spread and flattened morphology with numerous small projections. These results suggest that fibronectin associated with the endothelial basement membrane may be, in part, responsible for establishing an adhesive gradient that could be important in malignant cell extravasation.

Animals↗
PubMed1979

The surface glycoproteins of the HeLa cell. Internalization of wheat germ agglutinin-receptors.

The sensitivity of 125I-labeled sialoglycoproteins to neuraminidase digestion was used to monitor the loss of specific membrane glycoproteins from the cell surface in to the cytoplasmic compartment during lectin-mediated endocytosis. These studies demonstrated that a major portion of the surface glycoproteins had undergone internalization concurrently with wheat germ agglutinin in a time- and temperature-dependent process. The internalized 125I-labeled glycoproteins were associated with the small vesicle fraction and were present in the same relative proportion as they existed in the plasma membrane isolated from control untreated cells. Many of the 125I-labeled membrane proteins were shown to be receptors and were isolated after affinity chromatography of the solubilized plasma membranes on wheat germ agglutinin-agarose columns.

Agglutinins↗
PubMed1979

Interactions of tumor cells with vascular endothelial cell monolayers: a model for metastatic invasion.

The interactions of tumorigenic and nontumorigenic human and rodent cells with vascular endothelial cells and their underlying extracellular matrix were studied in culture. The abilities of various cells to attach to endothelial monolayers and cause morphologic changes, such as rupture of endothelial-endothelial cell interactions leading to retraction of endothelial cells and exposure of extracellular matrix, as well as their propensities to invade and underlap retracted endothelial monolayers and continue migration were assessed by time-lapse and phase-contrast microscopy as well as scanning and transmission electron microscopy. In general, highly malignant or highly invasive cells in vivo were capable of attachment, invasion, and migration under endothelial cells in vitro. This system may be useful for elucidating mechanisms of tumor cell arrest and extravasation.

Animals↗
PubMed1978

Isolation and characterization of surface glycoproteins from L-1210, P-388 and HeLa cells.

A method is described that permits the rapid extraction of the cell surface glycoproteins of two murine leukemic cells, the P-388 and the L-1210 cells as well as those of the human adenocarcinoma cells, the HeLa cells. Proof of the surface location of these glycoproteins is provided by labeling the intact cells; (a) with 125I by the lactoperoxidase iodination technique; (b) with 3H by the galactose oxidase-reductive tritiation method. Most of these glycoproteins were also shown to incorporate radioactive glucosamine and fucose. By these criteria as well as by the distribution of molecular weights, the surface glycoproteins of the two murine cells are indistinguishable; however, they differ markedly from the surface glycoproteins of HeLa cells. The extracts of the murine cells were shown to contain lectin receptor activity as determined by their ability to inhibit the lectin-induced agglutination of the intact cells.

Agglutination↗
PubMedDate not supplied

Regulation of cGMP-dependent current in On bipolar cells by calcium/calmodulin-dependent kinase.

The metabotropic receptor mGluR6 is localized to the dendrites of On bipolar cells and mediates synaptic input from photoreceptors. The binding of glutamate to the receptor activates a phosphodiesterase (PDE), which then hydrolyzes cGMP. A nonselective cationic conductance, believed to be gated directly by cGMP, is turned off as a result of the fall in cGMP levels, and the cell hyperpolarizes. Here we present evidence for regulation of the conductance by an additional mechanism that it is independent of cGMP. Whole-cell recordings were obtained from On bipolar cells in slices of tiger salamander retina. Dialysis of cells with 1 microM KN-62 or 10 microM KN-93, two inhibitors of type II calmodulin-dependent protein kinase (CaMKII), depressed cGMP-dependent currents. This depression persisted when hydrolysis of cGMP was prevented with IBMX, a broad-spectrum PDE inhibitor, suggesting that CaMKII acts downstream from the PDE in the cascade. The depression of cGMP-dependent currents was probably not due to a direct interaction of the inhibitors with the channels as neither 1 microM KN-62 or 10 microM KN-93 was found to have any effect on cyclic nucleotide-gated channels when applied directly to excised patches of rod outer segments. We propose that phosphorylation by CaMKII may be an important mechanism for regulating the cGMP-dependent conductance of On bipolar cells.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗