Characterization of root agravitropism induced by genetic, chemical, and developmental constraints

The patterns and rates of organelle redistribution in columella (i.e., putative statocyte) cells of agravitropic agt mutants of Zea mays are not significantly different from those of columella cells in graviresponsive roots. Graviresponsive roots of Z. mays are characterized by a strongly polar movement of 45Ca2+ across the root tip from the upper to the lower side. Horizontally-oriented roots of agt mutants exhibit only a minimal polar transport of 45Ca2+. Exogenously-induced asymmetries of Ca result in curvature of agt roots toward the Ca source. A similar curvature can be induced by a Ca asymmetry in normally nongraviresponsive (i.e., lateral) roots of Phaseolus vulgaris. Similarly, root curvature can be induced by placing the roots perpendicular to an electric field. This electrotropism increased with 1) currents between 8-35 mA, and 2) time between 1-9 hr when the current is constant. Electrotropism is reduced significantly by treating roots with triiodobenzoic acid (TIBA), an inhibitor of auxin transport. These results suggest that 1) if graviperception occurs via the sedimentation of amyloplasts in columella cells, then nongraviresponsive roots apparently sense gravity as do graviresponsive roots, 2) exogenously-induced asymmetries of a gravitropic effector (i.e., Ca) can induce curvature of normally nongraviresponsive roots, 3) the gravity-induced downward movement of exogenously-applied 45Ca2+ across tips of graviresponsive roots does not occur in nongraviresponsive roots, 4) placing roots in an electrical field (i.e., one favoring the movement of ions such as Ca2+) induces root curvature, and 5) electrically-induced curvature is apparently dependent on auxin transport. These results are discussed relative to a model to account for the lack of graviresponsiveness by these roots.     

Plasminogen activator and collagenase production by cultured capillary endothelial cells

Cultured bovine capillary endothelial (BCE) cells produce low levels of collagenolytic activity and significant amounts of the serine protease plasminogen activator (PA). When grown in the presence of nanomolar quantities of the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), BCE cells produced 5-15 times more collagenolytic activity and 2-10 times more PA than untreated cells. The enhanced production of these enzymes was dependent on the dose of TPA used, with maximal response at 10(-7) to 10(-8) M. Phorbol didecanoate (PDD), an analog of TPA which is an active tumor promoter, also increased protease production. 4-O-methyl-TPA and 4α-PDD, two analogs of TPA which are inactive as tumor promoters, had no effect on protease production. Increased PA and collagenase activities were detected within 7.5 and 19 h, respectively, after the addition of TPA. The TPA-stimulated BCE cells synthesized a urokinase-type PA and a typical vertebrate collagenase. BCE cells were compared with bovine aortic endothelial (BAE) cells and bovine embryonic skin (BES) fibroblasts with respect to their production of protease in response to TPA. Under normal growth conditions, low levels of collagenolyic activity were detected in the culture fluids from BCE, BAE, and BES cells. BCE cells produced 5-13 times the basal levels of collagenolytic activity in response to TPA, whereas BAE cells and BES fibroblasts showed a minimal response to TPA. Both BCE and BAE cells exhibited relatively high basal levels of PA, the production of which was stimulated approximately threefold by the addition of TPA. The observation that BCE cells and not BAE cells produced high levels of both PA and collagenase activities in response to TPA demonstrates a significant difference between these two types of endothelial cells and suggests that the enhanced detectable activities are a property unique to bovine capillary and microvessel and endothelial cells.     

Oxidation of thiol-proteases in the hippocampus of Alzheimer's disease

The hippocampus of Alzheimer's disease brain has been shown to be highly oxidized compared to age-matched controls. One of the most sensitive targets of oxidation is anionic sulfur which can be found within the active site of members of the cysteine-protease family. Thus, while members of the cysteine-protease family such as the calpains and caspases have been found to be in an active conformation in vulnerable brain regions in AD it is possible that their proteolytic activity is hampered due to the robust oxidative stress found at these locations. To address this issue, the amount of caseinolytic activity present in the hippocampus from post-mortem brain samples of AD and age-matched controls was determined. No difference in caseinolytic activity in the absence of exogenous reducing agent was observed between AD and control. However, after addition of the thiol-specific reducing agent, dithiothreitol (DTT), the amount of caseinolytic activity was significantly increased in AD compared to the DTT-mediated increase in control. This suggests that the cysteine proteases are more oxidized in AD brain and that latent proteolytic activity in AD brain can be released by antioxidants. Further testing revealed that the calcium-dependent caseinolytic activity was significantly lower in AD brain compared to controls. This is despite the fact that the major calcium-dependent thiol protease, calpain, is threefold more activated in AD brain based on autolytic activation measured by Western blotting. This calcium-dependent protease difference between AD and control brains was negated by addition of DTT. These data suggest that cysteine protease activity in AD brain is inactivated by oxidants, which is evident by the ability of thiol-specific reducing agents such as DTT to rescue and recover activity.     

Systematic screening of potential beta-cell imaging agents

The beta-cell loss seen in diabetes mellitus could be monitored clinically by positron emission tomography (PET) if imaging agents were sufficiently specific for beta-cells to overcome the high ratio of non-beta-cell to beta-cell tissue in pancreas. In this report, we present a screening assay for identifying beta-cell-specific compounds that is based on the relative accumulation and retention by islet, INS-1, and exocrine (PANC-1) cells of candidate molecules. Molecules thought to have a high affinity for beta-cells were tested and included glibenclamide, tolbutamide, serotonin, L-DOPA, dopamine, nicotinamide, fluorodeoxyglucose, and fluorodithizone. Glibenclamide and fluorodithizone were the most specific, but the specificity ratios fell well below those needed to attain robust signal to background ratio as a PET imaging agent for quantifying beta-cell mass. In vivo tests of the biodistribution of glibenclamide and fluorodithizone in rats indicated that the compounds were not specifically associated with pancreas, bearing out the predictions of the in vitro screen.     

Reflections on humanizing biomedicine

Although biomedicine is responsible for the "miracles" of modern medicine, paradoxically it has also led to a quality-of-care crisis in which many patients feel disenfranchised from the health-care industry. To address this crisis, several medical commentators make an appeal for humanizing biomedicine, which has led to shifts in the philosophical boundaries of medical knowledge and practice. In this paper, the metaphysical, epistemological, and ethical boundaries of biomedicine and its humanized versions are investigated and compared to one another. Biomedicine is founded on a metaphysical position of mechanistic monism, an epistemology of objective knowing, and an ethic of emotionally detached concern. In humanizing modern medicine, these boundaries are often shifted to a metaphysical position of dualism/holism, an epistemology of subject knowing, and an ethic of empathic care. In a concluding section, the question is discussed whether these shifts in the philosophical boundaries are adequate to resolve the quality-of-care crisis.     

Medical cure and progress: the case of type-1 diabetes

Medicine often defines progress in terms of medical cures; however, such cures are rare. At best, medical progress generally consists of managing patients more efficiently or competently, especially in terms of relieving their symptoms. Today, medicine stands on the edge of curing a number of diseases. However, before achieving such cures, biomedical scientists need to understand in finer detail the mechanisms of physiological and pathological processes, especially from a systems perspective, and students of biomedical studies need to understand more precisely what a medical cure is conceptually or philosophically. This article utilizes type-1 diabetes to explore the issues surrounding medical progress with respect to defining medical cure, especially in terms of distinguishing it from managing a patient's illness.     

Anticoagulantly active heparin-like molecules from cultured fibroblasts

Heparin-like molecules, isolated from cultured rat and human skin fibroblasts, accelerated the inactivation of purified human thrombin via purified human antithrombin. Only 15% of the biologic activity of the complex carbohydrates derived from human skin fibroblasts was expressed when the heparin-binding domain an antithrombin was chemically modified at the Trp 49 residue. Human skin fibroblasts were metabolically labeled with [35S]Na2SO4, and radiolabeled heparin-like molecules were isolated utilizing ion-exchange chromatography and fractionated into two separate populations employing immobilized antithrombin. The species which bound with high avidity to the affinity matrix represented about 12% of the radiolabeled heparin-like molecules, accounted for almost 95% of the initial anticoagulant activity, and exhibited a specific activity of 3.98 USP units/10(6) 35S-cpm.     

Hemostatic regulation and Whitehead's philosophy of organism

Biology as a scientific discipline has relied heavily upon advances in chemistry and physics. An inherent danger in this relationship is the reduction of living phenomena to physico-chemical terms. Whitehead's Philosophy of Organism is utilized to examine current methodologies within biology and to evaluate their appropriateness for future research. Hemostatic regulation is employed to illustrate the applications of organistic concepts to biological research. It is concluded that understanding of living entities and their properties as well as possibly life itself will require synthesis of the many analytical elements as informational structures.     

Cloned bovine aortic endothelial cells synthesize anticoagulantly active heparan sulfate proteoglycan

Cloned bovine aortic endothelial cells were cultured with [35S]Na2SO4 and proteolyzed extensively with papain. Radiolabeled heparan sulfate was isolated by DEAE-Sephacel chromatography. The mucopolysaccharide was then affinity fractionated into two separate populations utilizing immobilized antithrombin. The heparan sulfate, which bound tightly to the protease inhibitor, represented 0.84% of the mucopolysaccharide mass, accounted for greater than 99% of the initial anticoagulant activity, and exhibited a specific activity of 1.16 USP units/10(6) 35S-cpm. However, the heparan sulfate that interacted minimally with the protease inhibitor constituted greater than 99% of the mucopolysaccharide mass, represented less than 1% of the starting biologic activity, and possessed a specific anticoagulant potency of less than 0.0002 USP unit/10(6) 35S-cpm. An examination of the disaccharide composition of the two populations revealed that the high-affinity heparan sulfate contained a 4-fold or greater amount of GlcA----GlcN-SO3-3-O-SO3 (where GlcA is glucuronic acid), which is a marker for the antithrombin-binding domain of commercial heparin, as compared with the depleted material. Cloned bovine aortic endothelial cells were incubated with [35S]Na2SO4 as well as tritiated amino acids and completely solubilized with 4 M guanidine hydrochloride and detergents. The double-labeled proteoglycans were isolated by DEAE-Sephacel, Sepharose CL-4B, and octyl-Sepharose chromatography. These hydrophobic macromolecules were then affinity fractionated into two separate populations utilizing immobilized antithrombin. The heparan sulfate proteoglycans which bound tightly to the protease inhibitor represented less than 1% of the starting material and exhibited a specific anticoagulant activity as high as 21 USP units/10(6) 35S-cpm, whereas the heparan sulfate proteoglycan that interacted weakly with the protease inhibitor constituted greater than 99% of the starting material and possessed a specific anticoagulant potency as high as 0.02 USP unit/10(6) 35S-cpm. The high-affinity heparan sulfate proteoglycan is responsible for more than 85% of the anticoagulant activity of the cloned bovine aortic endothelial cells. Binding studies conducted with 125I-labeled antithrombin demonstrated that these biologically active proteoglycans are located on the surface of cloned bovine aortic endothelial cells.