Kinetics of smooth and skeletal muscle activation by laser pulse photolysis of caged inositol 1,4,5-trisphosphate

Inositol 1,4,5-trisphosphate (InsP3) can stimulate skinned smooth and skeletal muscle to contract by initiating Ca2+ release from the sarcoplasmic reticulum. Whether this process is an integral component of the in vivo muscle activation mechanism was tested by releasing InsP3 rapidly within skinned muscle fibers of rabbit main pulmonary artery and frog semitendinosus. InsP3 was liberated on laser pulse photolysis of a photolabile but biologically inactive precursor of InsP3 termed caged InsP3. Caged InsP3 is a mixture of compounds in which InsP3 is esterified with 1(2-nitrophenyl)diazoethane (probably at the P4- or P5-position). Photochemical release of InsP3 induced a full contraction in both muscles at physiological free Mg2+ concentrations, but only in the smooth muscle were the InsP3 concentration (0.5 microM) and the activation rate compatible with the in vivo physiological response. Endogenous InsP3-specific phosphatase activity was present in smooth muscle and had about 35-fold greater activity than that in the skeletal-muscle preparation. Caged InsP3 was not susceptible to phosphatases in either preparation.     

Endstopped neurons in the visual cortex as a substrate for calculating curvature

Neurons in the visual cortex typically respond selectively to the orientation, and velocity and direction of movement, of moving-bar stimuli. These responses are generally thought to provide information about the orientation and position of lines and edges in the visual field. Some cells are also endstopped, that is selective for bars of specific lengths. Hubel and Wiesel first observed that endstopped hypercomplex cells could respond to curved stimuli and suggested they might be involved in detection of curvature, but the exact relationship between endstopping and curvature has never been determined. We present here a mathematical model relating endstopping to curvature in which the difference in response of two simple cells gives rise to endstopping and varies in proportion to curvature. We also provide physiological evidence that endstopped cells in area 17 of the cat visual cortex are selective for curvature, whereas non-endstopped cells are not, and that some are selective for the sign of curvature. The prevailing view of edge and curve determination is that orientations are selected locally by the class of simple cortical cells and then integrated to form global curves. We have developed a computational theory of orientation selection which shows that measurements of orientation obtained by simple cells are not sufficient because there will be strong, incorrect responses from cells whose receptive fields (RFs) span distinct curves (Fig. 1). If estimates of curvature are available, however, these inappropriate responses can be eliminated. Curvature provides the key to structuring the network that underlies our theory and distinguishes it from previous lateral inhibition schemes.     

Inhibition of mitosis by an antibody to the mitotic calcium transport system

Calcium ions are important in the regulation of mitotic apparatus assembly and in the control of chromosome movement. Changes in intracellular free calcium concentration, [Ca2+]i are achieved by an intracellular calcium-transport system which is highly conserved in different cell types. A membrane-bound protein of relative molecular mass (Mr) 46,000 (46K) is part of this transport system and has been implicated in the regulation of the [Ca2+]i changes associated with the course of mitosis. A monoclonal antibody against this 46K protein inhibits Ca2+-uptake into isolated Ca2+-sequestering membranes and specifically labels membranes associated with the mitotic apparatus of sea urchin embryos. Here we investigate the relationship between the intracellular calcium transport system and mitosis by injection of this monoclonal antibody into living mitotic sea urchin embryos. We find that after injection the intracellular free calcium increases up to 10(-6) M, the mitotic apparatus is rapidly destroyed and the cell is irreversibly blocked in its development.     

Astrocytes induce blood-brain barrier properties in endothelial cells

The highly impermeable tight junctions between endothelial cells forming the capillaries and venules in the central nervous system (CNS) of higher vertebrates are thought to be responsible for the blood-brain barrier that impedes the passive diffusion of solutes from the blood into the extracellular space of the CNS. The ability of CNS endothelial cells to form a blood-brain barrier is not intrinsic to these cells but instead is induced by the CNS environment: Stewart and Wiley demonstrated that when avascular tissue from 3-day-old quail brain is transplanted into the coelomic cavity of chick embryos, the chick endothelial cells that vascularize the quail brain grafts form a competent blood-brain barrier; on the other hand, when avascular embryonic quail coelomic grafts are transplanted into embryonic chick brain, the chick endothelial cells that invade the mesenchymal tissue grafts form leaky capillaries and venules. It is, however, not known which cells in the CNS are responsible for inducing endothelial cells to form the tight junctions characteristic of the blood-brain barrier. Astrocytes are the most likely candidates since their processes form endfeet that collectively surround CNS microvessels. In this report we provide direct evidence that astrocytes are capable of inducing blood-brain barrier properties in non-neural endothelial cells in vivo.     

Active sliding between cytoplasmic microtubules

Microtubules are versatile cellular polymers that play a role in cell shape determination and mediate various motile processes such as ciliary and flagellar bending, chromosome movements and organelle transport. That a sliding microtubule mechanism can generate force has been demonstrated in highly ordered structures such as axonemes, and microtubule-based force generation almost certainly contributes to the function of mitotic and meiotic spindles. Most cytoplasmic microtubule arrays, however, do not exhibit the structural regularity of axonemes and some spindles, and often appear disorganized. Yet many cellular activities (such as shape changes during morphogenesis, axonal extension and spindle assembly) involve highly coordinated microtubule behaviour and possibly require force generated by an intermicrotubule sliding mechanism, or perhaps use sliding to move microtubules rapidly into a protrusion for stabilization. Here we show that active sliding between cytoplasmic microtubules can occur in microtubule bundles of the amoeba Reticulomyxa. A force-producing mechanism of this sort could be used by this organism to facilitate the extension of cell processes and to generate the dynamic movements of the cytoplasmic network.     

An immunohistochemical study of the clearance of apoptotic cellular fragments

We investigated the distribution and fate of apoptotic bodies during human development and in the adult, using an antibody (M30) that recognizes a neo-epitope formed early in the apoptotic cascade by caspase cleavage of cytokeratin 18. In the fetus, we found extensive accumulation of M30-positive, non-phagocytosed fragments in the red pulp of the spleen, subcutaneous and submucosal vessels, the interstitium of the lung, and the glomerular mesangium of the kidneys. In the liver, M30-immunoreactive fragments were found inside macrophages in the sinusoids. The number of these fragments and the intensity of the immunostaining increased with the gestational age of the fetus. In the adult, M30-positive fragments were barely detectable in normal tissues. However, many pathological situations, including both chronic degenerative processes and metastatic cancer, were associated with accumulation of M30-positive fragments in the red pulp of the spleen. In the liver and kidney, no fragments could be detected. Remarkably, 13 of the 16 patients with metastasized cancer showed pronounced accumulation of M30-positive fragments containing hematoxylin-reactive material in the red pulp of the spleen. In the non-cancerous cases, such DNA-containing fragments were only seen in 9 of 94 cases. The results show that when apoptotic activity is high, as during development in the fetus or during metastasis and other pathological processes in the adult, the phagocytic clearance of apoptotic bodies can be overloaded. These apoptotic fragments then accumulate in the spleen. The visual detection of apoptotic fragments is concluded to reflect increased cell turnover. Received 1 July 2002; accepted 1 July 2002     

Identification and characterisation of two allelic forms of human alcohol dehydrogenase 2

The human alcohol dehydrogenase system is comprised of multiple forms that catalyse the oxidation/reduction of a large variety of alcohols and aldehydes. A transition that results in an Ile308Val substitution was identified in the human ADH2 gene by single-strand conformation polymorphism analysis. Screening a Swedish population revealed that Val308 was the most frequent allele (73%), and site-directed mutagenesis was used to obtain both allelozymes, which were expressed in Escherichia coli for characterisation. Thermostability was assayed by activity measurements and circular dichroism spectroscopy. The results showed that the 308Val substitution decreases protein stability, as compared to the Ile308 variant, an effect also demonstrated during prolonged storage. Ethanol, octanol, 12-hydroxydodecanoic acid and all-trans retinol were used as model substrates and, generally, slightly higher Km values were observed with Val at position 308. Finally, homology modelling, from mouse ADH2, further supported the decreased stability of the Val308 variant and located position 308 in the subunit interface of the molecule and in the vicinity of the active-site pocket entrance. In conclusion, the Ile308Val substitution represents a novel functional polymorphism within the human alcohol dehydrogenase gene cluster that may affect the metabolism of ethanol and other substrates.     

Protein kinases mediate nitric oxide-induced apoptosis in the insect cell line IPLB-LdFB

The involvement of protein kinases (PKA, PKC and PKB) in nitric oxide (NO)-induced apoptosis with sodium nitroprusside plus N-acetyl-L-cysteine in the IPLB-LdFB cell line from the insect Lymantria dispar was investigated. The presence of protein kinase-like molecules was demonstrated by western blot analysis. The role of the kinases in programmed cell death was analysed in cytofluorimetric experiments by incubating the insect cells with H-89 (a specific inhibitor of PKA), calphostin C (an inhibitor of PKC) or wortmannin (an inhibitor of phosphatidylinositol 3-kinase). The results show that PKA is correlated with the induction and PKC and PKB with the prevention of NO-induced insect cell death. Moreover, NO-induced apoptosis involves the release of cytochrome c. Received 15 March 2002; accepted 25 March 2002     

RAGE is a multiligand receptor of the immunoglobulin superfamily: implications for homeostasis and chronic disease

Receptor for AGE (RAGE) is a member of the immunoglobulin superfamily that engages distinct classes of ligands. The biology of RAGE is driven by the settings in which these ligands accumulate, such as diabetes, inflammation, neurodegenerative disorders and tumors. In this review, we discuss the context of each of these classes of ligands, including advance glycation end-products, amyloid beta peptide and the family of beta sheet fibrils, S100/calgranulins and amphoterin. Implications for the role of these ligands interacting with RAGE in homeostasis and disease will be considered.