Antisense GAP-43 Inhibits the Evoked Release of Dopamine from PC12 Cells

Abstract: To investigate the role of the neuronal growth-associated protein GAP-43 (neuromodulin, B-50, F1, P-57) in neurotransmitter release, we transfected PC12 cells with a recombinant expression vector coding for antisense human GAP-43 cRNA. Two stable transfectants, designated AS1 and AS2, were selected that had integrated the recombinant sequence and expressed antisense GAP-43 RNA. Immunoblot analysis of proteins from AS1 and AS2 cells indicated that the level of GAP-43 in these cell lines was reduced. In the presence of extracellular calcium, a depolarizing concentration of K⁺ (56 m M ) evoked dopamine release from control cells, but not from AS1 and AS2 cells. Similarly, the calcium ionophore A23187 evoked dopamine release from control cells, but was ineffective in stimulating dopamine release from AS1 and AS2 cells. The antisense transfectants, as well as the control cells, contained appreciable quantities of dopamine and secretory granules with a normal appearance. Because the expression of antisense GAP-43 RNA in PC12 cells leads to a decrease in GAP-43 expression and to the loss of evoked dopamine release, these results provide evidence of a role for GAP-43 in calcium-dependent neurotransmitter release.     

Temperature dependence of growth,enzyme secretion and activity of psychrophilic Antarctic bacteria

Five psychrophilic Antarctic bacteria have been selected for their capacity to secrete exoenzymes into culture medium. These strains are able to grow from 0 to about 25° C. However, production of lipase fromMoraxella, -amylase fromAlteromonas haloplanctis, -lactamase fromPsychrobacter immobilis and protease fromBacillus is maximal at temperatures close to that of their environment (—2 to 4° C) and is strongly inhibited at higher temperatures. This thermal effect involves alterations in the secretory pathway in the upper range of temperatures, losses due to the enzyme thermal lability and in some cases to reduction in cell development. The apparent optimal activity temperature of these enzymes is between 30 and 40° C, i.e. about 20° C lower than that of their mesophilic counterparts.     

Tropomyosin from the striated muscles of carp (Cyprinus carpio) and of icefish (Channichthys rhinoceratus).

Tropomyosin of fast-twitch, slow-twitch and cardiac muscles of carp and icefish has been isolated by hydroxyapatite chromatography. The subunit distribution has been investigated by polyacrylamide gel electrophoresis and by peptide mapping. The purified skeletal muscle tropomyosins all belong to the alpha family and differ from higher vertebrate tropomyosin by the lack of beta subunits. Specific alpha isotypes are however encountered in fast-twitch fibres (alpha w subunit) and slow-twitch or intermediate (pink) fibres (alpha and alpha w subunits). The amino acid compositions and the paracrystals formed by the carp alpha w alpha w and alpha alpha w tropomyosins do not differ markedly from that of rabbit alpha alpha chains. They differ however by their capability to inhibit the ATPase activity of rabbit skeletal muscle acto-HMM system. A beta-like subunit is found in carp cardiac tropomyosin, in the proportion of 25% of the native protein, but not in icefish heart.     

Cytochemical reactivity of T mu lymphocytes in human lymphatic tissue for dipeptidylaminopeptidase IV

Summary The enzyme dipeptidylaminopeptidase IV (DAP IV; EC 3.4.14.-) was recently shown cytochemically to be confined, in blood and bone marrow, to human T cells bearing, Fc receptors for IgM (T lymphocytes). This observation, confirmed by direct biochemical tests, stimulated us to study the histochemical distribution of DAP IV in normal human lymphatic tissue. In cryostat sections of lymph node, tonsil and thymus, DAP IV was detectable only in lymphocytes, Hassal's corpuscles and the endothelia of vessels. The distribution pattern of DAP IV-positive lymphocytes accorded well with results obtained with human T cell antisera. Compared to cytochemical reactions for other enzymes, such as acid esterase, DAP IV has the advantage that it does not stain monocytes, B lymphocytes or other mononuclear cells. Further, it does not depend on a particular type of staining pattern like, for example, the dot-like reaction product of acid esterase in T lymphocytes. Since the reaction for DAP IV remains more or less unchanged in month-old sections, it is easily adaptable to routine work and has the potentiality of being applied to the diagnosis of T cell lymphomas.     

Alteration in the Wnt microenvironment directly regulates molecular events leading to pulmonary senescence

In the aging lung, the lung capacity decreases even in the absence of diseases. The progenitor cells of the distal lung, the alveolar type II cells (ATII), are essential for the repair of the gas‐exchange surface. Surfactant protein production and survival of ATII cells are supported by lipofibroblasts that are peroxisome proliferator‐activated receptor gamma (PPARγ)‐dependent special cell type of the pulmonary tissue. PPARγ levels are directly regulated by Wnt molecules; therefore, changes in the Wnt microenvironment have close control over maintenance of the distal lung. The pulmonary aging process is associated with airspace enlargement, decrease in the distal epithelial cell compartment and infiltration of inflammatory cells. qRT–PCR analysis of purified epithelial and nonepithelial cells revealed that lipofibroblast differentiation marker parathyroid hormone‐related protein receptor (PTHrPR) and PPARγ are reduced and that PPARγ reduction is regulated by Wnt4 via a β‐catenin‐dependent mechanism. Using a human in vitro 3D lung tissue model, a link was established between increased PPARγ and pro‐surfactant protein C (pro‐SPC) expression in pulmonary epithelial cells. In the senile lung, both Wnt4 and Wnt5a levels increase and both Wnt‐s increase myofibroblast‐like differentiation. Alteration of the Wnt microenvironment plays a significant role in pulmonary aging. Diminished lipo‐ and increased myofibroblast‐like differentiation are directly regulated by specific Wnt‐s, which process also controls surfactant production and pulmonary repair mechanisms.     

First record of lily thrips, Liothrips vaneeckei Priesner, in Australia (Thysanoptera: Phlaeothripidae)

The first Australian record of the lily thrips, Liothrips vaneeckei Priesner, is reported from a bulb farm in Warragul South, Victoria. It is an occasional pest of Lilium bulbs, both in the field and in storage, particularly in the USA and several European countries, and is also infrequently found in considerable numbers on the corms of orchids.     

Structural similarities and evolutionary relationships in chloride-dependent alpha-amylases

The alpha-amylase sequences contained in databanks were screened for the presence of amino acid residues Arg195, Asn298 and Arg/Lys337 forming the chloride-binding site of several specialized alpha-amylases allosterically activated by this anion. This search provides 38 alpha-amylases potentially binding a chloride ion. All belong to animals, including mammals, birds, insects, acari, nematodes, molluscs, crustaceans and are also found in three extremophilic Gram-negative bacteria. An evolutionary distance tree based on complete amino acid sequences was constructed, revealing four distinct clusters of species. On the basis of multiple sequence alignment and homology modeling, invariable structural elements were defined, corresponding to the active site, the substrate binding site, the accessory binding sites, the Ca(2+) and Cl(-) binding sites, a protease-like catalytic triad and disulfide bonds. The sequence variations within functional elements allowed engineering strategies to be proposed, aimed at identifying and modifying the specificity, activity and stability of chloride-dependent alpha-amylases.     

The value of preoperative Doppler sonography for planning free perforator flaps

The individual perforating vessels have a high degree of anatomical variation, therefore it is desirable to conduct a careful examination of them before undertaking a perforator flap operation. Because locating the vessels beforehand makes performing the operative procedure much easier, the aim of the present study was to assess the value of using simple acoustic Doppler sonography to plan a perforator flap operation. The vessel examinations were carried out before taking 46 free microvascular flaps from either the lower abdominal wall or the buttock for reconstructive breast surgery. The perforating vessels located were marked, and their position relative to the umbilicus or the most cranial point of the rima ani recorded using a coordinate system. In 40 patients, a perforator flap operation (deep inferior epigastric perforator flap, n = 32; superior gluteal artery perforator flap, n = 8) was actually carried out; in six of these patients, a myocutaneous flap was used because of the insufficient availability of perforating vessels. Before the operation, perforating vessels were marked for each patient, with an average of 7.3 for the deep inferior epigastric perforator flap and 6.5 for the superior gluteal artery perforator flap. Out of 286 vessels marked for later perforator flaps, 162 were identified during the operation. A preoperatively marked vessel was used in 37 of 40 patients. In the remaining patients, a vessel was used that had not been previously marked. The vertical and horizontal distance between the perforating vessels identified during the operation and the preoperative marks averaged 0.8 cm. The results show preoperative Doppler sonography to be useful for locating the position of individual perforating vessels, making it much easier to find them during the operation.     

Effects of forest age and disturbance on population persistence in the understory herb,Arisaema triphyllum (Araceae)

Long-lived understory herbs experience a highly dynamic forest over space and time, yet can persist for more than a century. To understand how these populations persist, we examined effects of forest age and disturbance on potential sexual reproduction and clonal growth in the sexually labile perennial, Arisaema triphyllum. Potential sexual reproduction (female:male ratio) was significantly greater in the Young and Old-Gap forest states compared with Old, closed-canopy sites, where it was virtually absent. In contrast, clonal growth (estimated by cormlet production) did not differ significantly among the three forest states. Of seven environmental variables measured, only light (positively) and plant density (negatively) contributed significantly to the variation in potential sexual reproduction, while no measured variables contributed significantly to the variation in number of cormlets. The larger sexual reproductive effort (flower+stalk biomass/total biomass) for males in the undisturbed, 100 yr old forest may explain the absence of females in these sites, while the invariant vegetative reproductive effort (cormlet biomass/total biomass) may explain the similarity in average number of cormlets per individual per season across forest states. These results suggest that potential sexual reproduction is resource-limited, while clonal growth may be resource-independent. By maintaining ramet production during unfavorable periods, A. triphyllum populations disperse temporally, waiting for conditions under which sexual reproduction may resume.