Cytokeratin provides a specific marker for human arachnoid cells grown in vitro

Cells from cranial and spinal arachnoid membranes of humans were grown in culture. Their growth characteristics, morphology and details of their cytoskeletal composition are described. Arachnoid membranes, obtained at autopsy, were finely minced and incubated in tissue culture medium. Monolayers of cells of homogeneous morphology grew from these tissue fragments. The cells were flat and polygonal. They divided slowly to form non-overlapping monolayers of low cell density. Electron microscopic examination of cultured arachnoid cells revealed numerous desmosome-like tight junctions and abundant intermediate filaments (tonofilaments). Both morphological features are characteristic of arachnoid cells in situ, but not of cells in the fibroblast-rich dura mater. Immunofluorescence microscopy with monoclonal antibodies demonstrated cytokeratin in the cytoplasm of primary cultures of arachnoid cells. Thus we demonstrated that these cultured cells retained certain of the specific differentiated properties of arachnoid cells in situ and that they are not fibroblasts (which lack tight junctions and cytokeratins). To our knowledge, there have been no previous reports of in vitro growth of arachnoid cells. This in vitro model should be useful in studying the response of arachnoid cells to a variety of substances thought to be involved in the chronic inflammatory condition of the meninges known as arachnoiditis.     

Effects of cyclic AMP and butyrate on cell cycle,DNA, RNA,and purine synthesis of cultured astrocytes

Dibutyryl cyclic monophosphate (dBcAMP) has been shown to inhibit growth, and alter the morphology of astrocytes. However, the potential contribution of its hydrolytic product, butyrate, in inducing some of the changes that have been attributed to dBcAMP, is not clear. DNA, RNA, and purine synthesis were therefore studied in primary astrocyte cultures after 24 hours of exposure to varying concentrations of butyrate, dBcAMP, and agents that increase intracellular cAMP levels. Progression of cells through cell cycle was also studied by flow cytometry. Dibutyryl cAMP partially arrested cells in Go/G1 phase of cell cycle while sodium butyrate increased the percentage population of cells in G2/M phase. DNA synthesis and de novo purine synthesis were inhibited after treatment with dBcAMP, sodium butyrate, and various drugs that increase intracellular cAMP levels. RNA synthesis was increased with cAMP but was not affected by sodium butyrate. Our study shows that at millimolar concentrations, butyrate is capable of altering the cell cycle and inhibiting DNA synthesis in primary astrocyte cultures, in a manner that is similar although not identical to the effects of dBcAMP.     

Molecular and symbiotic characterization of exopolysaccharide-deficient mutants of Rhizobium tropici strain CIAT899

We studied the symbiotic behaviour of 20 independent Tn5 mutants of Rhizobium tropici strain CIAT899 that were deficient in exopolysaccharide (EPS) production. The mutants produced non-mucoid colonies, were motile, grew in broth cultures at rates similar to those of the parent, and produced significantly less EPS than did CIAT899 in broth culture. A genomic library of strain CIAT899, constructed in pLA2917, was mobilized into all of the mutants, and cosmids that restored EPS production were identified. EcoRI restriction digests of the cosmids revealed nine unique inserts. Mutant complementation and hybridization analysis showed that the mutations affecting EPS production fell into six functional and physical linkage groups. On bean, the mutants were as efficient in nodulation and as effective in acetylene reduction as strain CIAT899, induced a severe interveinal chlorosis, and all but one were less competitive than CIAT899. On siratro, CIAT899 induced nodules that were ineffective in acetylene reduction, whereas the EPS-deficient mutants induced effective nodules. Microscopic examination of thin sections showed that nodules from both siratro and bean plants inoculated with either CIAT899 or an EPS-deficient mutant contained infected cells. These data indicate that EPS is not required for normal nodulation of bean by R. tropici, that it may contribute to competitiveness of R. tropici on bean, and that the loss of EPS production is accompanied by acquisition of the ability to reduce acetylene on siratro.     

Valve and seta (spine) morphogenesis in the centric diatomChaetoceros peruvianus Brightwell

Summary InChaetoceros peruvianus, the two very long, delicately tapered setae (spine-like processes) of the lower valve curve downwards gently until they are often almost parallel, while those emerging from the upper valve curve sharply downwards until oriented almost in the same direction as the setae of the lower valve. This curvature creates a deep pit between the bases of the upper valve's setae, where they emerge from the valve. In live cells, extension of setae is rapid and very sensitive to disturbance. After cleavage the new silica deposition vesicle (SDV) appears in the centre of the furrow and expands outwards over it. A distinct microtubule centre (MC) appears directly on top of the SDV. Microtubules (MTs) from the MC ensheath the nucleus, and others fan out over the SDV and plasmalemma. A little later, the MC in the lower daughter cell moves off the SDV, and its MTs now appear to mould the plasmalemma/ SDV into the deep pit between the base of the setae. In the upper daughter cell, the MC remains on the SDV. Initiation of setae is first observed as protuberances of bare cytoplasm growing from the sides of the daughter cells, through gaps in the parental valve. Many MTs initially line the plasmalemma of these protuberances as they grow outwards and the SDV also expands over the new surface. As the setae get longer, a unique complex of three organelles appears. Just behind the naked cytoplasm at the tip of the seta, a thin flat layer of fibrous material lines the plasmalemma. This, the first of the complex, is called the thin band. Immediately behind this is the second, a much thicker, denser fibrous band, the thick band. At the front edge of the SDV, 5–6 finger-like outgrowths of silicified wall grow forwards. These are interconnected by the elements of the thick band which thus apparently dictate the polygonal profile of the seta. These also appear to generate the spinules (tiny spines) that adorn the surface of the seta; the spiral pattern of the spinules indicates that this whole complex might differentiate one after the next, in order. Further back from the tip, evenly spaced transverse ribs are formed. These are connected to the third organelle in the complex, the striated band; our interpretation is that the striated band sets up the spacing of the ridges that regularly line the inner surface of the setae. During seta growth, this complex is apparently responsible for controlling the delicate tapering curvature of the very fine silica processes. Since the complex is always seen near the tip of the seta, we conclude that it migrates forwards steadily as the tip grows. While the thin and thick bands could slide continuously over the cell membrane, the striated band must be disassembled and then recycled forward during extension if it is indeed connected to the ridges lining the inside of the setae. We could find no indication that turgor pressure drives extension of the setae, in which event the activity of these organelles is responsible for growth using the justformed silica tube as the base from which extension is generated.     

Interconversion of the salicylic acid signal and its glucoside in tobacco

Salicylic acid (SA) has been proposed to play a role in the induction of pathogenesis-related (PR) proteins and systemic acquired resistance (SAR) in tobacco. Since SA is rapidly converted to salicylic acid β-glucoside (SAG) in tobacco, we have attempted to assess the role of SAG in pathogenesis by application of chemically synthesized SAG to tobacco leaves. SAG was as active as SA in induction of PR-1 gene expression. This induction was preceded by a transient release of SA, which occurred in the extracellular spaces. The existence of a mechanism that releases SA from SAG suggests a possible role for SAG in SAR.     

Fluorescent in situ hybridization of the telomere repeat sequence in hamster sperm nuclear structures

The flat, hooked-shaped architecture of the hamster sperm nucleus makes this an excellent model for in situ hybridization studies of the three dimensional structure of the genome. We have examined the structure of the telomere repeat sequence (TTAGGG)_n with respect to the various nuclear structures present in hamster spermatozoa, using fluorescent in situ hybridization. In fully condensed, mature sperm nuclei, the telomere sequences appeared as discrete spots of various sizes interspersed throughout the volume of the nuclei. While the pattern of these signals was non-random, it varied significantly in different nuclei. These discrete telomere foci were seen to gradually lengthen into linear, beaded signals as sperm nuclei were decondensed, in vitro, and were not associated with the nuclear annulus. We also examined the relationship of telomeres to the sperm nuclear matrix, a residual nuclear structure that retains the original size and shape of the nucleus. In these structures the DNA extends beyond the perimeter of the nucleus to form a halo around it, representing the arrangement of the chromosomal DNA into loop domains attached at their bases to the nuclear matrix. Telomere signals in these structures were also linear and equal in length to those of the decondensed nuclei, and each signal represented part of a single DNA loop domain. The telomeres were attached at one end to the nuclear matrix and extended into the halo. Sperm nuclear matrices treated with Eco RI retained the telomere signals. These data support sperm DNA packaging models in which DNA is coiled into discrete foci, rather than spread out linearly along the length of the sperm nucleus.