Selective protein transport through a thin cellulose coated porous membrane

A new composite membrane was designed and studied for permselectivity of various molecular weight proteins. The membrane is composed of a porous substrate membrane [Durapore; poly(vinylidene fluoride)] coated with a thin dense layer of regenerated cellulose. This composite membrane was fabricated by spin coating a cellulose acetate solution onto the membrane, followed by alkaline hydrolysis of the cellulose acetate coating to regenerate cellulose. The coated layer was physically characterized by scanning electron microscopy (SEM) and infrared (IR) spectroscopy. In addition, the water uptake into and permeation properties of macromolecules across the coated and uncoated membranes were studied. A typical composite membrane coating was 0.8 +/- 0.2 mum thick, resulting in a molecular weight cutoff of approximately 40,000 daltons. This composite membrane also demonstrated negligible diffusional lag time for permeants, due to the diffusional barrier. (c) 1994 John Wiley & Sons, Inc.     

Effects of annular cranial vault modification on the cranial base and face

Artificial modification of the cranial vault was practiced by a number of prehistoric and protohistoric populations, frequently during an infant's first year of life. We test the hypothesis that, in addition to its direct effects on the cranial vault, annular cranial vault modification has a significant indirect effect on cranial base and facial morphology. Two skeletal series from the Pacific Northwest Coast, which include both nonmodified and modified crania, were used: the Kwakiutl (62 nonmodified, 45 modified) and Nootka (28 nonmodified, 20 modified). Three-dimensional coordinates of 53 landmarks were obtained using a diagraph, and 36 landmarks were used to define nine finite elements in the cranial vault, cranial base, and face. Finite element scaling was used to compare average nonmodified and average modified crania, and the significance of the results were evaluated using a bootstrap test. Annular modification of the cranial vault produces significant effects on the morphology of the cranial base and face. Annular modification in the Kwakiutl resulted in restrictions of the cranial vault in the medial-lateral and superior-inferior dimensions and an increase in anterior-posterior growth. Similar dimensional changes are observed in the cranial base. The Kwakiutl face is increased anterior-posteriorly and reduced anterior-laterally to posterior-medially. Similar effects of modification are observed in the Nootka cranial vault and cranial base, though not in the face. These results demonstrate the developmental interdependence of the cranial vault, cranial base, and face. © 1993 Wiley-Liss, Inc.     

Enumeration of Vibrio vulnificus on membrane filters with a fluorescently labeled oligonucleotide probe specific for kingdom-level 16S rRNA sequences.

Vibrio vulnificus was enumerated on membrane filters after hybridization with a fluorescent oligonucleotide eubacterial probe. Cells were hybridized in liquid buffer or directly on membrane filters. There was no significant difference between fluorescent oligonucleotide direct counts and acridine orange direct counts (P > 0.05). Liquid buffer hybridization was preferable to direct filter hybridization.     

Expression instability and genetic disorders in transgenicNicotiana plumbaginifolia L. plants

The ease of integrative transformation with foreign genes and the extent of their expression and stability in successive generations determine the applicability of direct gene transfer. InNicotiana plumbaginifolia, one to ten copies of foreign DNA were integrated into the plant genome, resulting in simple to complex patterns of integration. Genetic analysis showed that in more than 50% of the cases, this DNA inserted at two or more loci in the genome. Of the 156 crosses performed between F₁ monogenic transformants, only eight combinations showed linkage of the inserted neomycin phosphotransferase genes (npt). The following instability events were registered: physical loss, alterations in the initial segregation rates in successive meiotic generations observed in either selfing or crossing (reduction or increase in number of segregating loci) and genomic disorders in crosses between transformants. Among them of particular interest were the discordant segregation values observed between corresponding R₁ and F₁ progenies in up to 9% of the evaluated transformants. In addition, 5% of the transformants showed a phenotypic loss of resistance. In the F₃ generation, 5 out of 15 transformants exhibited instability, which was transmitted to the F₄ generation. Further increases in instability rates were observed with higher numbers of insertion loci and in crosses between independent transgenic plants, reaching 100% when a trigenic partner was involved.N. plumbaginifolia exhibited more instability thanN. tabacum under equivalent experimental conditions. The molecular bases of such instability events are discussed in relation to DNA methylation, co-suppression and genomic imbalance.     

Desensitization by protein kinase C activation differentially uncouples formyl peptide receptors from effector enzymes in HL-60 granulocytes

The hypothesis that protein kinase C (PKC) participates in agonist-mediated desensitization of formyl peptide receptors in HL-60 granulocytes was tested. fMet-Leu-Phe and leukotriene B₄(LTB₄) produced homologous desensitization of agonist-stimulated intracellular calcium transients. Pre-treatment with the PKC activator, phorbol myristate acetate (PMA; 10 nM), abolished both fMet-Leu-Phe and LTB₄-stimulated calcium transients. Membranes prepared from control HL-60 granulocytes (NM) or cells treated with 10 nM PMA (PMA-M) demonstrated increased formyl peptide receptor and G protein density, as determined by radioligand binding and pertussis toxin- and cholera toxin-catalysed ADP ribosylation. fMet-Leu-Phe stimulation of guanosine 5′-[γ-thio]-triphosphate (GTPγS) binding and GTP hydrolysis and GDP inhibition of fMet-Leu-Phe binding were not different between NM and PMA-M. Pre-treatment with 10 nM PMA did not inhibit subsequent fMet-Leu-Phe-stimulated superoxide generation or phospholidase D activation. We conclude that PKC desensitizes fMet-Leu-Phe-stimulated phospholipase C, but not phospholipase D, responses and that PKC activation does not mediate agonist-induced desensitization of formyl peptide receptors.     

The E3L and K3L vaccinia virus gene products stimulate translation through inhibition of the double-stranded RNA-dependent protein kinase by different mechanisms.

Vaccinia virus has evolved multiple mechanisms to counteract the interferon-induced antiviral host cell response. Recently, two vaccinia virus gene products were shown to interfere with the activity of the double-stranded RNA-dependent protein kinase (PKR): the K3L gene product and the E3L gene product. We have evaluated the efficiency by which these gene products inhibit PKR and whether they act in a synergistic manner. The effects of the two vaccinia virus gene products were compared in an in vivo system in which translation of a reporter gene (dihydrofolate reductase or eukaryotic translation initiation factor 2 alpha [eIF-2 alpha]) was inhibited because of the localized activation of PKR. In this system, the E3L gene product, and to a lesser extent the K3L gene product, potentiated translation of the reporter gene and inhibited eIF-2 alpha phosphorylation. Analysis in vitro demonstrated that the E3L gene product inhibited PKR approximately 50- to 100-fold more efficiently than the K3L gene product. However, further studies demonstrated that the mechanism of action of these two inhibitors was different. Whereas the E3L inhibitor interfered with the binding of the kinase to double-stranded RNA, the K3L inhibitor did not. We propose that the K3L inhibitor acts through its homology to eIF-2 alpha to interfere with the interaction of eIF-2 alpha with PKR. The two inhibitors did not display a synergistic effect on translation or eIF-2 alpha phosphorylation. In addition, neither K3L nor E3L expression detectably altered cellular protein synthesis.     

Collagen fibrillogenesis in a three-dimensional fibroblast cell culture system

The purpose of this study was to follow collagen fibril formation in a newly developed three dimensional cell culture system. Human neonatal foreskin fibroblasts were grown on a nylon mesh in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% fetal calf serum and antibiotics. Fibrillogenesis was initiated by the addition of 50 micrograms/ml ascorbate to confluent cultures. Sample meshes were processed for electron microscopy or immuno-electron microscopy. Fibrils 20–30 nm in diameter, with 67 nm periodicity, were first detected five days after the addition of ascorbate. As cultures progressed, cells organized into parallel layers between which collagen fibers continued to form and increase in diameter. By day 50, fiber diameter ranged from 30 to 80 nm and large bundles were seen. No collagen fibril formation occurred in control cultures to which no ascorbate was added. However, large amounts of microfibrils were observed. Antibodies against the aminopropeptide of type I procollagen were found to bind to fibrils with diameters less than 34 nm while antibodies against the aminopropeptide of type III collagen bound primarily to fibers which ranged from 35–54 nm in diameter. We believe that this system, which morphologically resembles a normal dermis, will werve as an excellent model for the study of collagen fibrillogenesis.     

A nucleotide substitution in one of the β-tubulin genes of Trichoderma viride confers resistance to the antimitotic drug methyl benzimidazole-2-yl-carbamate

We characterized a Trichoderma viride strain that is resistant to the antimitotic drug methyl benzimidazole-2-yl-carbamate (MBC). This species has two -tubulin genes (tub1 and tub2) and by reverse genetics we showed that a mutation in the tub2 gene confers MBC resistance in this strain. Comparison of the tub2 sequence of the mutant strain with that of the wild type revealed that a single amino acid substitution of tyrosine for histidine at position 6 is responsible for the MBC tolerance. Furthermore, we showed that this gene can be used as a homologous dominant selectable marker in T. viride transformation. Both tubulin genes were completely sequenced. They differ by 48 residues and the degree of identity between their deduced amino acid sequences is 86.3%.