Markers for trans-Golgi Membranes and the Intermediate Compartment Localize to Induced Membranes with Distinct Replication Functions in Flavivirus-Infected Cells

Replication of the flavivirus Kunjin virus is associated with virus-induced membrane structures within the cytoplasm of infected cells; these membranes appear as packets of vesicles associated with the sites of viral RNA synthesis and as convoluted membranes (CM) and paracrystalline arrays (PC) containing the components of the virus-specified protease (E. G. Westaway, J. M. Mackenzie, M. T. Kenney, M. K. Jones, and A. A. Khromykh, J. Virol. 71:6650-6661, 1997). To determine the cellular origins of these membrane structures, we compared the immunolabelling patterns of several cell markers in relation to these sites by immunofluorescence and immunoelectron microscopy. A marker for the trans-Golgi membranes and the trans-Golgi network, 1,4-galactosyltransferase (GalT), was redistributed to large foci in the cytoplasm of Kunjin virus-infected cells, partially coincident with immunofluorescent foci associated with the putative sites of viral RNA synthesis. As determined by immunoelectron microscopy, the induced vesicle packets contained GalT, whereas the CM and PC contained a specific protein marker for the intermediate compartment (ERGIC53). A further indicator of the role of cellular organelles in their biogenesis was the observation that the Golgi apparatus-disrupting agent brefeldin A prevented further development of immunofluorescent foci of induced membranes if added before the end of the latent period but that once formed, these membrane foci were resistant to brefeldin A dispersion. Reticulum membranes emanating from the induced CM and PC were also labelled with the rough endoplasmic reticulum marker anti-protein disulfide isomerase and were obviously redistributed during infection. This is the first report identifying trans-Golgi membranes and the intermediate compartment as the apparent sources of the flavivirus-induced membranes involved in events of replication.     

COMPARISON OF ISOZYMES AMONG TYPHA SPECIES IN THE EASTERN UNITED STATES

Biochemical phenotsypes of four taxa of Typha from the eastern United States were determined by starch gel electrophoresis. The isozyme banding patterns of T. latifolia, T. angustifolia and T. domingensis are distinct and allow unambiguous species identification when morphological characters are inadequate or unsuitable. The fourth form, T. glauca, is not an F₁ hybrid, but it does appear to be intermediate between T. latifolia and T. angustifolia. The status of T. glauca and evolutionary relationships among the four forms may now be clarified by additional sampling because of the distinct and relatively invariant isozyme banding patterns which are described.     

Intermediate sucrose octa-acetate sensitivity suggests a third allele at mouse bitter taste locus Soa and Soa-Rua identity

Mice have been characterized as either tasters or non-tastersof the bitter compound sucrose octa-acetate(SOA). However, 11of 17 supposedly non-taster inbred strains were found to avoid1 mM SOA. All 17 strains were indifferent to 0.1 mM SOA. Tasterstrains avoided both concentrations. The intermediate phenotypewas dubbed demitaster. A consistent phenotypic dominance orderwas found in crosses among both inbred and outbred strains (taster> non-taster > demitaster). Demitasters were found (withtasters) in an outbred strain showing monogenic segregationfor SOA avoidance. This, plus monogenic segregation in a back-crossof taster to demitaster inbred strains, suggested a third alleleat the Soa locus (Soa^c). Demitaster allelism was supported bythe strong associations found in 15 strains between the threeSOA phenotypes and HindIII restriction fragment patterns forthe closely linked Prp (proline rich protein) loci. SOA demitasterstrains were also intermediate in raffinose undeca-acetate (RUA)avoidance. Furthermore, B6.SW-Soa² congenic mice avoided notonly SOA, but RUA and eight other acetylated sugars. A previouslyproposed separate RUA-sensitivity gene (Rua) thus appeared tobe redundant.     

Intraspinal Tumors in Children

Thirty cases of intraspinal neoplasms occurring during the first two decades of life are reviewed. Histologic examination showed 13 of these to be astrocytomas, 6 neuroblastomas, 5 sarcomas, 3 ependymomas, 2 neurofibromas and 1 a schwannoma. Orthopedic deformities developed or worsened in 60 percent of patients surviving longer than a year after diagnosis. In five patients some form of endocrine deficiency developed after irradiation. For treatment of radiosensitive extradural malignant lesions, biopsy followed by irradiation is advocated.     

RGD Surface Functionalization of the Hydrophilic Acrylic Intraocular Lens Material to Control Posterior Capsular Opacification

Posterior Capsular Opacification (PCO) is the capsule fibrosis developed on implanted IntraOcular Lens (IOL) by the de-differentiation of Lens Epithelial Cells (LECs) undergoing Epithelial Mesenchymal Transition (EMT). Literature has shown that the incidence of PCO is multifactorial including the patient''s age or disease, surgical technique, and IOL design and material. Reports comparing hydrophilic and hydrophobic acrylic IOLs have shown that the former has more severe PCO. On the other hand, we have previously demonstrated that the adhesion of LECs is favored on hydrophobic compared to hydrophilic materials. By combining these two facts and contemporary knowledge in PCO development via the EMT pathway, we propose a biomimetically inspired strategy to promote LEC adhesion without de-differentiation to reduce the risk of PCO development. By surface grafting of a cell adhesion molecule (RGD peptide) onto the conventional hydrophilic acrylic IOL material, the surface-functionalized IOL can be used to reconstitute a capsule-LEC-IOL sandwich structure, which has been considered to prevent PCO formation in literature. Our results show that the innovative biomaterial improves LEC adhesion, while also exhibiting similar optical (light transmittance, optical bench) and mechanical (haptic compression force, IOL injection force) properties compared to the starting material. In addition, compared to the hydrophobic IOL material, our bioactive biomaterial exhibits similar abilities in LEC adhesion, morphology maintenance, and EMT biomarker expression, which is the crucial pathway to induce PCO. The in vitro assays suggest that this biomaterial has the potential to reduce the risk factor of PCO development.     

Hospital admissions before and after shipyard closure.

To determine the effect of job loss on health an investigation was made of admissions to hospitals in 887 men five years before and three years after the closure of a Danish shipyard. The control group comprised 441 men from another shipyard. The information on hospital admissions was obtained from the Danish national register of patients. The relative risk of admission in the control group dropped significantly in terms of the number of men admitted from the study group from 1.29 four to five years before closure to 0.74 in the three years after closure. This was especially true of admissions due to accidents (1.33 to 0.46) and diseases of the digestive system (4.53 to 1.03). For diseases of the circulatory system, particularly cardiovascular diseases, the relative risk increased from 0.8 to 1.60, and from 1.0 to 2.6 respectively. These changes in risk of illness after redundancy are probably a consequence of a change from the effects of a high risk work environment to the effects of psychosocial stresses such as job insecurity and unemployment.     

Expression of the β-subunit of β-conglycinin in seeds of transgenic plants

Soybean (Glycine max (L.) Merr.) seeds contain the storage protein -conglycinin, encoded by a multigene family. -Conglycinin consists of three subunits; , , and . A genomic clone for a -subunit of -conglycinin has been characterized by restriction-enzyme mapping and hybrid selected in-vitro translation followed by immunoprecipitation. In order to determine the developmental regulation of this -subunit gene, its expression was studied in seeds of transgenic petunia (Petunia hybrida) and tobacco (Nicotiana tabacum L.) plants. The -subunit expressed in seeds of petunia and tobacco was recognized by anti--conglycinin serum at a relative molecular mass of 53 000, equivalent to that of the native protein. Separation of the petunia-seed proteins by isoelectric focusing followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis showed that multiple isoelectric forms of the -subunit were produced. There was approximately a twofold variation in the accumulation of the -subunit protein in the mature seeds of transgenic petunia plants, each containing a single -subunit gene. However, the level of protein accumulation in mature seeds and the amount of -subunit mRNA in developing seeds was not correlated. Accumulation of the -subunit protein in transgenic seeds was less than the -subunit protein that accumulated in transgenic petunia seeds containing a single -subunit gene and less than the amount of the -subunit in mature soybean seeds which contain 8–13 -subunit genes. In transgenic tobacco plants, the accumulation of the -subunit protein in seeds was generally well correlated with the number of genes that were incorporated in the different transformants.Abbreviations kb kilobase - kDa kilodalton - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Quantitative estimation of pancreatic polypeptide (PP) cells of islet tissue of pancreas in Australian mammals

Pancreases from three male and three female echidnas ( Tachyglossus aculeatus ), possums ( Trichosurus vulpecula ), grey kangaroos ( Macropus fuliginosus ), spinifex hopping mice ( Notomys alexis ) and water rats ( Hydromys chrysogaster ) were studied using a point-intercept method and Weibel graticule (Weibel, Kistler & Scherle, 1966) on sections stained by an immunoperoxidase method for pancreatic polypeptide (PP). PP cells were assessed in the head, neck, body and tail regions. There was a greater percentage proportion of PP cells in the head than in the other regions of echidna, possum and water rat. PP cells were not identified in grey kangaroo and spinifex hopping mouse, although control sections were positive.
These results show some agreement with a previously proposed hypothesis (Bonner-Weir & Weir, 1979) concerning the distribution of cell types in the mammalian endocrine pancreas.     

Prostaglandin biosynthesis by human decidual cells: effects of inflammatory mediators

There is substantial evidence that decidual activation, in association with infection, is linked with the onset of both preterm and term labor. We therefore undertook the present study to evaluate prostaglandin production and its potential regulation by inflammatory mediators in human decidual cells in primary monolayer culture. Upon attaining confluence, the cells were incubated with endotoxin, interleukin 1 alpha (IL1 alpha), interleukin 1 beta (IL1 beta); or tumor necrosis factor (TNF). Production of prostaglandin (PG) E2 and PGF2 alpha was determined using specific radioimmunoassays. Endotoxin and these cytokines all induced significant concentration-dependent increases in PGE2 and PGF2 alpha production. Our results suggest that term human decidual cells are responsive to endotoxin and cytokines and that generation of these substances in the decidua or nearby (eg. in response to infection) will lead to increased prostaglandin production and uterine contractions.     

The influence of fixation on the relative amount of cytoplasmic ribosomes in mouse epidermal basal keratinocytes

The nature and significance of so-called dark keratinocytes in the epidermis during chemical carcinogenesis is still a matter of concern and debate. Based on ultrastructural observations it has been suggested that dark cells most often are shrunken cells. Reports on skin carcinogenesis, however, claim that dark cells are a sign of ongoing tumor promotion and represent those stem cells in the epidermis from which the tumors originate. It is therefore important to find out whether these cells are simply injured and shrunken cells, or vital cells of great importance for carcinogenesis. Dark cells are assumed to be rich in ribosomes. There is evidence, however, that the observed number of dark cells is highly dependent on tissue fixation. In the present ultrastructural study, morphometric methods were used to compare the effects of two different fixation procedures on the amount of cytoplasmic ribosomes in dark cells from both untreated and carcinogen-treated hairless mouse epidermis. The results show that the ultrastructural features of both dark and clear cells vary considerably with different fixation procedures. In acetone-treated controls typical dark cells are only observed when the fixative has a lower osmotic activity than the plasma. With iso-osmolal fixation typical dark cells are not observed. After an abortive two-stage carcinogenesis treatment, in which a single application of 9,10-dimethyl-l,2-benzanthracene (DMBA) in acetone was followed by a single application of 12-O-tetradecanoyl-13-acetate (TPA) in acetone, signs of cell injury could be found after both fixation procedures. With DMBA/TPA and hypo-osmolal fixation the number of dark cells seemed to increase, whereas only signs of cell injury with occurrence of some heavily altered “clear cells” dominated the picture with iso-osmolal fixation. Morphometry showed that both the numerical and the volumetric densities of cytoplasmic ribosomes in basal keratinocytes varied most significantly with the fixation procedure used. The cytoplasmic volumes did not vary in a way that could explain these differences. One might therefore assume that the number of ribosomes depends on the fixative. Large swelling artifacts occurred when a fixative with low osmotic activity was used, leading to compression of neighboring cells. Hence, an increased ribosomal density reported previously in dark cells is probably related to such cell volume artifacts and does not reflect an actually increased quantity of ribosomes. With both fixation procedures, a single application of DMBA followed by one of TPA appeared to produce an increased number of ribosomes in basal keratinocytes. When hypo-osmolal fixation was used, however, treatment with DMBA/TPA did not influence the cytoplasmic volume or the numerical density of ribosomes, in dark cells. This might indicate that so-called dark keratinocytes following DMBA/TPA treatment are functionally inactive cells that appear more vulnerable than active cells to compression during hypo-osmolal fixation.