Age-dependent changes in the structure,composition and biophysical properties of a human basement membrane

Basement membranes (BMs) are considered to be uniform, approximately 100 nm-thin extracellular matrix sheets that serve as a substrate for epithelial cells, endothelial cells and myotubes. To find out whether BMs maintain their ultrastructure, protein composition and biophysical properties throughout life the natural aging history of the human inner limiting membranes (ILM) was investigated. The ILM is a BM at the vitreal surface of the retina that connects the retina with the vitreous. Transmission electron microscopy (TEM) showed that the ILM steadily increases in thickness from 70 nm at fetal stages to several microns at age 90. By the age of 20, the ILM loses its laminated structure to become an amorphous and very irregular extracellular matrix layer. Atomic force microscopy (AFM) showed that the native, hydrated ILMs are on average 4-fold thicker than the dehydrated ILMs as seen by TEM and that their thickness is prominently determined by its water-binding proteoglycans. The morphological changes are accompanied by age-related changes in the biochemical composition, whereby the relative concentrations of collagen IV and agrin increase, and the concentration of laminin decreases with age. Force-indentation measurements by AFM also showed that ILMs become increasingly stiffer with advancing age. The data suggest that BMs from other human tissues may undergo similar age-related changes.     

Ultrastructure of the ovary of <Emphasis Type="Italic">Amphilina japonica</Emphasis> Goto &; Ishii, 1936 (Cestoda) and its implications for phylogenetic studies

The ultrastructure of the ovary of the amphilinidean cestode Amphilina japonica Goto & Ishii, 1936 from the body-cavity of the American sturgeon Acipenser transmontanus Richardson is described using transmission electron microscopy. The characters of the ovary of Amphilina japonica are different from those of all other cestodes. The most important difference is in the nature of the relationship between the germ and accessory cells within the ovary. In A. japonica the oocytes and accessory cells form numerous different intercellular contacts (desmosome-like junctions and zonulae adherentes). Gap junctions are present between the narrow cytoplasmic processes of the accessory cells. Numerous micropinocytotic vesicles and vacuoles from the accessory cells discharge their content into spaces between the oocytes and the accessory cells. The accessory cells are closely associated with the oocytes during the early and middle stages of oogenesis. As the volume of oocytes increases, the accessory cells gradually lose their association with the oocyte surfaces. Peripherally located individual accessory cells of A. japonica give rise to a cellular epithelial layer of irregular shape and thickness which breaks down via numerous invaginations of the basal membrane and underlying basal matrix. The different arrangements of the interconnection of cell components in the Amphilinidea compared with the Gyrocotylidea and Eucestoda (the absence of specialised cell contacts and the syncytial nature of the accessory ‘interstitial’ cells) are evidence suggesting the presence of unrelated groups within the Cestoda. The nature of the association of the accessory and germ cells in ovary of A. japonica more closely resembles the ovary of non-platyhelminth invertebrates rather than that of other neodermatans.     

Screening for improved cell performance: selection of subclones with altered production kinetics or improved stability by cell sorting

One of the major problems in the biotechnology industry is the selection of cell lines well suited for production of biopharmaceutical proteins. Usually, the most important selection criterion is the cell specific production rate. Nevertheless, a good producer cell line should have a number of additional advantageous properties, which allow the cell line to perform well in the type of bioreactor chosen for the process. However, the time and work required to select for high production rates as well as the lack of methods to specifically select for other cellular properties, usually prevents researchers from including such criteria into their screening program.With the Single Cell Secretion Assay it is possible to measure the specific production rates of individual cells by catching secreted product in an artificial matrix applied to the cell surface. Flow cytometric cell sorting then allows selection of rare cells with high production rates, which occur at frequencies as low as 10(-6). By combining this method with culture conditions that bring out a desired cellular property, we were able to isolate subclones with similar production rates, but improved performance from a recombinant Chinese hamster ovary cell line producing a human monoclonal antibody. The two desired cellular properties screened for were a non-growth associated production kinetic and improved stability in the absence of selective pressure.     

Requirement for the p75 TNF-alpha receptor 2 in the regulation of airway hyperresponsiveness by gamma delta T cells

In a recent study, we found that TNF-alpha negatively regulates airway responsiveness through the activation of gammadelta T cells. The biological activities of TNF-alpha are mediated by two structurally related but functionally distinct receptors, p55 (TNFR1) and p75 (TNFR2), which are independently expressed on the cell surface. However, the relative importance of either TNFR in airway hyperresponsiveness (AHR) is unknown. To investigate the importance of these TNFRs in the development of allergen-induced AHR, p55-deficient and p75-deficient mice were sensitized to OVA by i.p. injection and subsequently challenged with OVA via the airways; airway responsiveness to inhaled methacholine was monitored. p75-deficient mice developed AHR to a similar degree as control mice. In contrast, p55-deficient mice, which were sensitized and challenged with OVA, failed to develop AHR. In p55-deficient mice, both the numbers of eosinophils and levels of IL-5 in bronchoalveolar lavage fluid were significantly lower than in sensitized/challenged control mice (p < 0.05). However, depletion of gammadelta T cells resulted in significant increases in AHR in the p55-deficient mice, whereas no significant effect of gammadelta T cell depletion was evident in the p75-deficient mice. These data indicate that, in the absence of TNFR1 (p55), where TNF-alpha uses the p75 pathway exclusively, the development of AHR is regulated by gammadelta T cells.     

Cloning and analysis of the mating type genes from Cochliobolus heterostrophus

Cochliobolus heterostrophus, a heterothallic Ascomycete, has a single mating type locus with two alternate forms called MAT-1 and MAT-2. MAT-1 was cloned by complementing a MAT-2 strain using a cosmid library from a MAT-1 strain and screening for a homothallic transformant. The cosmid recovered from this transformant was able to re-transform a MAT-2 strain to homothallism and MAT identity was proven by restriction fragment length polymorphism and conventional genetic mapping. All homothallic transformants could mate with either MAT-1 or MAT-2 strains, although the number of ascospores produced by self matings or crosses to MAT-2 strains was low. Progeny of selfed homothallic transformants were themselves homothallic. MAT-2 was cloned by probing a cosmid library from a MAT-2 strain with a fragment of insert DNA from the MAT-1 cosmid. A 1.5 kb subclone of either MAT-containing cosmid was sufficient to confer mating function in transformants. Examination of the DNA sequence of these subclones revealed that MAT-1 and MAT-2 contain 1297 by and 1171 bp, respectively, of completely dissimilar DNA flanked by DNA common to both mating types. Putative introns were found (one in each MAT gene) which, when spliced out, would yield open reading frames (ORFs) that occupied approximately 90% of the dissimilar DNA sequences. Translation of the MAT-1 ORF revealed similarity to the Neurospora crassa MATA, Podospora anserina mat–, and Saccharomyces cerevisiae MAT1 proteins; translation of the MAT-2 ORF revealed similarity to the N. crassa MATa, P. anserina mat+, and Schizosaccharomyces pombe mat-Mc proteins. These gene products are all proven or proposed DNA binding proteins. Those with similarity to MAT-2 are members of the high mobility group.The first three authors contributed equally to the work     

Colour receptors in the eye of the digger wasp,Sphex cognatus Smith: Evaluation by selective adaptation

Summary Pigment granule migration in pigment cells and retinula cells of the digger wasp Sphex cognatus Smith was analysed morphologically after light adaptation to natural light, dark adaptation and after four selective chromatic adaptations in the range between 358 nm and 580 nm and used as the index of receptor cell sensitivity. The receptor region of each ommatidium consists of nine retinula cells which form a centrally located rhabdom. Two morphologically and physiologically different visual units can be described, defined by the arrangement of the rhabdomeric microvilli, the topographical relationship of the receptor cells with respect to the eye axes and the unique retinula cell screening pigmentation. These two different sets of ommatidia (type A and B) are randomly distributed in a ratio of 13 throughout the eye (Ribi, 1978b). Chromatic adaptation experiments with wavelengths of 358 nm, 443 nm, 523 nm and 580 nm and subsequent histological examination reveal two UV receptors, two blue receptors and four yellow-green receptors in type A ommatidia and two UV receptors and six green to yellow-green receptors in type B ommatidia. The pigments in cells surrounding each ommatidium (two primary pigment cells, 20 secondary pigment cells and four pigmented cone extensions) were not affected significantly by the adaptation experiments.     

The significance of leaf pubescence for the absorption of global radiation by Tussilago farfara L.

Summary The spectral properties of juvenile and adult leaves of Tussilago farfara L. were measured for the wave range from 400 to 1350 nm. The significance of leaf pubescence for the absorption of global radiation was determined. The absorption is lowered by epidermal harrs only for the visible part of the spectrum (400–1350 nm). Absorptivities of the infrared do not vary whether hairs are present or not. The spectral properties of the upper and the lower surfaces without hairs are not equal. The hairs on the lower surface increase the amount of infrared radiation that is reflected by the upper surface. As shown by calculations of the absorbed radiation, pubescence has little influence on energy input. If leaves are put in an inverse position, the absorption of the global radiation energy is lowered by 15% compared with the same leaf in the regular position.     

Internal amino acid sequencing of proteins by in situ cyanogen bromide cleavage in polyacrylamide gels

A new method was developed for generating peptide fragments for amino acid sequence analysis from polyacrylamide-gel separated proteins. This method involves in situ CNBr treatment of proteins in the polyacrylamide gel after their separation by electrophoresis. Pure CNBr peptides were recovered either by solvent extraction followed by microbore column reversed-phase HPLC or, alternatively, by a second electrophoretic separation step (SDS-PAGE) followed by electrotransfer of the peptides onto polyvinylidene difluoride (PVDF) membranes. These approaches yielded sequence data at subnanomole levels for a wide range of CNBr fragments recovered from gel-separated proteins.     

Different potentials of gamma delta T cell subsets in regulating airway responsiveness: V gamma 1+ cells, but not V gamma 4+ cells, promote airway hyperreactivity, Th2 cytokines, and airway inflammation

Allergic airway inflammation and hyperreactivity are modulated by gammadelta T cells, but different experimental parameters can influence the effects observed. For example, in sensitized C57BL/6 and BALB/c mice, transient depletion of all TCR-delta(+) cells just before airway challenge resulted in airway hyperresponsiveness (AHR), but caused hyporesponsiveness when initiated before i.p. sensitization. Vgamma4(+) gammadelta T cells strongly suppressed AHR; their depletion relieved suppression when initiated before challenge, but not before sensitization, and they suppressed AHR when transferred before challenge into sensitized TCR-Vgamma4(-/-)/6(-/-) mice. In contrast, Vgamma1(+) gammadelta T cells enhanced AHR and airway inflammation. In normal mice (C57BL/6 and BALB/c), enhancement of AHR was abrogated only when these cells were depleted before sensitization, but not before challenge, and with regard to airway inflammation, this effect was limited to C57BL/6 mice. However, Vgamma1(+) gammadelta T cells enhanced AHR when transferred before challenge into sensitized B6.TCR-delta(-/-) mice. In this study Vgamma1(+) cells also increased levels of Th2 cytokines in the airways and, to a lesser extent, lung eosinophil numbers. Thus, Vgamma4(+) cells suppress AHR, and Vgamma1(+) cells enhance AHR and airway inflammation under defined experimental conditions. These findings show how gammadelta T cells can be both inhibitors and enhancers of AHR and airway inflammation, and they provide further support for the hypothesis that TCR expression and function cosegregate in gammadelta T cells.