The association of microtubules with the plasmalemma in epidermal tendon cells of the river crab

The mode of association of microtubules (MTs) with the plasmalemma in epidermal tendon cells of the river crab, Potamon dehaani was studied by thin-section electron microscopy. In the leg muscle, the tendon cells connect striated muscle cells with the cuticle, forming specialized junctions at both ends. At the muscle-tendon cell junction, the apposed plasmalemmas are interdigitated in a zig-zag pattern separated by a uniform space of about 50 nm, where the basal lamina is shared by two cells. At the tendon cell-cuticle junction, the plasmalemma of the tendon cell forms many conical invaginations, into which dense fibrous material extends from the cuticle. Inside the tendon cell, numerous microtubules run parallel to the direction of tension transmission and are arranged into parallel bundles of various sizes. Within such bundles, fine filamentous structures cross-link adjacent MTs. MTs span the entire length of the cell and attach at their both ends to the junctional domains of the plasmalemma. The junctional plasmalemma is characterized by formation of an electron-dense undercoat, through which MTs are connected with the plasmalemma proper. The ultrastructural features of MT association with the plasmalemma are basically the same at both junctions. At the junctions, MTs usually terminate with free ends and are linked laterally to the plasmalemmal undercoat with fine filamentous structures. These observations emphasize the role of the plasmalemmal undercoat as a device of the attachment of MTs to the plasmalemma.     

The matrix form of collagen and basal microporosity influence basal lamina deposition and laminin synthesis/secretion by stratified human keratinocytes in vitro

Summary The ability of the collagen matrix form to support the formation of a basal lamina by cultured normal human epidermal keratinocytes (NHEK) was determined using transmission electron microscopy. The collagen matrix forms tested in this study were a) a dry type I collagen film and b) a type I collagen gel. NHEK were grown for 14 days on the following five different substrates: plain plastic culture dishes without the addition of collagen (PP); plain plastic culture dishes overlaid with a dry, aldehyde-crosslinked type I collagen film (DCF-P); plain plastic culture dishes overlaid with an aldehyde-crosslinked type I collagen gel (GEL-P); Millipore Millicell CM microporous membranes overlaid with a dry, aldehyde-crosslinked type I collagen film (DCF-CM); and Millipore Millicell CM microporous membranes overlaid with an aldehyde-crosslinked type I collagen gel (GEL-CM). NHEK maintained for 2 wk on PP and DCF-P were unable to secrete a basal lamina. NHEK grown for 2 wk on the GEL-P and GEL-CM substrates, however, secreted a contiguous basal lamina at the GEL-NHEK interface. To determine if the appearance of this basal lamina correlated with laminin synthesis, laminin was immunoprecipitated from cellular extracts, as well as media from the apical and basal chambers. NHEK grown on the GEL-P substrate synthesized more laminin than did NHEK grown on the other four alternative substrates. In addition, NHEK grown on GEL-CM were able to direct more laminin to the basal compartment than NHEK grown on DCF-CM substrates. Taken together, the data indicate that the matrix form of collagen can influence basal lamina deposition, laminin synthesis, and laminin trafficking in NHEK.     

The commercial and agricultural applications of animal transgenesis

The potential for commercial application of transgenic technologies in domestic animals is discussed in relation to the areas where a significant impact on agriculture might be expected. These are the endocrine system, novel biochemical pathways, structural proteins of milk and of textile fibers, and the immune system. Manipulation of the endocrine system has been investigated for some years and it is clear that very accurate control is needed over gene expression if this approach is to prove commercially useful. The area most advanced in commercial application is the production of high-value pharmaceutical proteins in the mammary glands of domestic animals. Other applications that are discussed remain to be proven in larger animals despite being demonstrated laboratory test animals. These include a functional cysteine biosynthetic pathway and a functional glyoxylate cycle transferred from bacteria to mice, and alterations to the proteins of hair that change the physical properties of the resultant fibers. Research is also actively directed toward novel approaches for providing domestic animals with resistance to insects.     

Strong Enrichment of Aromatic Residues in Binding Sites from a Charge-neutralized Hyperthermostable Sso7d Scaffold Library

The Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus is an attractive binding scaffold because of its small size (7 kDa), high thermal stability (T_m of 98 °C), and absence of cysteines and glycosylation sites. However, as a DNA-binding protein, Sso7d is highly positively charged, introducing a strong specificity constraint for binding epitopes and leading to nonspecific interaction with mammalian cell membranes. In the present study, we report charge-neutralized variants of Sso7d that maintain high thermal stability. Yeast-displayed libraries that were based on this reduced charge Sso7d (rcSso7d) scaffold yielded binders with low nanomolar affinities against mouse serum albumin and several epitopes on human epidermal growth factor receptor. Importantly, starting from a charge-neutralized scaffold facilitated evolutionary adaptation of binders to differentially charged epitopes on mouse serum albumin and human epidermal growth factor receptor, respectively. Interestingly, the distribution of amino acids in the small and rigid binding surface of enriched rcSso7d-based binders is very different from that generally found in more flexible antibody complementarity-determining region loops but resembles the composition of antibody-binding energetic hot spots. Particularly striking was a strong enrichment of the aromatic residues Trp, Tyr, and Phe in rcSso7d-based binders. This suggests that the rigidity and small size of this scaffold determines the unusual amino acid composition of its binding sites, mimicking the energetic core of antibody paratopes. Despite the high frequency of aromatic residues, these rcSso7d-based binders are highly expressed, thermostable, and monomeric, suggesting that the hyperstability of the starting scaffold and the rigidness of the binding surface confer a high tolerance to mutation.     

A novel microscopy-based assay identifies extended synaptotagmin-1 (ESYT1) as a positive regulator of anoctamin 1 traffic

An attractive possibility to treat Cystic Fibrosis (CF), a severe condition caused by dysfunctional CFTR, an epithelial anion channel, is through the activation of alternative (non-CFTR) anion channels. Anoctamin 1 (ANO1) was demonstrated to be a Ca²⁺-activated chloride channel (CaCC) and thus of high potential to replace CFTR. Despite that ANO1 is expressed in human lung CF tissue, it is present at the cell surface at very low levels. In addition, little is known about regulation of ANO1 traffic, namely which factors promote its plasma membrane (PM) localization.Here, we generated a novel cellular model, expressing an inducible 3HA-ANO1-eGFP construct, and validated its usage as a microscopy tool to monitor for ANO1 traffic.We demonstrate the robustness and specificity of this cell-based assay, by the identification of siRNAs acting both as ANO1 traffic enhancer and inhibitor, targeting respectively COPB1 and ESYT1 (extended synaptotagmin-1), the latter involved in coupling of the endoplasmic reticulum to the PM at specific microdomains. We further show that knockdown of ESYT1 (and family members ESYT2 and ESYT3) significantly decreased ANO1 current density.This ANO1 cell-based assay constitutes an important tool to be further used in high-throughput screens and drug discovery of high relevance for CF and cancer.     

Effects of temperature on parameters of root growth relevant to nutrient uptake: Measurements on oilseed rape and barley grown in flowing nutrient solution

Summary Effects of root temperature on the growth and morphology of roots were measured in oilseed rape (Brassica napus L.) and barley (Hordeum vulgare L.). Plants were grown in flowing solution culture and acclimatized over several weeks to a root temperature of 5°C prior to treatment at a range of root temperatures between 3 and 25°C, with common shoot temperature. Root temperature affected root extension, mean radius, root surface area, numbers and lengths of root hairs. Total root length of rape plants increased with temperature over the range 3–9°C, but was constant at higher temperatures. Root length of barley increased with temperature in the range 3–25°C, by a factor of 27 after 20 days. Root radii had a lognormal distribution and their means decreased with increasing temperature from 0.14 mm at 3°C to 0.08 mm at 25°C. The density of root hairs on the root surface increased by a factor of 4 in rape between 3 and 25°C, but in barley the highest density was at 9°C. The contribution of root hairs to total root surface area was relatively greater in rape than in barley. The changes in root system morphology may be interpreted as adaptive responses to temperature stress on nutrient uptake, providing greater surface area for absorption per unit root weight or length.