Collagen and proteoglycan in a sea urchin ligament with mutable mechanical properties

Summary The problematic ligament of sea urchins is a connective tissue which crosses the ball-and-socket joint between spine and body wall. The problem of this ligament is that it is composed of parallel collagen fibrils, yet normally undergoes rapid and dramatic alterations in mechanical properties and in length. Previous work has suggested that the collagen fibrils of the ligament are able to slide past one another during length changes but are inhibited from sliding when the ligament is in catch. In this model of the ligament both the collagen fibrils and the interfibrillar matrix are mechanically important. We have found that the collagen fibrils of the spine ligament of the pencil urchin Eucidaris tribuloides are discontinuous and end by tapering within the body of the ligament. Intact fibrils that have been isolated from the ligament vary by more than an order of magnitude in length and in radius but have a constant length/radius (aspect) ratio of about 5300. This is the first determination of the aspect ratio of collagen fibrils from any source. The constant aspect ratio of the fibrils is consistent with their functioning as the discontinuous fiber phase in a fiber-reinforced composite material, while the high value of the aspect ratio indicates that the nonfibrillar matrix, which must act to transfer stress between fibrils, can produce a stiff and strong ligament even if it is several orders of magnitude weaker and more compliant than the fibrils. Moreover, the tensile properties of the ligament may be determined by the properties of the matrix. A prominent component of the interfibrillar matrix is a proteoglycan which associates with specific bands at the surface of the collagen fibrils through noncovalent binding of its core protein. The glycosaminoglycan moiety of this proteoglycan is partly comprised of chondroitin sulfate/dermatan sulfate polymers. These results are consistent with the sliding fibril hypothesis and suggest that the proteoglycan may be an important component of the stress-transfer matrix.     

Electron microscopical microprobe analysis of freeze dried and unstained mineralized epiphyseal cartilage

Summary Electron micrographs have been taken of unfixed, freeze dried, unstained epiphyseal cartilage. In the mineralized long septa round to elliptic clusters (up to 0.6 m in diameter) consisting mainly of dots and needles could be observed. The clusters were surrounded by microareas with a low contrast consisting mainly of ribbon plate-like crystallites. With the aid of scanning mode (STEM) of a transmission electron microscope, equipped with a Si(Li)detector system, both regions were analyzed for calcium and phosphorus by electronprobe X-ray microanalysis. In ten series of 106 measurements in each region, it could be determined by registration of the CaK and PKX-ray counts that the mineral content in the clusters was in the range of 30–100 % higher than in the light regions. The question of the sequence of the epiphyseal plate mineralization is discussed and whether the dense clusters represent the mineralized matrix vesicles.The authors thank the Deutsche Forschungsgemeinschaft for financial supportDedicated to Professor Dr. G. Pfefferkorn on the occasion of his 65th birthday     

Sea snake (Microcephalophis gracilis) hemoglobin: Primary structure and relationships to other forms

The hemoglobin of the sea snakeMicrocephalophis gracilis was purified and the primary structure of the and chains determined. This is the first sea snake hemoglobin structure characterized, and apparently also the first complete structure of any snake hemoglobin (an chain of a viper was known), allowing judgments of reptilian variants. Variations between the sea snake form and other reptilian forms are large (52–65 differences for the chains), of similar order as those between the sea snake and avian (56–65 differences) or human (58 differences) forms. Functionally, 19 residues at / contact areas and 7 at heme contacts are exchanged in relation to the human and chains. Four positions of the sea snake hemoglobin contain residues thus far unique to this form. However, all replacements appear compatible with conserved overall functional properties.     

Type-VI collagen in the human iris and ciliary body

Summary The distribution of type-VI collagen in the human iris and ciliary body was investigated by means of immunohistochemical techniques and compared with that of type-IV collagen, fibronectin and laminin. As has been described for other tissues, type-VI collagen surrounds type-I and-III collagen fibers. The aggregated from of type-IV collagen (the long-spacing or curly collagen), which has already been described in the trabecular meshwork and sclera, was also observed at the ciliary muscle tips surrounding the anterior elastic tendons of this muscle. In addition, staining for type-VI collagen was seen directly adjacent to the basement membranes of the ciliary muscle cells, the iris muscles, the uveal vascular endothelia and nerves, but not adjacent to the epithelial basement membranes. The staining did not form a discrete line like the immunoreaction for type-IV collagen, but bundles of marked fibrils extended into the surrounding connective tissue. We assume that type-VI collagen similar to type-VII collagen forms part of an anchoring system for these tissues. As type-VII collagen has been described only in connection with epithelial basement membranes, both type-VI and type-VII collagens may represent anchoring fibrils, however for different tissue components.     

Metal Cations Defibrillize the Amyloid Beta-Protein Fibrils

Amyloid beta-protein (A) is the major constituent of amyloid fibrils composing -amyloid plaques and cerebrovascular amyloid in Alzheimer's disease (AD). We studied the effect of metal cations on preformed fibrils of synthetic A by Thioflavin T (ThT) fluorescence spectroscopy and electronmicroscopy (EM) in negative staining. The amount of cross beta-pleated sheet structure of A 1–40 fibrils was found to decrease by metal cations in a concentration-dependent manner as measured by ThT fluorescence spectroscopy. The order of defibrillization of A 1–40 fibrils by metal cations was: Ca²⁺ and Zn²⁺ (IC₅₀ = 100 M) > Mg²⁺ (IC₅₀ = 300 M) > Al³⁺ (IC₅₀ =1.1 mM). EM analysis in negative staining showed that A 1–40 fibrils in the absence of cations were organized in a fine network with a little or no amorphous material. The addition of Ca²⁺, Mg²⁺, and Zn²⁺ to preformed A 1–40 fibrils defibrillized the fibrils or converted them into short rods or to amorphous material. Al³⁺ was less effective, and reduced the fibril network by about 80 % of that in the absence of any metal cation. Studies with A 1–42 showed that this peptide forms more dense network of fibrils as compared to A 1–40. Both ThT fluorescence spectroscopy and EM showed that similar to A 1–40, A 1–42 fibrils are also defibrillized in the presence of millimolar concentrations of Ca²⁺. These studies suggest that metal cations can defibrillize the fibrils of synthetic A.     

Three-dimensional organization of fibroblasts and collagen fibrils in rat tail tendon

Summary The orderly arrangement of fibroblasts and collagen in tendons and ligaments suggests that these cells may have precise relationships with one another and with the collagen fibrils. The spatial organization of rat tail tendon was therefore examined using scanning and transmission electron microscopy and by reconstructing a 35-m long segment of tendon from serial transmission electron micrographs. Fibroblasts were regularly arranged in columns and showed more intimate association in the longitudinal than in the transverse plane. Thin cytoplasmic sheets extended up to 3 m transversely, frequently forming junctional attachments with similar processes from adjacent cells or from the same cell. Longitudinal processes were longer, often extending for more than 20 m and forming junctional attachments with other cells in the same column. Such processes often exhibited invaginations in which there were single fibrils or small groups of fibrils; this arrangement may be indicative of fibril elongation or may serve to transmit tension between the fibroblast and the collagen fibrils. This organization has interesting implications for the growth and function of other fibrous connective tissue, such as the periodontal ligament.     

Comparative residual toxicities of pesticides to the predator Agistemus industani (Acari: Stigmaeidae) on citrus in Florida

Residual toxicities of registered and selected experimental pesticides used on citrus against Agistemus industani Gonzalez (Acari: Stigmaeidae) were compared. Pesticides considered highly toxic to A. industani were: abamectin 0.15 EC at 731ml/ha+FC 435-66 petroleum oil at 46.8l/ha, pyridaben 75WP at 469g/ha, ethion 4EC at 7.01l/ha+FC 435-66 petroleum oil at 46.8l/ha, propargite 6.55 EC at 3.51l/ha, chlorfenapyr 2SC at 1.46l/ha applied alone or in combination with FC 435-66 petroleum oil at 46.8l/ha, sulphur 80DF at 16.81kg/ha, dicofol 4EC at 7.01l/ha, fenbutatin oxide 50WP at 2.24kg/ha, benomyl 50WP at 2.24kg/ha, benomyl 50WP at 1.68kg/ha+ferbam 76 GF at 5.60kg/ha, ferbam 76GF at 11.21kg/ha, neem oil 90EC at 46.8l/ha, and copper hydroxide DF (40% metallic copper) at 4.48kg metallic copper/ha+FC 435-66 petroleum oil at 46.8l/ha. Pesticides that were moderately to slightly toxic included: copper sulphate 98% at 4.48kg metallic copper/ha+FC 435-66 petroleum oil at 46.8l/ha, fenbuconazole 2F at 280ml/ha+FC 435-66 petroleum oil at 46.8l/ha, FC 435-66 petroleum oil applied alone at 46.8l/ha or 23.4l/ha, and diflubenzuron 25WP at 1.40kg/ha. Pesticides that were non-toxic included: fenbuconazole 2F at 585ml/ha, malathion 57EC at 5.85l/ha, FC 435-66 petroleum oil at 46.8l/ha, carbaryl 80S at 3.36kg/ha, chlorpyrifos 4EC at 4.68l/ha, and formetanate 92SP at 1.12kg/ha. Understanding the toxic effects of field weathered pesticides against key predacious mite species is important for effective IPM. The results of this study provide a comparison of direct and indirect toxic effects of various pesticides to A. industani under field conditions.     

Binding of the galactose-specificPseudomonas aeruginosa lectin,PA-I,to glycosphingolipids and other glycoconjugates

The carbohydrate-binding specificity ofPseudomonas aeruginosa lectin I (PA-I) in iodinated or biotinylated form was studied. A large number of glycosphingolipids, as well as some glycoproteins and neoglycoproteins were used as ligands. Also, inhibition by free saccharides of PA-I binding to glycosphingolipids was tested. It was found that the lectin binds most strongly to terminal and nonsubstituted Gal3Gal- or Gal4Gal-structures.Abbreviations PA-I Pseudomonas aeruginosa lectin I - Cer ceramide - lactosylceramide Gal4GlcCer - iso globotriaosylcerami Gal3Gal4GlcCer - globotriaosylceramide Gal4Gal4GlcCer - globoside or globotetraosylceramide GalNAc3Gal4Gal4GlcCer - Forssman glycolipid GalNAc3GalNAc3Gal4Gal4GlcCer - P1 glycolipid Gal4Gal4GlcNAc3Gal4GlcCer - lactoneotetraosylceramide Gal4GlcNAc3Gal4GlcCer - B5 glycolipid Gal3Gal4GlcNAc3Gal4GlcCer - gangliotetraosylceramide Gal3GalNAc4Gal4GlcCer - GM1 Gal3GalNAc4(NeuAc3)Gal4GlcCer - RBC red blood cells - BSA bovine serum albumin - PBS phosphate-buffered saline - SDS sodium dodecyl sulfate - TLC thin-layer chromatography - HPLC high pressure liquid chromatography - MS mass spectrometry - FAB fast-atom bombardment - EI electron impact     

The expression of collagen mRNAs in normally developing neonatal rabbit long bones and after treatment of neonatal and adult rabbit tibiae with transforming growth factor-β2

Summary Normal transverse growth of long bones is by periosteal appositional bone formation, balanced by endosteal resorption. Changes in the distribution of cells that are expressing collagen mRNAs during growth were determined using digoxigeninlabelled riboprobes. In neonatal rabbit tibiae osteoblasts expressing type I collagen mRNA are found on periosteal, and at early stages on endosteal, bone surfaces and lining peripheral cavities. Occasional osteocytes express type I collagen mRNA very weakly. The pattern is disrupted when transforming growth factor-2 (TGF-2) is injected daily into the periosteum of neonatal animals; there is increased bone, and later cartilage, formation. Three injections of 20 ng TGF-2 onto the tibia of 3-day-old rabbits led to an increase of periosteal osteoblasts that express the mRNA for type I collagen. Some endosteal osteoblasts and osteocytes in newly-formed peripheral woven bone also express the mRNA. After five injections chondrocytes expressing type II collagen mRNA are found around the injection site. Similar injections of TGF-2 in old rabbits induce only fibrous tissue within which some cells express type I collagen mRNA. This precise localization of mRNAs shows that the expression of type I or II collagen mRNA is here restricted to osteoblasts and chondrocytes, respectively.     

Alzheimer’s Disease: Soluble Oligomeric Aβ(1–40) Peptide in Membrane Mimic Environment from Solution NMR and Circular Dichroism Studies

Amyloid beta peptide (A) is a small peptide present in normal cells and aggregated A is the main constituent of the extracellular amyloid plaques found in Alzheimers disease (AD) brain. Recent studies suggest that soluble A oligomers are neurotoxic rather than amyloid fibrils found in amyloid plaques. This study using multidimensional NMR spectroscopy and circular dichroism (CD) provides the first direct evidence that alterations in membrane structure can trigger the conversion of soluble -helical monomeric A into oligomeric A in a -sheet conformation.     

Melatonin enhances Th2 cell mediated immune responses: Lack of sensitivity to reversal by naltrexone or benzodiazepine receptor antagonists

Chronic administration of melatonin for 5 days to antigen-primed mice increased the production of pro-inflammatory cytokine IL10 but decreased the secretion of antiinflammatory cytokine TNF-. These results further confirm that melatonin activates Th2like immune response. Whether melatoninmediated Th2 response is dependent on opioid or central and peripheral benzodiazepine receptors was also examined. Hence, melatonin was administered to antigen-sensitised mice with either naltrexone (a opioid receptor antagonist) or flumazenil (a central benzodiazepine receptor antagonist) or PK11195 (a peripheral benzoidiazepine receptor antagonist). No significant difference in melatonin-induced Th2 cell response was observed by naltrexone, flumazenil or PK11195 treatment. These findings suggest that the Th2 cell response induced by melatonin in antigen sensitised mice neither dependent on endogenous opioid system nor is modulated through the central or peripheral benzodiazepine receptors.     

Freeze-fracture analysis of junctional complexes in the nephron of the garter snake,Thamnophis sirtalis

Summary Zonulae occludentes are shown by freeze-fracture to be pleomorphic along the garter snake nephron. In the neck and proximal segments the occluding junctions are moderately complex with frequent discontinuities in their junctional fibrils. Junctional depth and complexity are maximal in the distal and collecting segments and discontinuities in fibrils are absent. Comparison of these results with similar observations on other tissues indicates that the zonulae occludentes in the neck and proximal segments are intermediate to leaky and that they may be very tight in the distal and collecting segments. These findings suggest that in the garter snake nephron transepithelial flow of fluid may occur primarily by passive diffusion through the zonulae occludentes in the neck and proximal segments and by cell-mediated osmotic flow in the distal and collecting segments. Gap junctions occur only in the proximal tubule and are probably involved in low resistance, intercellular movement of ions.Supported by the National Research Council of Canada. The authors wish to acknowledge the generous provision of freeze-fracture facilities by Dr. M.W. Brightman, NINDS, National Institutes of Health, Bethesda, Maryland, U.S.A.     

In vivo effects of insulin and bis(maltolato)oxovanadium (IV) on PKB activity in the skeletal muscle and liver of diabetic rats

In this study, the in vivo effects of insulin and chronic treatment with bis(maltolato)oxovanadium (IV) (BMOV) on protein kinase B (PKB) activity were examined in the liver and skeletal muscle from two animal models of diabetes, the STZdiabetic Wistar rat and the fatty Zucker rat. Animals were treated with BMOV in the drinking water (0.75–1 mg/ml) for 3 (or 8) weeks and sacrificed with or without insulin injection. Insulin (5 U/kg, i.v.) increased PKB activity more than 10fold and PKB activity more than 3fold in both animal models. Despite the development of insulin resistance, insulininduced activation of PKB was not impaired in the STZdiabetic rats up to 9 weeks of diabetes, excluding a role for PKB in the development of insulin resistance in type 1 diabetes. Insulin-induced PKB activity was markedly reduced in the skeletal muscle of fatty Zucker rats as compared to lean littermates (fatty: 7fold vs. lean: 14fold). In contrast, a significant increase in insulinstimulated PKBa activity was observed in the liver of fatty Zucker rats (fatty: 15.7fold vs. lean: 7.6fold). Chronic treatment with BMOV normalized plasma glucose levels in STZdiabetic rats and decreased plasma insulin levels in fatty Zucker rats but did not have any effect on basal or insulininduced PKB and PKB activities. In conclusion (i) in STZdiabetic rats PKB activity was normal up to 9 weeks of diabetes; (ii) in fatty Zucker rats insulininduced activation of PKB (but not PKB) was markedly altered in both tissues; (iii) changes in PKB activity were tissue specific; (iv) the glucoregulatory effects of BMOV were independent of PKB activity.     

Immunohistochemical characterization of nurse cells in normal human thymus

Summary Lymphoepithelial complexes known as thymic nurse cells (TNC) have been isolated and described in the thymus of several animal species including man. Most of the investigations on TNC have been carried out in enzymatically digested thymuses in which TNC were isolated by differential sedimentation. In the present study we demonstrate TNC in immunohistochemically stained sections of human thymus as ring-shaped cells completely enclosing thymocytes and localized not only in the cortex, but also at the corticomedullary junction where they have not been previously described. TNC expressed epithelial markers [low and high molecular weight keratins identified by 35H11 and 34E12 monoclonal antibodies, a cortical antigen shared with neuroectodermal neoplasms recognized by the GE2 monoclonal antibody, and tissue polypeptide antigen (TPA:B₁)], class II histocompatibility antigens (HLA-DR), and thymosin ₁. Double staining experiments with the nuclear proliferation-associated antigen Ki-67 and the cortical epithelium marker GE2 showed that most thymocytes enclosed in these cortical TNC were not proliferating. The antigens expressed by TNC indicate that not only cortical, but also medullary epithelial cells are part of the TNC system. The possible role of TNC in the education and maturation of thymocytes is discussed.     

Electron microscopy and electron microscopical measurements of collagen mineralization in hard tissues

Summary The best focussed members of a through focus series of electron micrographs of ultra-thin sections of freeze-dried forming rat incisor dentine and glutaraldehyde-fixed 10 months old human femoral cortex were selected for measurements. The original micrographs were prepared at magnifications of x20000 or x40000 and were photographically enlarged to a magnification from x200000 to x600000. The number of dot-like calcium phosphate nuclei per hole zone and per overlapping zone of the 64 nm collagen macro-period was determined; we found a range of 6–8 nuclei per hole zone and 3–5 nuclei per overlapping zone. Thus a total of 9–13 nuclei per 64 nm macro-period was found; this is in agreement with the total number of cross bandings per macro-period reported by previous authors. The distances between neighbouring dot-like calcium phosphate nuclei were measured and found to lie in the range of 4.0 to 8.5 nm, which is the range in which most of the distances between the collagen cross-bandings appear. The distances between close, parallel calcium phosphate rows within the collagen fibres were found to lie in the range 4.6–8.0 nm which seems to be a good deal shorter than the distances between the typical microholes of the Hodge-Petruska collagen model. Our results and ideas concerning the active sites of nucleation also are in agreement with previous amino acid sequence analyses in respect of the occurrence of neighbouring polar amino acids which would be free to bind ions.We thank Professor S. Heuck for valuable bone specimens. We thank Frau G. Neinhardt, Frau R. Höhling and Mr. P. S. Reynolds for their valuable technical assistance and Mr. H. Pittorf for building the metal model of Fig. 10. We thank the Deutsche Forschungsgemeinschaft and the Medical Research Council for their financial support.     

Molecular switch of F0F1-ATP synthase,G-protein,and other ATP-driven enzymes

Exchange-out of amide tritium from labeled -subunit of ₃₃ complex of F₀F₁-ATP synthase was not accelerated by ATP, suggesting that hemagglutinin-type transition of coiled-coil structure did not occur in -subunit. Local topology of nucleotide binding site and switch II region of G-protein resemble those of F₁- subunit and other proteins which catalyze ATP-triggered reactions. Probably, binding of nucleotide to F₀F₁-ATP synthase induces conformational change of the switch II-like region with transforming subunit structure from open to closed form and this transformation results in loss of hydrogen bonds with the subunit, thus enabling the subunit to move.     

Flexibility on the karyotype evolution in bitterlings (Pisces, Cyprinidae)

In bitterlings (Acheilognathinae) C- and Ag-banding karyotypes of 6 species-subspecies collected in China and South Korea were analyzed. The chromosomal constitution of 2n=46 (4SM+42ST) in Rhodeus atremius fangi was quite different from that of 2n=48 (8M+20SM+20ST) in other species-subspecies in Rhodeus. It was concluded from the analysis of banded chromosomes that the increase in number of ST during the karyotype change from 2n=48 to 2n=46 was achieved by a series of pericentric inversions from 24 M-SM to 24 ST, and the decrease in the diploid number was caused by an additional tandem fusion of 4 ST chromosomes, forming a new ST pair in the 2n=46 karyotype. The karyotype of Tanakia koreensis, T. signifer, and Acheilognathus macropterus is 2n=48 (8M+20SM+20ST), 2n=48 (8M+20SM+14–16ST+4–6 A), 2n=44 (14M+16SM+14ST), respectively. In R. ocellatus ocellatus, T. koreensis, T. signifer and A. macropterus, karyotype changes from 2n=48 to 2n=44 due to centric fusion and inversion have also been estimated. It was suggested that C-banding heterochromatin was greatly concerned with the karyotype evolution in bitterlings.     

Temperature and moisture effects on C and N mineralization from surface applied clover residue

A better understanding of the effect of temperature (T) and moisture on soil microbial activity should improve our ability to predict N mineralization from soil organic matter and crop residues. The objective of this study was to evaluate the effects of water potential () and T on C and N mineralization from unamended Cecil loamy sand soil (clayey, kaolinitic, thermic Typic Kanhapludult) and from crimson clover (Trifolium incarnatum L.) residues applied on the soil surface. Cecil soil was packed into acrylic plastic cylinders, adjusted to -5.0, -1.5, -0.03, or -0.003 MPa, treated with clover residues on the surface or left unamended, and incubated at 10, 20, 28, or 35°C for 21 d. Headspace gas samples for CO2 and N2O determinations were taken periodically and NH3 evolved was trapped. Inorganic N in soil and residue extracts was analyzed after 21 d. When increased from -5.0 to -0.003 MPa, total CO2 evolved from unamended soil increased linearly with ln(-), whereas total CO2 evolved from clover residue increased exponentially with . In both cases the effect of was enhanced as T increased. Two-dimensional (T, ) equations were developed to describe these effects. Apparent net mineralized N from the clover residue increased with until it reached a maximum between -0.5 and -0.03 Mpa.     

Steroid-induced regulation of 3α,20β- and 3β,17β-hydroxysteroid dehydrogenase activity in wild type and mutants of Streptomyces hydrogenans

The effect of estradiol, hydrocortisone and progesterone on 3,20-and 3,17-hydroxysteroid dehydrogenase (HSD) in mutants of Streptomyces hydrogenans was compared to the steroid response of the wild type. Mutants were defective in arginine biosynthesis and/or aerial mycelial formation and lacked both enzymes or only 17-HSD. Some 17-HSD mutants had lost the ability to be induced by estradiol, by progesterone or by both. Some 20-HSD mutants had lost the ability to be induced by hydrocortisone, by progesterone or by both. Non-inducibility of 17-and 20-HSD by progesterone was not co-ordinate. An additional study of the growth phase-dependent enzyme activity of the wild type after induction with estradiol, hydrocortisone and progesterone was performed.Non-standard abbreviations 17-HSD 3,17-Hydroxysteroid dehydrogenase (EC 1.1.1.51) - 20-HSD 3,20-hydroxysteroid dehydrogenase (EC 1.1.1.53) - AO acridine orange - EBr ethidium bromide - EMS ethyl methanesulfonate - MNNG N-methyl-N-nitro-N-nitrosoguanidine