Transmission electron microscopy characterization of macromolecular domain cavities and microstructure of single-crystal calcite tooth plates of the sea urchin Lytechinus variegatus

The calcite plates and prisms in Lytechinus variegatus teeth form a complex biocomposite and employ a myriad of strengthening and toughening strategies. These crystal elements have macromolecule-containing internal cavities that may act to prevent cleavage. Transmission electron microscopy employing a small objective aperture was used to quantify several characteristics of these cavities. Cavity diameters ranged from 10 to 225 nm, the mean cavity diameter was between 50 and 60 nm, and cavities comprised approximately 20% of the volume of the crystal. Some cavities exhibited faceting and trace analysis identified these planes as being predominately of {1014} type. Through focus series of micrographs show the cavities were homogeneously distributed throughout the foil. The electron beam decomposed a substance within cavities and this suggests that these cavities are filled with a hydrated organic phase.     

Phosphorylation of phosphophoryn is crucial for its function as a mediator of biomineralization

Phosphoproteins of the organic matrix of bone and dentin have been implicated as regulators of the nucleation and growth of the inorganic Ca-P crystals of vertebrate bones and teeth. One such protein identified in the dentin matrix is phosphophoryn (PP). It is highly acidic in nature because of a high content of aspartic acid and phosphate groups on serines. The 244-residue carboxyl-terminal domain of rat PP, predominantly containing the aspartic acid-serine repeats, has been cloned, and the corresponding protein has been expressed recombinantly in Escherichia coli. This portion of PP, named DMP2 (dentin matrix protein 2), is not phosphorylated by the bacteria and thus provided a means to study the function of the phosphate groups, the major post-translational modification of native PP. The recombinant DMP2 (rDMP2) possessed much lower calcium binding capacity than native PP. Small angle x-ray scattering experiments demonstrated that PP folds to a compact globular structure upon calcium binding, whereas rDMP2 maintained an unfolded structure. In vitro nucleation experiments showed that PP could nucleate plate-like apatite crystals in pseudophysiological buffer, whereas rDMP2 failed to mediate the transformation of amorphous calcium phosphate to apatite crystals under the same experimental conditions. Collagen binding experiments demonstrated that PP favors the formation of collagen aggregates, whereas in the presence of rDMP2 thin fibrils are formed. Overall these results suggested that the phosphate moieties in phosphophoryn are important for its function as a mediator of dentin biomineralization.     

X-ray absorption microtomography (microCT) and small beam diffraction mapping of sea urchin teeth

Two noninvasive X-ray techniques, laboratory X-ray absorption microtomography (microCT) and X-ray diffraction mapping, were used to study teeth of the sea urchin Lytechinus variegatus. MicroCT revealed low attenuation regions at near the tooth's stone part and along the carinar process-central prism boundary; this latter observation appears to be novel. The expected variation of Mg fraction x in the mineral phase (calcite, Ca(1-x)Mg(x)CO(3)) cannot account for all of the linear attenuation coefficient decrease in the two zones: this suggested that soft tissue is localized there. Transmission diffraction mapping (synchrotron X-radiation, 80.8 keV, 0.1 x 0.1mm(2) beam area, 0.1mm translation grid, image plate area detector) simultaneously probed variations in 3-D and showed that the crystal elements of the "T"-shaped tooth were very highly aligned. Diffraction patterns from the keel (adaxial web) and from the abaxial flange (containing primary plates and the stone part) differed markedly. The flange contained two populations of identically oriented crystal elements with lattice parameters corresponding to x=0.13 and x=0.32. The keel produced one set of diffraction spots corresponding to the lower x. The compositions were more or less equivalent to those determined by others for camarodont teeth, and the high Mg phase is expected to be disks of secondary mineral epitaxially related to the underlying primary mineral element. Lattice parameter gradients were not noted in the keel or flange. Taken together, the microCT and diffraction results indicated that there was a band of relatively high protein content, of up to approximately 0.25 volume fraction, in the central part of the flange and paralleling its adaxial and abaxial faces. X-ray microCT and microdiffraction data used in conjunction with protein distribution data will be crucial for understanding the properties of various biocomposites and their mechanical functions.     

Nematodes associated with Ips cembrae (Coleoptera: Curculionidae): comparison of generations,sexes and sampling methods

A study of nematodes associated with the large larch bark beetle Ips cembrae (Heer 1836) was carried out at three locations in the Czech Republic. The proportion of beetles infested by endoparasitic nematodes (representatives of genera Contortylenchus, Parasitylenchus, Cryptaphelenchus and Parasitorhabditis) ranged from 29.9 to 50.9%. Significant differences were determined in nematode infestation levels among locations, generations and sampling methods. No differences were found in infestation rates between males and females. The percentage of bark beetles with phoretic nematodes ranged from 18 to 42.9%. Phoretic nematodes directly found under elytra, on wings and between body segments of the bark beetles belong to the genus Micoletzkya. However, adults and juveniles of other two phoretic species Laimaphelenchus penardi and Bursaphelenchus sp. were found in the gallery frass of I. cembrae. Infestation by phoretic nematodes positively correlated with the presence of mites under elytra.     

Endoplasmic reticulum protein HSP47 binds specifically to the N-Terminal globular domain of the amino-propeptide of the procollagen I α1(I)-chain

Hsp47, an endoplasmic reticulum-resident heat shock protein in fibroblasts has gelatin-binding properties. It had been hypothesized that it functions as a chaperone regulating procollagen chain folding and/or assembly, but the mechanism of the hsp47-procollagen I interaction was not clear. Hsp47 could bind to both denatured and native procollagen I. A series of competition studies were carried out in which various collagens and collagen domain peptides were incubated with³⁵[S]-methionine-labeled murine 3T6 cell lysates prior to mixing with gelatin-Sepharose 4B beads. The gelatin-bound proteins were collected and analyzed by gel electrophoresis and autoradiography. Collagenase digested procollagen I had the same effect as denatured intact procollagen, indicating that the propeptides were the major interaction sites. The addition of intact pro α1 (l)-N-propeptide at 25 μg/ml compeletely inhibited hsp47 binding to the gelatin-Sepharose. Even the pentapeptide VPTDE, residues 86–90 of the pro α1 (l)-N-propeptide, inhibits hsp47-gelatin binding. These data implicating the pro α1 (l)-N-propeptide domain were confirmed by examination of polysome-associated pro α chains. The nascent pro α1(l)-chains with intact N-propeptide regions could be precipitated by monoclonal hsp47 antibody 11D10, but could not be precipitated by monoclonal anti-pro α1 (l)-N-propeptide antibody SP1.D8 unless dissociated from the hsp47. GST-fusion protein constructs of residues 23–108 (NP1), 23–151 (NP2), and 23–178 (NP3) within the pro α1 (l)-N-propeptide were coupled to Sepharose 4B and used as affinity beads for collection of hsp47 from 3T6 cell lysates. NP1 and NP2 both showed strong specific binding for lysate hsp47. Finally, the interaction was studied in membrane-free in vitro cotranslation systems in which the complete pro α1(l)- and pro α2(l)-chain RNAs were translated alone and in mixtures with each other and with hsp47 RNA. There was no interaction evident between pro α2(l)-chains and hsp47, whereas there was strong interaction between pro α1 (l)-chains and nascent hsp47. SP1.D8 could not precipitate pro α1 (l)-chains from the translation mix if nascent hsp47 was present. These data all suggest that if hsp47 has a “chaperone” role during procollagen chain processing and folding it performs this specific role via its preferential interaction with the proα1 (l) chain, and the pro α1 (l) amino-propeptide region in particular. © 1995 Wiley-Liss, Inc.     

Latent membrane protein of Epstein-Barr virus induces cellular phenotypes independently of expression of Bcl-2.

The stable expression of the Epstein-Barr virus (EBV) latent membrane protein (LMP) in certain EBV-negative Burkitt's lymphoma cell lines correlates with an increased expression of the oncogene Bcl-2 (S. Henderson, M. Rowe, C. Gregory, D. Croom-Carter, F. Wang, R. Longnecker, E. Kieff, and A. Rickinson, Cell 65:1107-1115, 1991). This finding is consistent with a model in which Bcl-2 contributes to the immortalization of B cells mediated by EBV. We therefore asked whether the expression of Bcl-2 protein correlates with the induction of three cellular phenotypes induced by or associated with LMP. The expression of Bcl-2 in primary B cells infected with the B95-8 strain of EBV varied between 1 and 1.8 times that in uninfected cells when 50% of the cells were infected, expressed LMP, and incorporated 20-fold more [3H]thymidine than did uninfected cells. This finding indicates that induced proliferation of these primary cells is not sufficient to induce Bcl-2. We found that BALB/c 3T3 cells and their derivatives transformed by LMP do not express Bcl-2 detectably. The expression of LMP at high levels in lymphoid cells is cytotoxic and correlates with an increased expression of Bcl-2 following stable selection for the introduced LMP gene; 2 days after transfection, control vector- and LMP-transfected populations, however, express equal levels of Bcl-2 protein. We also analyzed transient expression of LMP in an EBV-negative Burkitt's lymphoma cell line. Infection of BJAB cells with the B95-8 strain of EBV results in an increase in Bcl-2 expression with a time course similar to that of LMP expression, and LMP alone transiently induces an increase in Bcl-2 expression in these cells. We interpret these observations to indicate that increased expression of Bcl-2 is unlikely to contribute to the ability of EBV to immortalize primary B cells and that both the transformation of rodent cells and the cytotoxicity mediated by LMP are independent of Bcl-2.     

Depletion of metal in the rat hippocampal mossy fibre system by intravital chelation with dithizone

Summary Adult albino rats were given dithizone by repeated intraperitoneal injections followed by a standard test dose. After a standard interval of 15 or 60 minutes the animals were killed by vascular perfusion with a buffered acidified formaldehyde solution. The brains were immediately removed, frozen, and cut at a constant thickness of 160 microns on a freezing microtome.The red stain of metal dithizonate known to be particularly marked in the hippocampal mossy fibre system was observed and recorded by a technique based on photography of sections. It was found that dithizone in doses of 25 mg per kg body weight, injected hourly for 12 hours prevented the dithizone staining of the mossy fibre system caused by the test dose in control animals. The result is interpreted as evidence of true depletion of metal.This study was supported in part by U.S.P.H.S. Grant NS 07998. This aid is gratefully acknowledged. Einar Hansen, Thorkild Nielsen, Birgit Örum, Albert Meier, Karin Sörensen and Inger Madsen rendered valuable technical assistance.     

Dithizone and sulphide silver staining of the amygdala in the cat

Summary In material obtained following the intravital injection of dithizone and sections prepared by the sulphide silver technique of Timm, a differential staining of the nuclear subgroups of the amygdala was observed. With both stains, the areas of maximum density were as follows: 1. the lateral border of the lateral nucleus; 2. the ventromedial part of the lateral nucleus; 3. an area in the parvocellular part of the basal nucleus; 4. a round area laterally in the central nucleus; 5. the cortical nucleus. It is suggested that the histochemical specificity of the stain is related to one or more of the afferent systems to the amygdala. The areas projecting to the amygdaloid complex are considered briefly and the possibility that the stain may be associated with the terminals of fibres arising in the pyriform cortex is discussed.Sponsored by the Norwegian Research Council for Science and the Humanities, United States Public Health RO 5 TW-91 and NB 02215, and the Medical Research Council of Canada M. T. 870.     

Intracellular location of triple helix formation of collagen. Enzyme probe studies.

Primary cultures of chick embryo fibroblasts were used to study ribosomal events in the processing of procollagen. Polyribosomes from radiolabeled cells were subjected to enzyme probe analysis using collagenase and pepsin digestion to assess both the amount of procollagen present on the polyribosomes and the conformation of the molecule. The peptides rendered dialyzable by each enzyme treatment were analyzed for radioactive proline and hydroxyproline. Approximately 30% of the nascent proteins were collagenous. Although some hydroxyproline was dialyzable in the pepsin-treated material, a low ratio of hydroxyproline to proline (0.04) indicated that considerable amounts of noncollagenous proteins were digested. Polyribosomal material, previously treated with pepsin, was digested with purified collagenase. Similarly, collagenase-digested polyribosomes were treated with pepsin. The pepsin pretreatment released noncollagenous protein and served to purify the remaining ribosomally bound pepsin-resistant collagenous protein. Collagenase treatment of the pepsin-resistant ribosomally bound peptides released peptides with a hydroxyproline to proline ratio of 0.65, indicating that considerable hydroxylation of proline occurs on nascent ribosomally bound procollagen. This finding combined with the well documented stabilizing effect of hydroxyproline on the collagen triple helix and the demonstrated resistance of ribosomally bound procollagen to pepsin digestion indicates that the collagen triple helix may well form on the polyribosome.     

Fasting reduces the response of liver glycogen phosphorylase to physiological levels of epinephrine in rats

The effect of 24 h fasting on the response of rat liver glycogen phosphorylase activity to an i.v. bolus of 2.75, 5.50 or 22.00 nmol kg-1 of epinephrine was studied. Even the lowest dose increased activity of the a form of the enzyme in the liver of anesthetized, fed rats to approximately 70 - 80% of total enzyme activity two minutes after administration. Further increased epinephrine doses failed to potentiate the enzyme response significantly, but shortened the time necessary for attaining the response, and delayed the return of enzyme activity to control values. No activation of phosphorylase was demonstrable after 2.75 nmol kg-1 of the hormone injected to fasted rats, but after increasing the hormone dose to 5.50 nmol kg-1 the enzyme response was the same as in the corresponding fed group at 2 min, and after administering the highest dose both at 1 and 2 min. According to these results, an increased threshold to epinephrine should be added to the already described effects of fasting, i.e. decreased phosphorylase a and total enzyme activity and shortened response to catecholamines. The efficacy of the i.v. bolus of 5.50 nmol kg-1 of epinephrine in increasing plasma epinephrine level to the theoretical value of 27.5 pmol ml-1 was proven by measuring plasma epinephrine which increased during the first minute after hormone administration to 24.5 + 5.9 pmol ml-1, to decrease during an additional minute with a half life of cca 22.2 seconds.