Occurrence of osteoblast necroses during ossification of long bone cortices in mouse fetuses

Previous investigations concerned with in vitro osteogenesis and mineralization have revealed some indication of a participation of cell necroses in the course of calcification. These observations were confirmed by in vivo investigations on desmoid ossification in fetal mouse calvariae, where abundant necrotic osteoblasts were found at the mineralization border and in the osteoid. In the present study, ossification of long bone cortices from fetal mice was investigated by use of electron microscopy. Specimens obtained from the collection of the Institute of Anatomy, Free University of Berlin (mouse fetuses, forearm; rat fetuses, forearm) were reinvestigated for control purposes. In all cases, mineralization of osteoid was accompanied by cell necroses. Cell degeneration was characterized by swelling of the endoplasmic reticulum and loss of the plasma membrane resulting in freely distributed vesicular structures. Cell debris was incorporated within the mineral. Initially, cell necroses in the perichondrium occurred in the region surrounding the hypertrophic cartilage and the matrix of which showed spots of endochondral mineralization. Necrotic osteoblasts occurred simultaneously with mineralization of the osteoid. During further ossification of the long bone cortices, the number of necrotic cells increased markedly. In addition to necrotic cells, healthy osteoblasts, osteocytes and perichondral tissue were present, indicating that an artifact can be excluded. The importance of cell necroses in the process of mineralization is as yet unclear. Possibly, the cells act as calcium and/or phosphate stores, which are liberated by cell death to increase the amount of mineral constituents at sites of mineralization.     

Isolation and expression in Escherichia coli of hepB and hepC, genes coding for the glycosaminoglycan-degrading enzymes heparinase II and heparinase III, respectively, from Flavobacterium heparinum.

Upon induction with heparin, Flavobacterium heparinum synthesizes and secretes into its periplasmic space heparinase I (EC 4.2.2.7), heparinase II, and heparinase III (heparitinase; EC 4.2.2.8). Heparinase I degrades heparin, and heparinase II degrades both heparin and heparan sulfate, while heparinase III degrades heparan sulfate predominantly. We isolated the genes encoding heparinases II and III (designated hepB and hepC, respectively). These genes are not contiguous with each other or with the heparinase I gene (designated hepA). hepB and hepC were found to contain open reading frames of 2,316 and 1,980 bp, respectively. Enzymatic removal of pyroglutamate groups permitted sequence analysis of the amino termini of both mature proteins. It was determined that the mature forms of heparinases II and III contain 746 and 635 amino acids, respectively, and have calculated molecular weights of 84,545 and 73,135, respectively. The preproteins have signal sequences consisting of 26 and 25 amino acids. Truncated hepB and hepC genes were used to produce active, mature heparinases II and III in the cytoplasm of Escherichia coli. When these enzymes were expressed at 37 degrees C, most of each recombinant enzyme was insoluble, and most of the heparinase III protein was degraded. When the two enzymes were expressed at 25 degrees C, they were both present predominantly in a soluble, active form.     

E-NTPDase and E-ADA activities are altered in lymphocytes of patients with indeterminate form of Chagas' disease

Trypanosoma cruzi infection triggers a chronic inflammatory process in human host and purinergic system ecto-enzymes play an important role in modulating the inflammatory and immune responses. In this study, it was investigated ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase; EC 3.6.1.5; CD39) and ecto-adenosine deaminase (E-ADA; EC 3.5.4.4) activities in lymphocytes from patients with indeterminate form of Chagas' disease (IFCD). Twenty-five IFCD patients and 25 healthy subjects (control group) were selected. The peripheral lymphocytes were isolated and E-NTPDase and E-ADA activities were determined. Adenine nucleotides and adenosine levels were determined in serum by HPLC and the E-NTPDase1 expression in lymphocytes by Western blot analysis. E-NTPDase (ATP and ADP as substrates) and E-ADA (adenosine as substrate) activities were decreased in lymphocytes from IFCD patients (P<0.05 and P<0.01, respectively), while the E-NTPDase1 expression presented no changes in these patients. Serum ATP levels showed to be decreased (P<0.05) and both AMP (P<0.01) and adenosine (P<0.001) levels were increased in the IFCD group. The enzymatic alterations observed are in agreement with the immune response against T. cruzi infection in IFCD patients, since the decreased extracellular ATP and the increased adenosine levels trigger a Th2 anti-inflammatory response, which it is associated to adaptation of host to parasite, preventing clinical progress of disease.     

Proton NMR studies of transforming and nontransforming H-ras p21 mutants

One- and two-dimensional nuclear magnetic resonance spectroscopy (1D and 2D NMR) and site-directed mutagenesis were used to study the influence of mutations on the conformation of the H-ras oncogene product p21. No severe structural differences between the different mutants, whether they were transforming or nontransforming, could be detected. Initially, selective incorporation of 3,5-deuterated tyrosyl residues into p21 and 2D NMR were used to identify the resonances representing the spin systems of the imidazole rings of the three histidyl residues in the protein, of six of the nine tyrosyl rings, and of four of the five phenylalanyl rings. The spin systems of the phenyl rings of Phe28, Phe78, and Phe82 could be assigned by using mutant proteins, since no severe structure-induced spectral changes in the aromatic part of the spectra of the mutant proteins were detected. Sequence-specific assignments of the histidine imidazole resonances could be obtained by comparison of the distance information obtained by nuclear Overhauser enhancement spectroscopy (NOESY) experiments with the crystal structure. The change in the chemical shift values of the Hl' proton and the alpha-phosphate of the bound GDP in the NMR spectra of the p21(F28L) mutant and the 28-fold increase in the GDP dissociation rate constants of this mutant suggest a strong interaction between Phe28 and the p21-bound nucleotide. In solution, the p21-bound GDP.Mg2+ has an anti conformation, and the phenyl ring of Phe28 is close to the ribose of the bound GDP.Mg2+.     

Light-microscopic histochemistry of non-specific alkaline phosphatase using lanthanide-citrate complexes

Summary New lanthanide methods for the histochemical detection of non-specific alkaline phosphatase in the light microscope are described and compared with already existing techniques for the light microscopical demonstration of this enzyme. To avoid formation of insoluble lanthanide hydroxide at alkaline pH citrate complexes with the capture ions cerium, lanthanum and didymium were used. A molar ratio of 11 mM citrate/14 mM capture reagent is proposed. For preincubated sections, pretreatment in chloroform-acetone and fixation in glutaraldehyde, for non-preincubated sections fixation in glutaraldehyde yielded the best results. 4-Methylumbelliferyl and 5-Br-4-Cl-3-indoxyl phosphate were found to be the most suitable substrates. For routine purposes 4-nitrophenyl, 1-naphthyl, 2-naphthyl and 2-glycerophosphate were also sufficient; naphthol AS phosphates were inferior but still suitable. After incubation for 5–60 min at 37° C lanthanide phosphate was converted into lead phosphate which was visualized as lead sulfide. At pH 9.2–9.5 enzyme activity was demonstrated at many sites such as intestinal, uterine, placental, renal and epididymal microvillous zones, plasma membranes of arterial, sinus and capillary endothelial cells, vaginal and urethral epithelium, smooth muscle cells, myoepithelial cells as well as excretory duct cells of salivary and lacrimal glands and in secretory granules of laryngeal glands. In comparison with Gomori's calcium, Mayahara's lead, Burstone's and Pearse's azo-coupling, McGadey's tetrazolium salt and Gossrau's azoindoxyl coupling technique the lanthanide methods detected alkaline phosphatase activities at identical or additional sites depending on the respective procedure. However, in contrast to the other methods especially the cerium citrate procedure yielded a more precisely localized and more stable reaction product, can be used with all available alkaline phosphatase substrates including those up till now less suitable or unsuitable for light microscopic alkaline phosphatase histochemistry.     

Comparative measurements of the xylem pressure ofNicotiana plants by means of the pressure bomb and pressure probe

Determination of the pressure in the water-conducting vessels of intactNicotiana rustica L. plants showed that the pressure probe technique gave less-negative values than the Scholander-bomb method. Even though absolute values of the order of −0.1 MPa could be directly recorded in the xylem by means of the pressure probe, pressures between zero and atmospheric were also frequently found. The data obtained by the pressure probe for excised leaves showed that the Scholander bomb apparently did not read the actual tension in the xylem vessles ofNicotiana plants. The possibility that the pressure probe gave false readings was excluded by several experimental controls. In addition, cavitation and leaks either during the insertion of the microcapillary of the pressure probe, or else during the measurements were easily recognized when they occurred because of the sudden increase of the absolute xylem tension to that of water vapour or to atmospheric, respectively. Tension values of the same order could also be measured by means of the pressure probe in the xylem vessels of pieces of stem cut from leaves and roots under water and clamped at both ends. The magnitude of the absolute tension depended on the osmolarity of the bathing solution which was adjusted by addition of appropriate concentrations of polyethylene glycol. Partial and uniform pressurisation of plant tissues or organs, or of entire plants (by means of the Scholander bomb or of a hyperbaric chamber, respectively) and simultaneous recording of the xylem tension using the pressure probe showed that a 1∶1 response in xylem pressure only occurred under a few circumstances. A 1∶1 response required that the xylem vessels were in direct contact with an external water reservoir and/or that the tissue was (pre-)infiltrated with water. Corresponding pressure-probe measurements in isolated vascular bundles ofPlantago major L. orP. lanceolata L. plants attached to a Hepp-type osmometer indicated that the magnitude of the tension in the xylem vessels was determined by the external osmotic pressure of the reservoir. These and other experiments, as well as analysis of the data using classical thermodynamics, indicated that the turgor and the internal osmotic pressure of the accessory cells along the xylem vessels play an important role in the maintenance of a constant xylem tension. This conclusion is consistent with the cohesion theory. In agreement with the literature (P.E. Weatherley, 1976, Philos. Trans. R. Soc. London Ser. B23, 435–444; 1982, Encyclopedia of plant physiology, vol. 12B, 79-109), it was found that the tension in the xylem of intact plants under normal and elevated ambient pressure (as measured with the pressure probe) under quasi-stationary conditions was independent of the transpiration rate over a large range, indicating that the conductance of the flow path must be flow-dependent.     

Wild mouse lemurs revisit artificial feeding platforms: implications for field experiments on sensory and cognitive abilities in small primates

Dealing effectively with space to find important resources in a natural environment is a fundamental ability necessary for survival. Evidence has already been provided that wild gray mouse lemurs revisit stationary feeding sites regularly. In this study, we explore to what extent two sympatric mouse lemur species, Microcebus murinus and M. ravelobensis, revisited artificial feeding sites during a period of food scarcity. As the tested populations are marked with individual transponders, we built up artificial feeding platforms equipped with a transponder reader at nine different locations where mouse lemurs had been previously caught. We baited them with a liquid reward and recorded the visitors' ID, the time and frequency of their visits, as well as all encounters that occurred on the platforms. Only mouse lemurs visited platforms and a total of sixteen individuals across both species were identified. Mouse lemurs visited a platform with a frequency of 2.02 (+/-0.95, range: 1-3.4) times in a night and they revisited it on several consecutive nights following their first visit (percentage of revisits 90.6%+/-11.7, range: 73.3-100%). First visits on a platform occurred on average 44 min (+/-35; range: 13-131) after sunset. We identified encounters between mouse lemurs on platforms: all of them were agonistic and within a species. Within a dyad, chasers were significantly heavier than chasees (N=7 dyads). Our design of platform experiments offers the advantage of observing wild individually known small primates in their natural environment and of setting up controlled experiments to gain insight into their sensory and cognitive abilities.     

Determinants of Versican-V1 Proteoglycan Processing by the Metalloproteinase ADAMTS5

Proteolysis of the Glu⁴⁴¹-Ala⁴⁴² bond in the glycosaminoglycan (GAG) β domain of the versican-V1 variant by a disintegrin-like and metalloproteinase domain with thrombospondin type 1 motif (ADAMTS) proteases is required for proper embryo morphogenesis. However, the processing mechanism and the possibility of additional ADAMTS-cleaved processing sites are unknown. We demonstrate here that if Glu⁴⁴¹ is mutated, ADAMTS5 cleaves inefficiently at a proximate upstream site but normally does not cleave elsewhere within the GAGβ domain. Chondroitin sulfate (CS) modification of versican is a prerequisite for cleavage at the Glu⁴⁴¹-Ala⁴⁴² site, as demonstrated by reduced processing of CS-deficient or chondroitinase ABC-treated versican-V1. Site-directed mutagenesis identified the N-terminal CS attachment sites Ser⁵⁰⁷ and Ser⁵²⁵ as essential for processing of the Glu⁴⁴¹-Ala⁴⁴² bond by ADAMTS5. A construct including only these two GAG chains, but not downstream GAG attachment sites, was cleaved efficiently. Therefore, CS chain attachment to Ser⁵⁰⁷ and Ser⁵²⁵ is necessary and sufficient for versican proteolysis by ADAMTS5. Mutagenesis of Glu⁴⁴¹ and an antibody to a peptide spanning Thr⁴³²-Gly⁴⁴⁵ (i.e. containing the scissile bond) reduced versican-V1 processing. ADAMTS5 lacking the C-terminal ancillary domain did not cleave versican, and an ADAMTS5 ancillary domain construct bound versican-V1 via the CS chains. We conclude that docking of ADAMTS5 with two N-terminal GAG chains of versican-V1 via its ancillary domain is required for versican processing at Glu⁴⁴¹-Ala⁴⁴². V1 proteolysis by ADAMTS1 demonstrated a similar requirement for the N-terminal GAG chains and Glu⁴⁴¹. Therefore, versican cleavage can be inhibited substantially by mutation of Glu⁴⁴¹, Ser⁵⁰⁷, and Ser⁵²⁵ or by an antibody to the region of the scissile bond.     

Web-based analysis of the mouse transcriptome using Genevestigator

Background  

Gene function analysis often requires a complex and laborious sequence of laboratory and computer-based experiments. Choosing an effective experimental design generally results from hypotheses derived from prior knowledge or experimentation. Knowledge obtained from meta-analyzing compendia of expression data with annotation libraries can provide significant clues in understanding gene and network function, resulting in better hypotheses that can be tested in the laboratory.     

Cholesteryl ester hydrolase activity is abolished in HSL macrophages but unchanged in macrophages lacking KIAA1363

Cholesteryl ester (CE) accumulation in macrophages represents a crucial event during foam cell formation, a hallmark of atherogenesis. Here we investigated the role of two previously described CE hydrolases, hormone-sensitive lipase (HSL) and KIAA1363, in macrophage CE hydrolysis. HSL and KIAA1363 exhibited marked differences in their abilities to hydrolyze CE, triacylglycerol (TG), diacylglycerol (DG), and 2-acetyl monoalkylglycerol ether (AcMAGE), a precursor for biosynthesis of platelet-activating factor (PAF). HSL efficiently cleaved all four substrates, whereas KIAA1363 hydrolyzed only AcMAGE. This contradicts previous studies suggesting that KIAA1363 is a neutral CE hydrolase. Macrophages of KIAA1363^−/− and wild-type mice exhibited identical neutral CE hydrolase activity, which was almost abolished in tissues and macrophages of HSL^−/− mice. Conversely, AcMAGE hydrolase activity was diminished in macrophages and some tissues of KIAA1363^−/− but unchanged in HSL^−/− mice. CE turnover was unaffected in macrophages lacking KIAA1363 and HSL, whereas cAMP-dependent cholesterol efflux was influenced by HSL but not by KIAA1363. Despite decreased CE hydrolase activities, HSL^−/− macrophages exhibited CE accumulation similar to wild-type (WT) macrophages. We conclude that additional enzymes must exist that cooperate with HSL to regulate CE levels in macrophages. KIAA1363 affects AcMAGE hydrolase activity but is of minor importance as a direct CE hydrolase in macrophages.