Validation of Bone Conversion in Osteoconductive and Osteoinductive Bone Substitutes

Bone reconstruction can be performed with an autogeneic graft from various donor regions. Osteoconductive and osteoinductive bone substitutes originate from substances of diverse chemical and morphological types and can have a synthetic or a biological derivation. Alongside autogeneic bone transplants and allogenic and xenogeneic bone implants, alloplastic bone replacements of synthetic or semi-synthetic origin are being used for defect reconstruction. In an animal model in rabbits five bone substitutes and one autogeneic graft were surgically incorporated into identical bone defects (10times 10 mm in size) in six anatomically defined regions of the skull. With scintigraphic and histological methods, the metabolic dynamics of the bone is examined as it reacts to the transplantation of autogeneic bone or to implanted bone replacement material. The different autogeneic, xenogeneic and alloplastic bone replacement materials can be differentiated according to the functional quality of the new tissue and the dynamics of the bone conversion thus induced. In the comparison of mineralized, osteoconductive bone subsitutes (TCP, HA, calcium carbonate ceramics) with demineralized, osteoinductive implants (DBM new, DBM old) and autogeneic bone grafts, the bone inducing matrices show the largest quantity of new bone formation, making possible a volume-constant reconstruction. This revised version was published online in July 2006 with corrections to the Cover Date.     

Semiochemicals from ovaries of gravid females attract ovipositing female houseflies,Musca domestica

Chemical signals originating from the ovaries of gravid females of Musca domestica (Diptera: Cyclorrhapha: Muscidae) attract ovipositing females to common egg-laying sites. Behavioral experiments indicated that females preferred to oviposit in fermented wheat bran containing ovaries from reproductively mature houseflies. Females preferred to oviposit in fermented wheat bran than wet wheat bran. This effect was additive with the attraction to housefly ovaries. Solvent extracts from housefly ovaries were attractive to gravid females. Extracts obtained with hexane were most attractive to gravid females for egg laying, and extracts obtained with ethyl acetate attracted more egg laying than extracts obtained by dichloromethane. Gas chromatography (GC) and gas chromatography coupled with mass spectrometry (GC-MS) analysis showed that tricosane and (Z)-9-tricosene were the main components of the hexane extracts. Both tricosane and (Z)-9-tricosene were shown to elicit dose-dependent aggregation of gravid females in oviposition bioassays, but high doses of either chemical were not attractive.     

Fluorescence resonance energy transfer analysis of protein-protein interactions in single living cells by multifocal multiphoton microscopy

Two variants of an endo-beta-1,4-mannanase from the digestive tract of blue mussel, Mytilus edulis, were purified by a combination of immobilized metal ion affinity chromatography, size exclusion chromatography in the absence and presence of guanidine hydrochloride and ion exchange chromatography. The purified enzymes were characterized with regard to enzymatic properties, molecular weight, isoelectric point, amino acid composition and N-terminal sequence. They are monomeric proteins with molecular masses of 39216 and 39265 Da, respectively, as measured by MALDI-TOF mass spectrometry. The isoelectric points of both enzymes were estimated to be around 7.8, however slightly different, by isoelectric focusing in polyacrylamide gel. The enzymes are stable from pH 4.0 to 9.0 and have their maximum activities at a pH about 5.2. The optimum temperature of both enzymes is around 50-55 degrees C. Their stability decreases rapidly when going from 40 to 50 degrees C. The N-terminal sequences (12 residues) were identical for the two variants. They can be completely renatured after denaturation in 6 M guanidine hydrochloride. The enzymes readily degrade the galactomannans from locust bean gum and ivory nut mannan but show no cross-specificity for xylan and carboxymethyl cellulose. There is no binding ability observed towards cellulose and mannan.     

Norepinephrine-induced cardiac hypertrophy and fibrosis are not due to mast cell degranulation

The norepinephrine (NE)-induced hypertrophy of the left ventricle (LV) in the rat is preceded by increased interleukin (IL)-6 expression and associated with LV fibrosis. We have examined whether the elevated level of IL-6 may be due to mast cell degranulation. Therefore we tested the effect of cromoglycate sodium salt (cromolyn), an inhibitor of mast cell degranulation with anti-inflammatory and membrane-stabilizing activity, on the increased expression of IL-6 mRNA and of mRNAs of proteins involved in the remodelling of the extracellular matrix (ECM) which is induced by NE (0.1 mg/kg·h). After 4 h, the NE-induced increase in IL-6 mRNA expression was not influenced by cromolyn (20 mg/kg·h). Cromolyn-infusion for 3 days did not affect the extent of LV hypertrophy induced by NE, as measured by the LV weight/body weight (LVW/BW) ratio and by atrial natriuretic peptide (ANP) expression. Cromolyn induced a slight depression of the NE-induced elevation of the matrix metalloproteinase (MMP)-2. However, it did not affect the NE-induced elevated levels of mRNAs of collagen I and III and the tissue inhibitor of matrix metalloproteinase (TIMP)-2. Since cromolyn did not reduce the NE-effects in rat hearts in vivo we conclude that mast cell degranulation seems not to be involved in them.     

Identification of immunologically related proteins in sieve-tube exudate collected from monocotyledonous and dicotyledonous plants

The mature, functional sieve-tube system in higher plants is dependent upon protein import from the companion cells to maintain a functional long-distance transport system. Soluble proteins present within the sieve-tube lumen were investigated by analysis of sieve-tube exudates which revealed the presence of distinct sets of polypeptides in seven monocotyledonous and dicotyledonous plant species. Antibodies directed against sieve-tube exudate proteins from Ricinus communis L. demonstrated the presence of shared antigens in the phloem sap collected from Triticum aestivum L., Oryza sativa L., Yucca filamentosa L., Cucurbita maxima Duch., Robinia pseudoacacia L. and Tilia platyphyllos L. Specific antibodies were employed to identify major polypeptides. Molecular chaperones related to Rubisco-subunit-binding protein and cyclophilin, as well as ubiquitin and the redox proteins, thioredoxin h and glutaredoxin, were detected in the sieve-tube exudate of all species examined. Actin and profilin, a modulator of actin polymerization, were also present in all analyzed phloem exudates. However, some proteins were highly species-specific, e.g. cystatin, a protease-inhibitor was present in R. communis but was not detected in exudates from other species, and orthologs of the well-known squash phloem lectin, phloem protein 2, were only identified in the sieve-tube exudate of R. communis and R. pseudoacacia. These findings are discussed in terms of the likely roles played by phloem proteins in the maintenance and function of the enucleate sieve-tube system of higher plants. Received: 12 February 1998 / Accepted: 16 March 1998     

A covalent two-step immobilization technique using itaconic anhydride

β-Glucosidase from almonds (EC 3.2.1.21) was covalently immobilized by a two-step technique. In the first step, double bonds were introduced into the β-glucosidase by derivatization with itaconic anhydride. In separate studies with α-N-protected l-amino acids, it was established that itaconic anhydride acylated mainly primary amino groups of lysines and, to a much lesser extent hydroxyl groups of tyrosines and sulfhydryl groups of cysteines. The acylated β-glucosidase showed no loss of activity and the K _m decreased from 3.6 mM to 2.6 mM when p-nitrophenyl β-d-glucopyranoside was used as the substrate. In the second step, the derivatized β-glucosidase was co-polymerized radically with N,N′-methylenebisacrylamide in buffer solution. The resulting acrylamide immobilizate possessed a much better storage stability at 30–56 °C when compared to β-glucosidase immobilized on Eupergit C. However, the specific activity was higher with the Eupergit immobilizate. Free and acrylamide-immobilized β-glucosidase were used for glucosylation of chloramphenicol by transglucosylation in 20% (v/v) acetonitrile at 37 °C. The acrylamide immobilizate demonstrated a great enhancement of stability and approximately 50% more chloramphenicol β-glucoside was obtained after 5 h. Received: 22 September 1997 / Accepted: 28 October 1997     

Production of polyhydroxybutyrate by Ralstonia eutropha from protein hydrolysates

Polyhydroxybutyrate (PHB) was produced by Ralstonia eutropha DSM 11348 (formerly Alicaligenes eutrophus) in media containing 20–30 g l⁻¹ casein peptone or casamino acids as sole sources of nitrogen. In fermentations using media based on casein peptone, permanent growth up to a cell dry mass of 65 g l⁻¹ was observed. PHB accumulated in cells up to 60%–80% of dry weight. The lowest yields were found in media without any trace elements or with casamino acids added only. The residual cell dry masses were limited to 10–15 g l⁻¹ and did not contain PHB. The highest productivity amounted to 1.2 g PHB l⁻¹ h⁻¹. The mean molecular mass of the biopolymer was determined as 750 kDa. The proportion of polyhydroxyvalerate was less than 0.2% in PHB. The bioprocess was scaled up to a 300-l plant. During a fermentation time of 39 h the cells accumulated PHB to 78% w/w. The productivity was 0.98 g PHB l⁻¹ h¹. Received: 8 July 1998 / Accepted: 26 August 1998     

Dynamics of a Kalahari long-lived mega-lake system: hydromorphological and limnological changes in the Makgadikgadi Basin (Botswana) during the terminal 50 ka

The Kalahari features a long-lived lacustrine system which may exist since the Early Pleistocene. The emergence of an extant cichlid fish radiation from this (palaeo-) lake during the Middle Pleistocene indicates an ancient lake character. The early history of the system remains speculative, but it is established that lake extensions matching modern Lake Victoria in size have occurred during the Late Pleistocene. It has been assumed that the hydrographical dynamics chiefly depended on the inflow from the Okavango River and thus on ITCZ-controlled precipitation. Our studies, which focused the hydromorphological and palaeolimnological development of the Makgadikgadi Basin during the last 50 ka, suggest that from c. 46–16 ka it did not receive water from the Okavango River but from palaeo-rivers located in the northern and south-western catchment. A northward shift of the winter rainfall zone during the Last Glacial Maximum sustained a high lake level for a period of c. 6 ka. During Heinrich Event 1 (17–16 ka) the lake probably desiccated abruptly and completely. Higher lake levels, controlled by water from the Okavango river system, were reached again during the Holocene before the lake dried up in the middle of the last millennium.     

Analyzing female gametophyte development and function: There is more than one way to crack an egg

In flowering plants, gametes are formed in specialized haploid structures, termed gametophytes. The female gametophyte is a few-celled structure that integrates such diverse functions as pollen tube attraction, sperm cell release, gamete fusion and seed initiation. These processes are realized by distinct cell types, which ensure reproductive success in a coordinated manner. In the past decade, much progress has been made concerning the molecular nature of the functions carried out by the different cell types. Here, we review recent work that has shed light on female gametophyte development and function with a particular focus on approaches that have led to the isolation of genes involved in these processes.     

ADAM10 is the physiologically relevant, constitutive ��-secretase of the amyloid precursor protein in primary neurons

The amyloid precursor protein (APP) undergoes constitutive shedding by a protease activity called α‐secretase. This is considered an important mechanism preventing the generation of the Alzheimer's disease amyloid‐β peptide (Aβ). α‐Secretase appears to be a metalloprotease of the ADAM family, but its identity remains to be established. Using a novel α‐secretase‐cleavage site‐specific antibody, we found that RNAi‐mediated knockdown of ADAM10, but surprisingly not of ADAM9 or 17, completely suppressed APP α‐secretase cleavage in different cell lines and in primary murine neurons. Other proteases were not able to compensate for this loss of α‐cleavage. This finding was further confirmed by mass‐spectrometric detection of APP‐cleavage fragments. Surprisingly, in different cell lines, the reduction of α‐secretase cleavage was not paralleled by a corresponding increase in the Aβ‐generating β‐secretase cleavage, revealing that both proteases do not always compete for APP as a substrate. Instead, our data suggest a novel pathway for APP processing, in which ADAM10 can partially compete with γ‐secretase for the cleavage of a C‐terminal APP fragment generated by β‐secretase. We conclude that ADAM10 is the physiologically relevant, constitutive α‐secretase of APP.