Age-dependent effect of limb immobilization and remobilization on rat bone

The effect of unilateral hindlimb immobilization and subsequent free remobilization on bone tissue in rats was examined. Right hindlimb of intensively growing (G), young adult (Y) and adult (A) male rats was immobilized by taping for two weeks. Bone tissue was investigated post mortem in experimental and age-matched control rats, either directly after immobilization (Imm) or after two or four weeks of remobilization (Re2, Re4). Apparent density (d(app)) and mineralization (Min) were estimated in femora and pelvis. The mechanical state of bone tissue in femora was evaluated using an ultrasonic method. Additionally, activity of serum alkaline phosphatase, and serum calcium and phosphorus were measured in each group. Min and d(app) in Imm bones were changed in G rats, while in Y and A only d(app) in Imm femora was affected. Velocity of ultrasound was significantly lower in immobilized femora in each age group, indicating decreased elasticity of bone tissue. The differences between immobilized and control limbs were still significant in Re2 and Re4 groups in G rats. In Y rats the differences between experimental and control bones increased during remobilization. It is concluded that deterioration of bone initiated during two weeks of unloading, last within at least four weeks of free remobilization, despite restoration of normal activity.     

Characterization of zona pellucida glycoprotein 3 (ZP3) and ZP2 binding sites on acrosome-intact mouse sperm

There is considerable evidence that mouse fertilization requires the binding of sperm to two of the three glycoproteins that form the zona pellucida (ZP), ZP3 and ZP2. Despite the biologic importance of this binding, no one has demonstrated that sperm express separate, saturable, and specific binding sites for ZP3 and for ZP2. Such a demonstration is a prerequisite for defining the distribution, numbers, affinities, and regulation of function of ZP3 and ZP2 binding sites on sperm. The experiments reported herein used fluorochrome-labeled ZP3 and ZP2 and quantitative image analysis to characterize the saturable binding of ZP3 and ZP2 to distinct sites on living, capacitated, acrosome-intact mouse sperm. Approximately 20% of the ZP3 binding sites were found over the acrosomal cap, and the remaining sites were located over the postacrosomal region of the head. In contrast, ZP2 binding sites were detected only over the postacrosomal region. Saturation analysis estimated numbers and affinities of the binding sites for ZP3 (B(max) approximately 185 000 sites per sperm; K(d) approximately 67 nM) and ZP2 (B(max) approximately 500 000 sites per sperm; K(d) approximately 200 nM). Use of unlabeled ZP3, ZP2, and ZP1 as competitive inhibitors of the binding of fluorochrome-labeled ZP3 and ZP2 demonstrated that ZP3 and ZP2 bound specifically to their respective sites on sperm. Finally, we demonstrate that extracellular calcium as well as capacitation and maturation of sperm are required for these sites to bind their respective ligands.     

Reaction diffusion model of the enzymatic erosion of insoluble fibrillar matrices

Predicting the time course of in vivo biodegradation is a key issue in the design of an increasing number of biomedical applications such as sutures, tissue analogs and drug-delivery devices. The design of such biodegradable devices is hampered by the absence of quantitative models for the enzymatic erosion of solid protein matrices. In this work, we derive and simulate a reaction diffusion model for the enzymatic erosion of fibrillar gels that successfully reproduces the main qualitative features of this process. A key aspect of the proposed model is the incorporation of steric hindrance into the standard Michaelis-Menten scheme for enzyme kinetics. In the limit of instantaneous diffusion, the model equations are analogous to the standard equations for enzymatic degradation in solution. Invoking this analogy, the total quasi-steady-state approximation is used to derive approximate analytical solutions that are valid for a wide range of in vitro conditions. Using these analytical approximations, an experimental-theoretical method is derived to unambiguously estimate all the kinetic model parameters. Moreover, the analytical approximations correctly describe the characteristic hyperbolic dependence of the erosion rate on enzyme concentration and the zero-order erosion of thin fibers. For definiteness, the analysis of published experimental results of enzymatic degradation of fibrillar collagen is demonstrated, and the role of diffusion in these experiments is elucidated.     

Production and morphology of the hybrids Aegilops kotschyi x Secale cereale and Ae. biuncialis x S. cereale

Hybrids between Aegilops kotschyi and Ae. biuncialis with Secale cereale were synthesized. Five Ae. kotschyi and four Ae. biuncialis accessions, as well as one inbred and four self-compatible forms of Secale cereale were used for crossing. The hybrids were produced directly from cultured embryos or through embryo callus culture. Sixty hybrids, 11 involving Ae. kotschyi and 49 Ae. biuncialis, had a stable somatic chromosome number 2n = 3x = 21. The plants showed good vegetative vigour and tillering capacity. Morphologically the hybrids were intermediate between their parents and completely sterile. In vitro propagation of Ae. kotschyi and Ae. biuncialis x S. cereale hybrids revealed that their capacity for callus production and plantlet regeneration - varies.     

Differentiation of Lactobacillus Species by Molecular Typing

A total of 64 type, reference, clinical, health food, and stock isolates of microaerophilic Lactobacillus species were examined by restriction fragment length polymorphisms. Of particular interest were members of six of the eight species most commonly recovered from the vaginas of healthy premenopausal women, namely, Lactobacillus jensenii, L. casei, L. rhamnosus, L. acidophilus, L. plantarum, and L. fermentum. Six main groupings were identified on the basis of ribotyping. This technique was able to classify fresh isolates to the species level. In the case of the ribotype A grouping for L. rhamnosus, differences between strains were evident by chromosome typing (chromotyping). Many isolates did not possess plasmids. Six L. rhamnosus strains isolated from four different health food products appeared to be identical to L. rhamnosus ATCC 21052. The molecular typing system is useful for identifying and differentiating Lactobacillus isolates. Studies of strains of potential importance to the urogenital flora should include molecular characterization as a means of comparing genetic traits with those of strains whose characteristics associated with colonization and antagonism against pathogens have been defined.Lactobacilli colonizing human tissues have long been considered important for the maintenance of a healthy gastrointestinal tract (4) and urogenital tract (20, 21, 22, 25). The disruption of the lactobacillus flora has been associated with many urogenital infections, and as these afflict over 150 million women worldwide each year, this area of study is an important one. Indeed, many patients resort to taking health food products containing lactobacilli as a means of trying to maintain a healthy intestinal (and in some cases vaginal) flora.The typing of lactobacilli has generally been conducted by cell and colony morphology and biochemical tests. These techniques type bacteria based on their ability to ferment sugars and produce acids such as lactic acid and acetic acid (9). Unfortunately, these typing methods are not completely accurate, and strains which show intermediate characteristics are frequently encountered (9, 33).Many studies emphasize that the classification of lactobacilli is unsatisfactory and does not reflect the real phylogenetic relatedness of different strains and species (6, 19, 30). Several new genetic and chemotaxonomic approaches have been used during the last 14 years with an aim of improving the classification and identification of lactobacilli: for example, analysis of plasmid content (18), sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of whole-cell protein (19) and of total soluble cell protein (8), sequencing of rRNA (5, 6, 19), restriction endonuclease fingerprinting (14, 30), and DNA-DNA hybridization (6, 17, 19, 27). All of these approaches have improved the taxonomic knowledge of the generic and suprageneric relationships of lactobacilli. However, no analysis of urogenital or health food isolates of lactobacilli by molecular typing has been reported.Plasmid typing of Lactobacillus strains has been suggested as a taxonomic tool in a number of reports (27, 31), but there is evidence to suggest that it is not very effective (2, 12, 15, 28), since plasmids can be absent or unstable.Chromosome typing (restriction endonuclease fingerprinting of chromosomal DNA) (chromotyping) has been applied to the discrimination of strains of lactobacilli and has been found more specific and reproducible than plasmid content analysis (14). However, one of the disadvantages of chromotyping is that comparing electrophoretic patterns consisting of up to 100 bands is difficult. Hence, chromotyping alone may not be widely used for typing large numbers of strains.A sensitive but rapid method for species differentiation involves the use of ribotyping (1, 27), which combines Southern hybridization of chromosomal DNA fingerprints with the use of Escherichia coli rRNA probes, thereby discriminating between various species and individual strains of lactobacilli. The method involves the separation and identification of the rRNA genes present within the bacterial genome, which exist in various copy numbers (10, 13), making it possible to delineate species based on differences in the restriction fragment length polymorphisms of the rRNA genes. Of particular importance is the fact that specific regions of the rRNA genes have remained well conserved because of their functional importance, thus allowing the detection of a broad range of bacteria with 16S and 23S rRNA of E. coli as probes.The aim of this study was to develop a method to test the efficacy of ribotyping of Lactobacillus type and reference strains and to use this method to characterize a number of clinical, health food, and laboratory isolates.     

Covalent and three-dimensional structure of the cyclodextrinase from Flavobacterium sp. no. 92.

Starting with oligopeptide sequences and using PCR, the gene of the cyclodextrinase from Flavobacterium sp. no. 92 was derived from the genomic DNA. The gene was sequenced and expressed in Escherichia coli; the gene product was purified and crystallized. An X-ray diffraction analysis using seleno-methionines with multiwavelength anomalous diffraction techniques yielded the refined 3D structure at 2.1 A resolution. The enzyme hydrolyzes alpha(1,4)-glycosidic bonds of cyclodextrins and linear malto-oligosaccharides. It belongs to the glycosylhydrolase family no. 13 and has a chain fold similar to that of alpha-amylases, cyclodextrin glycosyltransferases, and other cyclodextrinases. In contrast with most family members but in agreement with other cyclodextrinases, the enzyme contains an additional characteristic N-terminal domain of about 100 residues. This domain participates in the formation of a putative D2-symmetric tetramer but not in cyclodextrin binding at the active center as observed with the other cyclodextrinases. Moreover, the domain is located at a position quite different from that of the other cyclodextrinases. Whether oligomerization facilitates the cyclodextrin deformation required for hydrolysis is discussed.     

OXYGEN MICROSENSOR STUDIES ON ZOOXANTHELLATE CLIONAID SPONGES FROM THE COSTA BRAVA,MEDITERRANEAN SEA1

Photosynthetic properties of two symbiotic demosponges were compared using Clark‐type oxygen microsensors. The putatively distinct sponge species, Cliona viridis (Schmidt, 1862) and Cliona nigricans (Schmidt, 1862) were discriminated by their mean megasclere lengths of 296 and 387 μm, respectively. Photosynthetic behavior was used to generate additional taxonomic information. Sponge–dinoflagellate symbioses were well adapted to low light due to the hosts' endolithic lifestyle. Both sponges reached light compensation and saturation at similar light levels with means close to 10 and 30 μmol photons·m^?2·s^?1, respectively. The gross photosynthetic activity was closely related to symbiont cell density in the sponge surface tissue. Mean symbiont densities, chl a content, and gross photosynthesis were about six times higher in C. viridis than in C. nigricans, with respective values of 3000 and 440 symbiont·mm^?2, 1.3 and 0.2 μg chl a·g^?1, and 5.4 and 1.0 μmol O₂·cm^?3·s^?1 gross photosynthesis. Net photosynthesis and respiration could not be calculated accurately from the oxygen gradients, because significant gas exchange occurs through the pumping activity. Thus, assumptions of diffusional oxygen exchange via the surface do not hold for sponges. Combined data of this study indicate that the metabolic activity of C. viridis depends on photosynthetic activity of its symbionts, whereas C. nigricans appears to have a higher pumping intensity and is more actively filter feeding. The difference in photosynthetic activities is not caused by different light adaptations but provides new evidence against the conspecifity of C. viridis and C. nigricans.     

Comparison of protein oxidation and aldehyde formation during oxidative stress in isolated mitochondria

Oxidative stress is known to cause oxidative protein modification and the generation of reactive aldehydes derived from lipid peroxidation. Extent and kinetics of both processes were investigated during oxidative damage of isolated rat liver mitochondria treated with iron/ascorbate. The monofunctional aldehydes 4-hydroxynonenal (4-HNE), n-hexanal, n-pentanal, n-nonanal, n-heptanal, 2-octenal, 4-hydroxydecenal as well as thiobarbituric acid reactive substances (TBARS) were detected. The kinetics of aldehyde generation showed a lag-phase preceding an exponential increase. In contrast, oxidative protein modification, assessed as 2,4-dinitrophenylhydrazine (DNPH) reactive protein-bound carbonyls, continuously increased without detectable lag-phase. Western blot analysis confirmed these findings but did not allow the identification of individual proteins preferentially oxidized. Protein modification by 4-HNE, determined by immunoblotting, was in parallel to the formation of this aldehyde determined by HPLC. These results suggest that protein oxidation occurs during the time of functional decline of mitochondria, i.e. in the lagphase of lipid peroxidation. This protein modification seems not to be caused by 4-HNE.     

Cytochrome c oxidase deficiency associated with the first stop-codon point mutation in human mtDNA.

We have identified the first stop-codon point mutation in mtDNA to be reported in association with human disease. A 36-year-old woman experienced episodes of encephalopathy accompanied by lactic acidemia and had exercise intolerance and proximal myopathy. Histochemical analysis showed that 90% of muscle fibers exhibited decreased or absent cytochrome c oxidase (COX) activity. Biochemical studies confirmed a severe isolated reduction in COX activity. Muscle immunocytochemistry revealed a pattern suggestive of a primary mtDNA defect in the COX-deficient fibers and was consistent with either reduced stability or impaired assembly of the holoenzyme. Sequence analysis of mtDNA identified a novel heteroplasmic G-->A point mutation at position 9952 in the patient's skeletal muscle, which was not detected in her leukocyte mtDNA or in that of 120 healthy controls or 60 additional patients with mitochondrial disease. This point mutation is located in the 3' end of the gene for subunit III of COX and is predicted to result in the loss of the last 13 amino acids of the highly conserved C-terminal region of this subunit. It was not detected in mtDNA extracted from leukocytes, skeletal muscle, or myoblasts of the patient's mother or her two sons, indicating that this mutation is not maternally transmitted. Single-fiber PCR studies provided direct evidence for an association between this point mutation and COX deficiency and indicated that the proportion of mutant mtDNA required to induce COX deficiency is lower than that reported for tRNA-gene point mutations. The findings reported here represent only the second case of isolated COX deficiency to be defined at the molecular genetic level and reveal a new mutational mechanism in mitochondrial disease.     

Production of poly-β-hydroxybutyrate by Alcaligenes eutrophus on different carbon sources

Poly--hydroxybutyrate was produced in shake cultures by Alcaligenes eutrophus H16 on fructose, xylose, and fumaric, itaconic, lactic and propionic acids in a three-stage process. The maximum polymer concentration of 6.9 g l^–1 (69% of cell dry matter) was obtained with 20g l^–1 of fructose with a volumetric productivity of about 0.22 g l^–1 h^–1 at 24h. Up to about 3 g l^–1 (about 50% of cell dry matter) of polymer was also produced on lactic and propionic acids as the sole carbon source during the production phase. In multivatiate optimization employing an orthogonal 2³-factorial central composite experimental design with fructose as the substrate in a single-stage process, the optimal initial fructose concentration decreased from 35 g l^–1 to 24 g l^–1 when the incubation time was increased from about 35 h to 96 h. The optimal shaking speed range was 90–113 rpm. Correspondence to: S. Linko