Electron-microscopic study of the maturing rat red nucleus. I. The large-neuron population

The maturing large neurons of the rat red nucleus in animals ranging in age from 1 to 21 days of postnatal life were studied ultrastructurally. Days 1--6 were characterized by rapid morphologic maturation occurring concomitantly with the onset of synaptogenesis. Morphogenesis was confined to the soma, while the first synaptic contacts were also formed in relationship to the soma. Days 6--9 demonstrated continued somal morphogenesis exemplified by cytoplasmic expansion and by the conspicuous presence of perisomatic and growth cone processes. Proximal dendritic morphogenesis was initiated, and synaptogenesis became complex with synaptic sites occurring in relation to the neuronal soma, the perisomatic processes and proximal dendrites. Days 9--15 were characterized by the completion of somal and proximal dendritic morphogenesis and by a massive degree of synaptogenic activity. During this interval, the soma lost perisomatic and growth cone processes, while somatic spines appeared. By the end of this period the neuronal soma and the proximal dendrites appeared mature in terms of both morphology and synaptic input. Complete neuronal maturation was ultimately attained by day 21 of postnatal life.     

4SALE – A tool for synchronous RNA sequence and secondary structure alignment and editing

Background  

In sequence analysis the multiple alignment builds the fundament of all proceeding analyses. Errors in an alignment could strongly influence all succeeding analyses and therefore could lead to wrong predictions. Hand-crafted and hand-improved alignments are necessary and meanwhile good common practice. For RNA sequences often the primary sequence as well as a secondary structure consensus is well known, e.g., the cloverleaf structure of the t-RNA. Recently, some alignment editors are proposed that are able to include and model both kinds of information. However, with the advent of a large amount of reliable RNA sequences together with their solved secondary structures (available from e.g. the ITS2 Database), we are faced with the problem to handle sequences and their associated secondary structures synchronously.     

Combinatorial effects of the phytoestrogen genistein and of estradiol in uterus and liver of female Wistar rats

The knowledge about safety of phytoestrogens on proliferative endpoints in the endometrium is rather limited, particularly when low amounts of estrogens are present like in postmenopausal women. Therefore, we now studied how genistein (GEN) exposure affects proliferative endpoints in the endometrium in estrogenized animals. We investigated the effects of GEN (10 mg/(kg day) BW) on uterine proliferation and on general uterine response markers in intact female rats and ovariectomized (OVX) female rats co-treated with different doses of estradiol (E2; 1 or 4 μg/(kg day) BW). In parallel we investigated generalized hepatic effects of GEN in this co-stimulatory protocol. In agreement to our previous results, GEN treatment of OVX animals for 3 days results in a faint stimulation of the uterine wet weight. In intact animals and in OVX animals co-treated with E2 no effects of GEN on uterine wet weight were detectable. GEN treatment did not affect the uterine epithelial height in intact animals but resulted in a decrease of the protein and mRNA expression of the proliferation marker PCNA. In OVX animals co-treated with E2, GEN antagonized the E2 stimulated increase of the uterine epithelial height and epithelial PCNA expression. Besides PCNA, GEN effects on the uterine mRNA expression of IGF-1, IGF-1R, Complement C3, estrogen receptor- (ER) and -β (ERβ), as well as progesterone receptor were investigated in intact and OVX co-treated animals. Overall there was a tendency in all combinatorial groups that GEN counteracts E2 function in uterine tissue. Surprisingly, while investigating estrogenic response markers in liver, we observed very strong effects of GEN on hepatic marker gene expression. GEN significantly down-regulated CaBP9K and IGFBP1 mRNA levels in intact animals. In OVX animals hepatic CABP9K and IGFBP1 mRNA levels were not affected by E2 treatment. GEN treatment, even in combination with E2, decreased the hepatic CaBP9K expression below the levels observed in untreated animals. Interestingly co-treatment of OVX rats with low dose E2 and GEN resulted in a significant increase of IGFBP1 mRNA expression. Summarising our results we conclude that (1) GEN treatment in the presence of E2 is safe regarding proliferative responses in the endometrium of adult animals; (2) the observation of differences of the GEN activity in intact and OVX/E2 substituted animals can be taken as a hint that GEN may interact mechanistically with progestins which has to be proven in detail in future investigations and (3) the detection of strong effects of the phytoestrogen GEN on hepatic gene expression may point to the need of future investigations to rule out the possibility of adverse responses in this organ.     

Osteoblast-targeted disruption of glucocorticoid signalling does not delay intramembranous bone healing

Objective

Glucocorticoids at pharmacological doses have been shown to interfere with fracture repair. The role of endogenous glucocorticoids in fracture healing is not well understood. We examined whether endogenous glucocorticoids affect bone healing in an in vivo model of cortical defect repair.

Methods

Experiments were performed using a well characterised mouse model in which intracellular glucocorticoid signalling was disrupted in osteoblasts through transgenic overexpression of 11β-hydroxysteroid-dehydrogenase type 2 (11β-HSD2) under the control of a collagen type I promoter (Col2.3-11β-HSD2). Unicortical bone defects (∅0.8 mm) were created in the tibiae of 7-week-old male transgenic mice and their wild-type littermates. Repair was assessed via histomorphometry, immunohistochemistry and microcomputed tomography (micro-CT) analysis at 1-3 weeks after defect creation.

Results

At week 1, micro-CT images of the defect demonstrated formation of mineralized intramembranous bone which increased in volume and density by week 2. At week 3, healing of the defect was nearly complete in all animals. Analysis by histomorphometry and micro-CT revealed that repair of the bony defect was similar in Col2.3-11β-HSD2 transgenic animals and their wild-type littermates at all time-points.

Conclusion

Disrupting endogenous glucocorticoid signalling in mature osteoblasts did not affect intramembranous fracture healing in a tibia defect repair model. It remains to be shown whether glucocorticoid signalling has a role in endochondral fracture healing.     

A new pathway for the synthesis of oligosaccharides by the use of non-Leloir glycosyltransferases

The growing recognition of the roles of carbohydrates in fundamental biological processes and their potential application as functional foods and new therapeutics have generated a need for larger amounts of different carbohydrate structures. Leloir glycosyltransferases catalyze the synthesis of complex oligosaccharides. However they are difficult or expensive to obtain, and require expensive nucleotide activated sugars. In contrast non-Leloir pathway enzymes use sucrose, which is known to be a high energy donor of d-glucose for glucosyltransferases like dextransucrase, or a donor of d-fructose for fructosyltransferases like inulin- and levansucrases for the synthesis of polysaccharides. Here we present the synthesis and kinetic studies of oligosaccharides using non-Leloir glycosyltransferases and sucrose analogues as new substrates, like β-d-fructofuranosyl-α-d-galactopyranoside (Gal-Fru) by a fructosyltransferase (FTF) from B. subtilis NCIMB 11871. The sucrose analogues carry a high binding energy in the glycosidic bond similar to that of sucrose. Thus, β-d-Fructofuranosyl-α-d-galactopyranoside (Gal-Fru) and β-d-Fructofuranosyl-α-d-fucopyranoside (d-Fuc-Fru) have been shown to be substrates for fructosyltransferases, which produce oligo- or polysaccharides, also in the presence of acceptors.     

Genome sequences of the biotechnologically important Bacillus megaterium strains QM B1551 and DSM319

Bacillus megaterium is deep-rooted in the Bacillus phylogeny, making it an evolutionarily key species and of particular importance in understanding genome evolution, dynamics, and plasticity in the bacilli. B. megaterium is a commercially available, nonpathogenic host for the biotechnological production of several substances, including vitamin B(12), penicillin acylase, and amylases. Here, we report the analysis of the first complete genome sequences of two important B. megaterium strains, the plasmidless strain DSM319 and QM B1551, which harbors seven indigenous plasmids. The 5.1-Mbp chromosome carries approximately 5,300 genes, while QM B1551 plasmids represent a combined 417 kb and 523 genes, one of the largest plasmid arrays sequenced in a single bacterial strain. We have documented extensive gene transfer between the plasmids and the chromosome. Each strain carries roughly 300 strain-specific chromosomal genes that account for differences in their experimentally confirmed phenotypes. B. megaterium is able to synthesize vitamin B(12) through an oxygen-independent adenosylcobalamin pathway, which together with other key energetic and metabolic pathways has now been fully reconstructed. Other novel genes include a second ftsZ gene, which may be responsible for the large cell size of members of this species, as well as genes for gas vesicles, a second β-galactosidase gene, and most but not all of the genes needed for genetic competence. Comprehensive analyses of the global Bacillus gene pool showed that only an asymmetric region around the origin of replication was syntenic across the genus. This appears to be a characteristic feature of the Bacillus spp. genome architecture and may be key to their sporulating lifestyle.     

Aberrant axial mineralization precedes spinal ankylosis: a molecular imaging study in ank/ank mice

Introduction

The diagnosis of ankylosing spondylitis is made from a combination of clinical features and the presence of radiographic evidence that may be detected only after many years of inflammatory back pain. It is not uncommon to have a diagnosis confirmed 5 to 10 years after the initial onset of symptoms. Development of a more-sensitive molecular imaging technology to detect structural changes in the joints would lead to earlier diagnosis and quantitative tracking of ankylosis progression. Progressive ankylosis (ank/ank) mice have a loss of function in the Ank gene, which codes for a regulator of PPi transport. In this study, we used these ank/ank mutant mice to assess a noninvasive, quantitative measure of joint ankylosis with near-infrared (NIR) molecular imaging in vivo.

Methods

Three age groups (8, 12, and 18 weeks) of ank/ank (15 mice) and wild-type littermates (12 +/+ mice) were assessed histologically and radiographically. Before imaging, OsteoSense 750 (bisphosphonate pamidronate) was injected i.v. Whole-body images were analyzed by using the multispectral Maestro imaging system.

Results

OsteoSense 750 signals in the paw joints were higher in ank/ank mice in all three age groups compared with controls. In the spine, significantly higher OsteoSense 750 signals were detected early, in 8-week-old ank/ank mice compared with controls, although minimal radiographic differences were noted at this time point. The molecular imaging changes in the ank/ank spine (8 weeks) were supported by histologic changes, including calcium apatite crystals at the edge of the vertebral bodies and new syndesmophyte formation.

Conclusions

Changes in joint pathology of ank/ank mice, as evaluated by histologic and radiographic means, are qualitative, but only semiquantitative. In contrast, molecular imaging provides a quantitative assessment. Ankylosis in ank/ank mice developed simultaneously in distal and axial joints, contrary to the previous notion that it is a centripetal process. NIR imaging might be feasible for early disease diagnosis and for monitoring disease progression in ankylosing spondylitis.     

RNase MRP RNA and RNase P activity in plants are associated with a Pop1p containing complex

RNase P processes the 5'-end of tRNAs. An essential catalytic RNA has been demonstrated in Bacteria, Archaea and the nuclei of most eukaryotes; an organism-specific number of proteins complement the holoenzyme. Nuclear RNase P from yeast and humans is well understood and contains an RNA, similar to the sister enzyme RNase MRP. In contrast, no protein subunits have yet been identified in the plant enzymes, and the presence of a nucleic acid in RNase P is still enigmatic. We have thus set out to identify and characterize the subunits of these enzymes in two plant model systems. Expression of the two known Arabidopsis MRP RNA genes in vivo was verified. The first wheat MRP RNA sequences are presented, leading to improved structure models for plant MRP RNAs. A novel mRNA encoding the central RNase P/MRP protein Pop1p was identified in Arabidopsis, suggesting the expression of distinct protein variants from this gene in vivo. Pop1p-specific antibodies precipitate RNase P activity and MRP RNAs from wheat extracts. Our results provide evidence that in plants, Pop1p is associated with MRP RNAs and with the catalytic subunit of RNase P, either separately or in a single large complex.     

Industrial carbohydrate biotransformations

Nearly all major industrial processes which involve carbohydrates, include biotechnological transformations. This is due to the complex nature of carbohydrates where stereo- and regioselectivity are highly complex and difficult to control. Enzymes and microorganisms work highly selectively and efficiently in water solution, and provide high yield in general. The article focuses on different types of reactions, including large-scale processes. Topics are hydrolytic reactions, including starch processing, oxidation and reduction transformations including organic acids, such as gluconic and ketogluconic acids and vitamin C synthesis, and isomerization and transfer reactions, which are established on a very large scale to produce glucose/fructose syrups and sucrose isomers. The article will further discuss some mechanistic aspects which are relevant for technology and present selected details of industrial-scale processing. Finally an outlook outlines perspectives of future processes.