Characteristics of aleveolar bone associated with physiological movement of molar in mice: a histological and histochemical study

Mouse molars undergo distal movement, during which new bone is formed at the mesial side of the tooth root whereas the preexisting bone is resorbed at the distal side of the root. However, there is little detailed information available regarding which of the bones that surround the tooth root are involved in physiological tooth movement. In the present study, we therefore aimed to investigate the precise morphological differences of the alveolar bone between the bone formation side of the tooth root, using routine histological procedures including silver impregnation, as well as by immunohistochemical analysis of alkaline phosphatase and tartrate-resistant acid phosphatase activity, and immunohistochemical analysis of the expression of the osteocyte markers dentin matrix protein 1, sclerostin, and fibroblast growth factor 23. Histochemical analysis indicated that bone formation by osteoblasts and bone resorption by osteoclasts occurred at the bone formation side and the bone resorption side, respectively. Osteocyte marker immunoreactivity of osteocytes at the surface of the bone close to the periodontal ligament differed at the bone formation and bone resorption sides. We also showed different specific features of osteocytic lacunar canalicular systems at the bone formation and bone resorption sides by using silver staining. This study suggests that the alveolar bone is different in the osteocyte nature between the bone formation side and the bone resorption side due to physiological distal movement of the mouse molar.

High affinity amino acid transporters specifically expressed in xylem parenchyma and developing seeds of Arabidopsis

Arabidopsis amino acid transporters (AAPs) show individual temporal and spatial expression patterns. A new amino acid transporter, AAP8 was isolated by reverse transcription-PCR. Growth and transport assays in comparison to AAP1-5 characterize AAP8 and AAP6 as high affinity amino acid transport systems from Arabidopsis. Histochemical promoter-beta-glucuronidase (GUS) studies identified AAP6 expression in xylem parenchyma, cells requiring high affinity transport due to the low amino acid concentration in xylem sap. AAP6 may thus function in uptake of amino acids from xylem. Histochemical analysis of AAP8 revealed stage-dependent expression in siliques and developing seeds. Thus AAP8 is probably responsible for import of organic nitrogen into developing seeds. The only missing transporter of the family AAP7 was nonfunctional in yeast with respect to amino acid transport, and expression was not detectable. Therefore, AAP6 and -8 are the only members of the family able to transport aspartate with physiologically relevant affinity. AAP1, -6 and -8 are the closest AAP paralogs. Although AAP1 and AAP8 originate from a duplicated region on chromosome I, biochemical properties and expression pattern diverged. Overlapping substrate specificities paired with individual properties and expression patterns point to specific functions of each of the AAP genes in nitrogen distribution rather than to mere redundancy.

Novel mitochondrial tRNA(Ile) m.4282A>G gene mutation leads to chronic progressive external ophthalmoplegia plus phenotype

BACKGROUND/AIM To investigate the underlying pathomechanism in a 33-year-old female Caucasian patient presenting with chronic progressive external ophthalmoplegia (CPEO) plus symptoms. METHODS Histochemical analysis of skeletal muscle and biochemical measurements of individual oxidative phosphorylation (OXPHOS) complexes. Genetic analysis of mitochondrial DNA in various tissues with subsequent investigation of single muscle fibres for correlation of mutational load. RESULTS The patient's skeletal muscle showed 20% of cytochrome c oxidase-negative fibres and 8% ragged-red fibres. Genetic analysis of the mitochondrial DNA revealed a novel point mutation in the mitochondrial tRNA(Ile) (MTTI) gene at position m.4282G>A. The heteroplasmy was determined in blood, buccal cells and muscle by restriction fragment length polymorphism (RFLP) combined with a last fluorescent cycle. The total mutational load was 38% in skeletal muscle, but was not detectable in blood or buccal cells of the patient. The phenotype segregated with the mutational load as determined by analysis of single cytochrome c oxidase-negative/positive fibres by laser capture microdissection and subsequent LFC-RFLP. CONCLUSIONS We describe a novel MTTI transition mutation at nucleotide position m.4282G>A associated with a CPEO plus phenotype. The novel variant at position m.4282G>A disrupts the middle bond of the D-stem of the tRNA(Ile) and is highly conserved. The conservation and phenotype-genotype segregation strongly suggest pathogenicity and is in good agreement with the MTTI gene being frequently associated with CPEO. This novel variant broadens the spectrum of MTTI mutations causing CPEO.

Developmental control of H+/amino acid permease gene expression during seed development of Arabidopsis

Long distance transport of amino acids is mediated by several families of differentially expressed amino acid transporters. The two genes AAP1 and AAP2 encode broad specificity H+-amino acid co-transporters and are expressed to high levels in siliques of Arabidopsis, indicating a potential role in supplying the seeds with organic nitrogen. The expression of both genes is developmentally controlled and is strongly induced in siliques at heart stage of embryogenesis, shortly before induction of storage protein genes. Histochemical analysis of transgenic plants expressing promoter-GUS fusions shows that the genes have non-overlapping expression patterns in siliques. AAP1 is expressed in the endosperm and the cotyledons whereas AAP2 is expressed in the vascular strands of siliques and in funiculi. The endosperm expression of AAP1 during early stages of seed development indicates that the endosperm serves as a transient storage tissue for organic nitrogen. Amino acids are transported in both xylem and phloem but during seed filling are imported only via the phloem. AAP2, which is expressed in the phloem of stems and in the veins supplying seeds, may function in uptake of amino acids assimilated in the green silique tissue, in the retrieval of amino acids leaking passively out of the phloem and in xylem-to-phloem transfer along the path. The promoters provide excellent tools to study developmental, hormonal and metabolic control of nitrogen nutrition during development and may help to manipulate the timing and composition of amino acid import into seeds.

High-resolution MALDI-FT-ICR MS imaging for the analysis of metabolites from formalin-fixed, paraffin-embedded clinical tissue samples.

We present the first analytical approach to demonstrate the in situ imaging of metabolites from formalin-fixed, paraffin-embedded (FFPE) human tissue samples. Using high-resolution matrix-assisted laser desorption/ionization Fourier-transform ion cyclotron resonance mass spectrometry imaging (MALDI-FT-ICR MSI), we conducted a proof-of-principle experiment comparing metabolite measurements from FFPE and fresh frozen tissue sections, and found an overlap of 72% amongst 1700 m/z species. In particular, we observed conservation of biomedically relevant information at the metabolite level in FFPE tissues. In biomedical applications, we analysed tissues from 350 different cancer patients and were able to discriminate between normal and tumour tissues, and different tumours from the same organ, and found an independent prognostic factor for patient survival. This study demonstrates the ability to measure metabolites in FFPE tissues using MALDI-FT-ICR MSI, which can then be assigned to histology and clinical parameters. Our approach is a major technical, histochemical, and clinicopathological advance that highlights the potential for investigating diseases in archived FFPE tissues.