Continuous culture of novel mitochondrial cells lacking nuclei

We isolated stable cell lines, designated as mitochondrial cells, from cybrids obtained by fusing mitochondria-less HeLa cells with platelets from patients with Leigh syndrome, a subtype of mitochondrial encephalomyopathy. The cells contain a pathogenic point mutation, T9176C, in the mitochondrial DNA. Hematoxylin-eosin staining, confocal fluorescent microscopy and flow cytometry in fixed or living cells showed that the majority of these mitochondrial cells lack nuclear DNA and nuclei, but contain active mitochondria. Despite the absence of nuclear DNA, these cells can be continuously generated in culture. Therefore, it is likely that they arise from the minority of cells which possess a nucleus.     

Possibility of cytoplasmic pre-tRNA splicing: the yeast tRNA splicing endonuclease mainly localizes on the mitochondria

Pre-tRNA splicing has been believed to occur in the nucleus. In yeast, the tRNA splicing endonuclease that cleaves the exon-intron junctions of pre-tRNAs consists of Sen54p, Sen2p, Sen34p, and Sen15p and was thought to be an integral membrane protein of the inner nuclear envelope. Here we show that the majority of Sen2p, Sen54p, and the endonuclease activity are not localized in the nucleus, but on the mitochondrial surface. The endonuclease is peripherally associated with the cytosolic surface of the outer mitochondrial membrane. A Sen54p derivative artificially fixed on the mitochondria as an integral membrane protein can functionally replace the authentic Sen54p, whereas mutant proteins defective in mitochondrial localization are not fully active. sen2 mutant cells accumulate unspliced pre-tRNAs in the cytosol under the restrictive conditions, and this export of the pre-tRNAs partly depends on Los1p, yeast exportin-t. It is difficult to explain these results from the view of tRNA splicing in the nucleus. We rather propose a new possibility that tRNA splicing occurs on the mitochondrial surface in yeast.     

The PAR-1-activating peptide facilitates pepsinogen secretion in rats

Protease-activated receptor-2 (PAR-2) is abundantly expressed in gastric mucosal chief cells, facilitating pepsinogen secretion. In the present study, we investigated whether PAR-1, a thrombin receptor, could modulate pepsinogen secretion in rats. The PAR-1-activating peptide TFLLR-NH(2) as well as the PAR-2-activating peptide SLIGRL-NH(2), administered i.v. repeatedly at 1-h intervals, significantly increased gastric pepsinogen secretion over 2-4 h (after two to four doses). In contrast, the control peptide FTLLR-NH(2), given in the same manner, had no such effect. Thus, PAR-1, like PAR-2, might function to facilitate pepsinogen secretion, suggesting a novel role of the thrombin-PAR-1-pathway in the stomach.     

The in vivo roles of STEF/Tiam1, Rac1 and JNK in cortical neuronal migration

The coordinated migration of neurons is a pivotal step for functional architectural formation of the mammalian brain. To elucidate its molecular mechanism, gene transfer by means of in utero electroporation was applied in the developing murine brain, revealing the crucial roles of Rac1, its activators, STEF/Tiam1, and its downstream molecule, c-Jun N-terminal kinase (JNK), in the cerebral cortex. Functional repression of these molecules resulted in inhibition of radial migration of neurons without affecting their proper differentiation. Interestingly, distinct morphological phenotypes were observed; suppression of Rac1 activity caused loss of the leading process, whereas repression of JNK activity did not, suggesting the complexity of the signaling cascade. In cultured neurons from the intermediate zone, activated JNK was detected along microtubules in the processes. Application of a JNK inhibitor caused irregular morphology and increased stable microtubules in processes, and decreased phosphorylation of microtubule associated protein 1B, raising a possibility of the involvement of JNK in controlling tubulin dynamics in migrating neurons. Our data thus provide important clues for understanding the intracellullar signaling machinery for cortical neuronal migration.     

The impact of a single-nucleotide mutation of <Emphasis Type="Italic">bgl2</Emphasis> on cellulase induction in a <Emphasis Type="Italic">Trichoderma reesei</Emphasis> mutant

Background

The filamentous fungus Trichoderma reesei (anamorph of Hypocrea jecorina) produces increased cellulase expression when grown on cellulose or its derivatives as a sole carbon source. It has been believed that β-glucosidases of T. reesei not only metabolize cellobiose but also contribute in the production of inducers of cellulase gene expression by their transglycosylation activity. The cellulase hyper-producing mutant PC-3-7 developed in Japan has enhanced cellulase production ability when cellobiose is used as the inducer. The comparative genomics analysis of PC-3-7 and its parent revealed a single-nucleotide mutation within the bgl2 gene encoding intracellular β-glucosidase II (BGLII/Cel1a), giving rise to an amino acid substitution in PC-3-7, which could potentially account for the enhanced cellulase expression when these strains are cultivated on cellulose and cellobiose.

Results

To analyze the effects of the BGLII mutation in cellulase induction, we constructed both a bgl2 revertant and a disruptant. Enzymatic analysis of the transformant lysates showed that the strain expressing mutant BGLII exhibited weakened cellobiose hydrolytic activity, but produced some transglycosylation products, suggesting that the SNP in bgl2 strongly diminished cellobiase activity, but did not result in complete loss of function of BGLII. The analysis of the recombinant BGLII revealed that transglycosylation products might be oligosaccharides, composed probably of glucose linked β-1,4, β-1,3, or a mixture of both. PC-3-7 revertants of bgl2 exhibited reduced expression and inducibility of cellulase during growth on cellulose and cellobiose substrates. Furthermore, the effect of this bgl2 mutation was reproduced in the common strain QM9414 in which the transformants showed cellulase production comparable to that of PC-3-7.

Conclusion

We conclude that BGLII plays an important role in cellulase induction in T. reesei and that the bgl2 mutation in PC-3-7 brought about enhanced cellulase expression on cellobiose. The results of the investigation using PC-3-7 suggested that other mutation(s) in PC-3-7 could also contribute to cellulase induction. Further investigation is essential to unravel the mechanism responsible for cellulase induction in T. reesei.

     

Deformation of the Outer Hair Cells and the Accumulation of Caveolin-2 in Connexin 26 Deficient Mice

BackgroundMutations in GJB2, which encodes connexin 26 (Cx26), a cochlear gap junction protein, represent a major cause of pre-lingual, non-syndromic deafness. The degeneration of the organ of Corti observed in Cx26 mutant—associated deafness is thought to be a secondary pathology of hearing loss. Here we focused on abnormal development of the organ of Corti followed by degeneration including outer hair cell (OHC) loss.MethodsWe investigated the crucial factors involved in late-onset degeneration and loss of OHC by ultrastructural observation, immunohistochemistry and protein analysis in our Cx26-deficient mice (Cx26^f/fP0Cre).ResultsIn ultrastructural observations of Cx26^f/fP0Cre mice, OHCs changed shape irregularly, and several folds or notches were observed in the plasma membrane. Furthermore, the mutant OHCs had a flat surface compared with the characteristic wavy surface structure of OHCs of normal mice. Protein analysis revealed an increased protein level of caveolin-2 (CAV2) in Cx26^f/fP0Cre mouse cochlea. In immunohistochemistry, a remarkable accumulation of CAV2 was observed in Cx26^f/fP0Cre mice. In particular, this accumulation of CAV2 was mainly observed around OHCs, and furthermore this accumulation was observed around the shrunken site of OHCs with an abnormal hourglass-like shape.ConclusionsThe deformation of OHCs and the accumulation of CAV2 in the organ of Corti may play a crucial role in the progression of, or secondary OHC loss in, GJB2-associated deafness. Investigation of these molecular pathways, including those involving CAV2, may contribute to the elucidation of a new pathogenic mechanism of GJB2-associated deafness and identify effective targets for new therapies.     

Clinical Report on the First Prototype of a Photoacoustic Tomography System with Dual Illumination for Breast Cancer Imaging

Photoacoustic tomography is a recently developed imaging modality that can provide high spatial-resolution images of hemoglobin distribution in tissues such as the breast. Because breast cancer is an angiogenesis-dependent type of malignancy, we evaluated the clinical acceptability of breast tissue images produced using our first prototype photoacoustic mammography (PAM) system in patients with known cancer. Post-excisionally, histological sections of the tumors were stained immunohistochemically (IHC) for CD31 (an endothelial marker) and carbonic anhydrase IX (CAIX) (a marker of hypoxia). Whole-slide scanning and image analyses were used to evaluate the tumor microvessel distribution pattern and to calculate the total vascular perimeter (TVP)/area for each lesion. In this clinical study, 42 lesions were primarily scanned using PAM preoperatively, three of which were reported to be benign and were excluded from statistical analysis. Images were produced for 29 out of 39 cancers (visibility rate = 74.4%) at the median depth of 26.5 (3.25–51.2) mm. Age, menopausal status, body mass index, history of neoadjuvant treatment, clinical stage and histological tumor angiogenesis markers did not seem to affect the visibility. The oxygen saturation level in all of the measured lesions was lower than in the subcutaneous counterpart vessels (Wilcoxon test, p value<0.001), as well as in the counterpart contralateral normal breast region of interest (ROI) (Wilcoxon test, p value = 0.001). Although the oxygen saturation level was not statistically significant between CAIX-positive vs. -negative cases, lesional TVP/area showed a positive correlation with the oxygen saturation level only in the group that had received therapy before PAM. In conclusion, the vascular and oxygenation data obtained by PAM have great potential for identifying functional features of breast tumors.     

Multi-Acupuncture Point Injections and Their Anatomical Study in Relation to Neck and Shoulder Pain Syndrome (So-Called Katakori) in Japan

Katakori is a symptom name that is unique to Japan, and refers to myofascial pain syndrome-like clinical signs in the shoulder girdle. Various methods of pain relief for katakori have been reported, but in the present study, we examined the clinical effects of multi-acupuncture point injections (MAPI) in the acupuncture points with which we empirically achieved an effect, as well as the anatomical sites affected by liquid medicine. The subjects were idiopathic katakori patients (n = 9), and three cadavers for anatomical investigation. BL-10, GB-21, LI-16, SI-14, and BL-38 as the WHO notation were selected as the acupuncture point. Injections of 1 mL of 1% w/v mepivacaine were introduced at the same time into each of these points in the patients. Assessment items were the Pain Relief Score and the therapeutic effect period. Dissections were centered at the puncture sites of cadavers. India ink was similarly injected into each point, and each site that was darkly-stained with India ink was evaluated. Katakori pain in the present study was significantly reduced by MAPI. Regardless of the presence or absence of trigger points, pain was significantly reduced in these cases. Dark staining with India ink at each of the points in the anatomical analysis was as follows: BL-10: over the rectus capitis posterior minor muscle and rectus capitis posterior major muscle fascia; GB-21: over the supraspinatus muscle fascia; LI-16: over the supraspinatus muscle fascia; SI-14: over the rhomboid muscle fascia; and BL-38: over the rhomboid muscle fascia. The anatomical study suggested that the drug effect was exerted on the muscles above and below the muscle fascia, as well as the peripheral nerves because the points of action in acupuncture were darkly-stained in the spaces between the muscle and the muscle fascia.