Oral alkalinizing supplementation suppressed intrarenal reactive oxidative stress in mild-stage chronic kidney disease: a randomized cohort study.

BACKGROUND: The beneficial effects of oral supplements with alkalinizing agents in patients with chronic kidney disease (CKD) have been limited to the severe stages. We investigated whether two types of supplements, sodium bicarbonate (SB) and potassium citrate/sodium citrate (PCSC), could maintain renal function in patients with mild-stage CKD. METHODS: This was a single-center, open-labeled, randomized cohort trial. Study participants with CKD stages G2, G3a, and G3b were enrolled between March 2013 and January 2019 and randomly assigned by stratification according to age, sex, estimated glomerular filtration rate (eGFR), and diabetes. They were followed up for 6 months (short-term study) for the primary endpoints and extended to 2 years (long-term study) for the secondary endpoints. Supplementary doses were adjusted to achieve an early morning urinary pH of 6.8-7.2. We observed renal dysfunction or new-onset cerebrovascular disease and evaluated urinary surrogate markers for renal injury. RESULTS: Overall, 101 participants were registered and allocated to three groups: standard (n = 32), SB (n = 34), and PCSC (n = 35). Two patients in the standard group attained the primary endpoints (renal stones and overt proteinuria) but were not statistically significant. There was one patient in the standard reduced eGFR during the long-term study (p = 0.042 by ANOVA). SB increased proteinuria (p = 0.0139, baseline vs. 6 months), whereas PCSC significantly reduced proteinuria (p = 0.0061, baseline vs. 1 year, or p = 0.0186, vs. 2 years) and urinary excretion of 8-hydroxy-2'-deoxyguanosine (p = 0.0481, baseline vs. 6 months). CONCLUSION: This study is the first to report supplementation of PCSC reduced intrarenal oxidative stress in patients with mild-stage CKD.

Purification and characterization of protease M, a yeast mitochondrial nucleotide-stimulated metal protease: its identification as CYM1 gene product, a mitochondrial presequence peptidase.

A chelator-sensitive protease in the mitochondrial matrix of the yeast, Saccharomyces cerevisiae (Biochem. Biophys. Res. Commun. 144, 277, 1987), was purified and characterized. The purified enzyme, termed protease M, specifically hydrolyzes peptide substrates on the N-side of the paired basic residues. When mastoparan was used as substrate, it cleaved Ala8-Leu9 and Lys11-Lys12 bonds as well as the N-side of Lys11-Lys12 residues. Nucleotide triphosphates stimulated the activity 3-fold at 2.5 mM. The genomic DNA sequence showed that protease M was a gene product of CYM1 known as mitochondrial presequence protease homologue in S. cerevisiae, encoding a 989-amino acid-long precursor protein. The N-terminal sequence of the purified enzyme indicated that protease M has 16-residue signal sequence and the 'mature' protein consists of 973 amino acids with a molecular mass of 110 kDa. Protease M contained consensus sequence motifs of ATP-binding site very near the carboxyl terminus. The alignment of the two ATP-binding motifs is an inverted version of the common alignment. Gene disruption of the enzyme generates mixed subunits in tetrameric MnSOD formed with 23-kDa mature and 24-kDa partial presequence-containing subunits. This report describes newly identified enzyme properties of the CYM1 gene product, protease M and abnormal MnSOD complex formation of the disruption mutant.

Localization and ER membrane insertion of parathyroid hormone-related protein analyzed without effects of reporter proteins.

Parathyroid hormone-related protein (PTHrP) is transported to both the secretory pathway and the nucleus/nucleolus by its dual targeting signals, that is, an N-terminal signal peptide and nuclear targeting signal. Curiously, reporter proteins such as enhanced green fluorescent protein strongly affect the localization of the fusion protein. Here, we report a novel methionine tag for 35 S-labelling added to the C-terminus of its prepro-form, which has no methionine and cysteine residue other than the initiation methionine that enables analyses of the molecular mechanism of its dual localization without the effects of the reporter proteins. Mutational analyses including insertion of a glycosylation site for the tagged PTHrP revealed that the evolutionarily conserved regions in the signal peptide and the pro-region facilitate the redirection of ppPTHrP from the secretory pathway to the nuclear targeting pathway.

Evolutionary well-conserved region in the signal peptide of parathyroid hormone-related protein is critical for its dual localization through the regulation of ER translocation.

Parathyroid hormone-related protein (PTHrP) has two different targeting signals: an N-terminal signal peptide for the endoplasmic reticulum (ER) targeting and an internal nuclear localization signal. The protein not only functions as a secretory protein, but is also found in the nucleus and/or nucleolus under certain conditions. PTHrP signal peptide is less hydrophobic than most signal peptides mainly due to its evolutionarily well-conserved region (QQWS). The substitution of four tandem leucine residues for this conserved region resulted in a significant inhibition of the signal peptide cleavage. At the same time, proportion of nuclear and/or nucleolar localization decreased, probably due to tethering of the protein to the ER membrane by the uncleaved mutant signal peptide. Almost complete cleavage of the signal peptide accompanied by a lack of nuclear/nucleolar localization was achieved by combining the hydrophobic h-region and an optimized sequence of the cleavage site. In addition, mutational modifications of the distribution of charged residues in and around the signal peptide affect its cleavage and/or nuclear/nucleolar localization of the protein. These results indicate that the well-conserved region in the signal peptide plays an essential role in the dual localization of PTHrP through ER targeting and/or the membrane translocation.

Cleavage of the ER-targeting signal sequence of parathyroid hormone-related protein is cell-type-specific and regulated in Cis by its nuclear localization signal.

Prepro-parathyroid hormone-related protein (ppPTHrP) has two targeting signals, an N-terminal signal sequence and a nuclear localization signal (NLS). In fact, the protein is not only secreted from the cell but also found in the nucleus and/or nucleolus. In order to understand the function of the PTHrP signal sequence for the dual localization, the signal sequence cleavage of a series of ppPTHrP deletion mutants fused to Escherichia coli leader peptidase was analysed in vitro and in several cell lines. Efficiency of the PTHrP signal sequence cleavage was intrinsically low in the in vitro reconstitution system. In cultured cells, cleavage efficiency of the PTHrP signal sequence varied significantly, being lowest in COS-1 cells, but rising in HeLa, HEK293 and CV-1 cells. However, virtually complete signal sequence cleavage was observed in CHO cells. In addition, the NLS of PTHrP had a negative effect on its own signal sequence cleavage, which could be enhanced by deletion of the spacer sequence between the signal sequence and the NLS. There was a roughly inverse relationship between the signal sequence cleavage and the nuclear localization of PTHrP. Thus, the final destination of PTHrP could be regulated at the ER membrane.

Prolyl-isomerase Pin1 accumulates in lewy bodies of parkinson disease and facilitates formation of alpha-synuclein inclusions.

Parkinson disease (PD) is a relatively common neurodegenerative disorder that is characterized by the loss of dopaminergic neurons and by the formation of Lewy bodies (LBs), which are cytoplasmic inclusions containing aggregates of alpha-synuclein. Although certain post-translational modifications of alpha-synuclein and its related proteins are implicated in the genesis of LBs, the specific molecular mechanisms that both regulate these processes and initiate subsequent inclusion body formation are not yet well understood. We demonstrate in our current study, however, that the prolyl-isomerase Pin1 localizes to the LBs in PD brain tissue and thereby enhances the formation of alpha-synuclein immunoreactive inclusions. Immunohistochemical analysis of brain tissue from PD patients revealed that Pin1 localizes to 50-60% of the LBs that show an intense halo pattern resembling that of alpha-synuclein. By utilizing a cellular model of alpha-synuclein aggregation, we also demonstrate that, whereas Pin1 overexpression facilitates the formation of alpha-synuclein inclusions, dominant-negative Pin1 expression significantly suppresses this process. Consistent with these observations, Pin1 overexpression enhances the protein half-life and insolubility of alpha-synuclein. Finally, we show that Pin1 binds synphilin-1, an alpha-synuclein partner, via its Ser-211-Pro and Ser-215-Pro motifs, and enhances its interaction with alpha-synuclein, thus likely facilitating the formation of alpha-synuclein inclusions. These results indicate that Pin1-mediated prolyl-isomerization plays a pivotal role in a post-translational modification pathway for alpha-synuclein aggregation and in the resultant Lewy body formations in PD.

A new type of O(2)(-)-generating tool for oxidative stress studies by remodeling neutrophil NADPH oxidase.

The effects of reactive oxygen species on cells have attracted much attention in relation to redox regulation and oxidative stress-related diseases. Superoxide (O(2)(-)) is the reactive oxygen species primarily formed in biological systems. However, no convenient O(2)(-)-generating device has been available for use in cell or tissue culture. The neutrophil NADPH oxidase, a professional enzyme for killing bacteria, has a high ability to produce O(2)(-). However, the cell-free activation process requires several protein factors and an anionic amphiphile, and moreover, the activation is transient. To utilize the enzyme as an O(2)(-) generator, we improved the cell-free activation method by remodeling regulatory components, optimizing lipid composition, and modifying the mixing conditions. We established a new method to produce an active enzyme that is stable, efficient, and preservable. As an application, we examined the effect of the device on cultured HEK293 cells and observed that it caused cell death. This system has several advantages over the xanthine oxidase system often used. The new device will be useful for studies of oxidative stress and related diseases.

[Severe chlamydia pneumoniae pneumonia requiring mechanical ventilation and steroid therapy in an elderly patient].

A 75-year-old man first developed dyspnea and low-grade fever in late March. A chest X-ray film showed infiltration in the right lower lung field and blood gas analysis revealed severe hypoxemia. Accordingly, he was diagnosed as having pneumonia and was admitted to our hospital on March 11, 2003. Mechanical ventilation for progressive respiratory failure was started immediately after admission, and he was treated with antibiotics. Chlamydia pneumoniae pneumonia was diagnosed due to an increase of the Chlamydia pneumoniae antibody titer. He had prolonged respiratory failure despite antibiotic therapy. Therefore, steroid therapy was started on day 15 for respiratory failure. At 21 days after admission, the infiltration was found to be decreased on chest X-ray films and improvement of hypoxemia allowed extubation. In conclusion, when severe community-acquired pneumonia occurs in elderly patients, we should remember the possibility of atypical pneumonia such as that due to Chlamydia pneumoniae infection.

Immunohistochemical study of synphilin-1 in brains of patients with dementia with Lewy bodies - synphilin-1 is non-specifically implicated in the formation of different neuronal cytoskeletal inclusions.

Dementia with Lewy bodies brains were immunohistochemically investigated using anti-synphilin-1 antibodies. The alpha-synuclein-positive brainstem type and well-defined cortical type Lewy bodies (LB) were positive for synphilin-1, while ill-defined LB and LB-related neurites were negative, suggesting that synphilin-1 does not directly associate with alpha-synuclein. Synphilin-1-positive LB were double-positive for phosphorylated neurofilament. In addition, tau-positive neurofibrillary tangles (NFT) were positive for synphilin-1, while neuropil threads were negative. Immunoelectron microscopically, synphilin-1 was located on filamentous components in cortical type LB and on paired helical filaments in NFT. It is likely that synphilin-1 accumulates in the cell body according to the axonal transport blockage, and associates with abnormal cytoskeltons during the formation of LB or NFT, suggesting that synphilin-1 is non-specifically implicated in the formation of different neuronal cytoskeletal inclusions.

Progression and staging of Lewy pathology in brains from patients with dementia with Lewy bodies.

Using alpha-synuclein-immunohistochemistry, 27 brains of dementia with Lewy bodies (DLB) were investigated to identify the progression of Lewy pathology including Lewy bodies (LB) and LB-related neurites in the cerebrum. The numbers of alpha-synuclein-positive LB and LB-related neurites were semiquantitatively evaluated in the amygdala, hippocampus, entorhinal cortex, transentorhinal cortex, insular cortex, middle temporal cortex and superior frontal cortex. The results indicated that Lewy pathology within the neuron progresses first in the axonal terminal, subsequently in the cell body and finally in the dendrite, that Lewy pathology in the cerebral cortex progresses first in layers V-VI, subsequently in layer III and finally in layer II, and that Lewy pathology in the cerebrum progresses first in the amygdala, subsequently in the limbic cortex and finally in the neocortex. In addition, Lewy pathology was graded from stage I to stage IV based on the progression of Lewy pathology. The 27 brains examined were classified into 3 brains showing stage I, 11 showing stage II, 7 showing stage III and 6 showing stage IV. Comparing these stages with the pathological subtypes of DLB brains, brains of the subtype showing severe Alzheimer pathology corresponded to brains showing an advanced stage, suggesting that Alzheimer pathology exacerbates Lewy pathology.