Molecular cloning and sequence analysis of cDNA for the catalytic subunit 1 alpha of rat kidney type 1 protein phosphatase, and detection of the gene expression at high levels in hepatoma cells and regenerating livers as compared to rat livers.

A cDNA clone containing the full coding sequence of a type 1 protein phosphatase catalytic subunit 1 alpha has been isolated from a rat kidney lambda gt 10 library. The protein sequence deduced from the cDNA contains 330 amino acid residues with a molecular mass of 38 kDa. The cDNA clone from rat kidney was 89% identical at the nucleotide level in the coding region to type 1 protein phosphatase 1 alpha from rabbit skeletal muscle. However, the two protein sequences were completely identical. The type 1 alpha protein phosphatase from rat kidney shows 49% homology of amino acid sequence to the rat type 2A alpha protein phosphatase. Thus, the protein sequence of type 1 alpha protein phosphatase was completely conserved between rat and rabbit. The mRNA levels of type 1 protein phosphatase were determined in rat liver, AH13, a strain of rat hepatoma, and regenerating rat liver by Northern blot analysis using the cDNA fragment as a probe, under which conditions a single mRNA of 1.5 kb was detected. The mRNA levels of AH13 were remarkably increased when compared to those of normal ivers, whereas the mRNA levels of regenerating livers were slightly but significantly increased. These results demonstrate a marked increase in gene expression of type 1 protein phosphatase in hepatoma cells, suggesting an important role of the type 1 protein phosphatase in hepatocarcinogenesis.     

Central hypertensive actions of angiotensin I, II and III in conscious rats

The effects of intracerebroventricular administrations of three natural angiotensins, angiotensin I (ANG I 3.8 X 10-11-9.4 X10-10 mol/kg body weight), II (9.6 X 10-12-2.4 X 10-10 mol/kg body weight) and III (2.7 X 10-10 2.5 X 10-9 mol/kg body weight) on systemic blood pressure were investigated in conscious rats. Angiotensin II (ANG II), ANG I and angiotensin III (ANG III), increased blood pressure in a dose-related manner. The order of potency of angiotensins was ANG II greater than ANG I greater than ANG III. The intraventricular administration of a converting enzyme inhibitor (SQ 14225, 6.9 X10-8 mol/kg) abolished the central effect of ANG I, while an angiotensin II analogue ([Sar1-Ala8]ANG II, 1.1 X 10-8 mol/kg) administered intraventricularly inhibited the central pressor effects of these three angiotensins. These results suggest that ANG II is a main mediator of the renin-angiotensin system in the central nervous system.     

Effect of vasoactive substances on natriuresis induced by atrial natriuretic peptide in isolated perfused rat kidneys

We studied the interaction between synthetic atrial natriuretic peptide (ANP) and various vasoactive substances, which included isoproterenol (ISO), aminophylline (AMI), and dibutyryl cyclic AMP (dBcAMP) as vasodilators, and angiotensin II (AII) and norepinephrine (NE) as vasoconstrictors, and prazosin as an alpha-blocker in isolated perfused rat kidneys (IPK). When 10(-9) mol of ANP was administered in 75 ml of a perfusate, the renal vascular resistance (RVR) was transiently decreased for 5 min, and increased thereafter. Simultaneously, ANP increased the glomerular filtration rate (GFR), urine flow (UV), absolute Na excretion (UNaV) and absolute K excretion (UKV). All of the above mentioned effects of ANP were significantly inhibited by administering ISO, AMI or dBcAMP. On the other hand, the administration of AII and NE significantly enhanced the increases in UV and UNaV and the fractional excretion of Na induced by ANP, although AII and NE had no influence on the changes in RVR and GFR induced by ANP. Prazosin did not modify the renal effects of ANP. These results suggest that the natriuretic effect of ANP is inhibited by agents that increase cyclic AMP in vascular smooth muscle cells. It is also suggested that the natriuretic effects of ANP can be explained by an increase in GFR and changes in intrarenal hemodynamics, rather than by the direct effect of ANP on renal tubules.     

Pyridoxine stimulates filaggrin production in human epidermal keratinocytes

Molecular Biology Reports - Pyridoxine (PN), one of the vitamers of vitamin B6, plays an important role in the maintenance of epidermal function and is used to treat acne and rough skin. Clinical...     

GDP Induces PANC-1 Human Pancreatic Cancer Cell Death Preferentially under Nutrient Starvation by Inhibiting PI3K/Akt/mTOR/Autophagy Signaling Pathway

Pancreatic tumors are hypovascular, which leads to a poor nutrient supply to support the aggressively proliferating tumor cells. However, human pancreatic cancer cells have extreme resistance to nutrition starvation, which enables them to survive under severe metabolic stress conditions within the tumor microenvironment, a phenomenon known as “austerity” in cancer biology. Discovering agents which can preferentially inhibit the cancer cells’ ability to tolerate starvation conditions represents a new generation of anticancer agents. In this study, geranyl 2,4-dihydroxy-6-phenethylbenzoate (GDP), isolated from Boesenbergia pandurata rhizomes, exhibited potent preferential cytotoxicity against PANC-1 human pancreatic cancer cells under nutrition starvation conditions. GDP also possessed PANC-1 cell migration and colony formation inhibitory activities under normal nutrient-rich conditions. Mechanistically, GDP inhibited PI3K/Akt/mTOR/autophagy survival signaling pathway, leading to selective PANC-1 cancer cell death under the nutrition starvation condition. Therefore, GDP is a promising anti-austerity agent for drug development against pancreatic cancer.     

Global overexpression of divalent metal transporter 1 delays crocidolite-induced mesothelial carcinogenesis in male mice

Abstract

Exposure to asbestos fiber is central to mesothelial carcinogenesis, for which iron overload in or near mesothelial cells is a key pathogenic mechanism. Alternatively, iron chelation therapy with deferasirox or regular phlebotomy was significantly preventive against crocidolite-induced mesothelial carcinogenesis in rats. However, the role of iron transporters during asbestos-induced carcinogenesis remains elusive. Here, we studied the role of divalent metal transporter 1 (DMT1; Slc11a2), which is a Fe(II) transporter, that is present not only on the apical plasma membrane of duodenal cells but also on the lysosomal membrane of every cell, in crocidolite-induced mesothelial carcinogenesis using DMT1 transgenic (DMT1Tg) mice. DMT1Tg mice show mucosal block of iron absorption without cancer susceptibility under normal diet. We unexpectedly found that superoxide production was significantly decreased upon stimulation with crocidolite both in neutrophils and macrophages of DMT1Tg mice, and the macrophage surface revealed higher iron content 1?h after contact with crocidolite. Intraperitoneal injection of 3?mg crocidolite ultimately induced malignant mesothelioma in ～50% of both wild-type and DMT1Tg mice (23/47 and 14/28, respectively); this effect was marginally (p?=?0.069) delayed in DMT1Tg mice, promoting survival. The promotional effect of nitrilotriacetic acid was limited, and the liver showed significantly higher iron content both in DMT1Tg mice and after crocidolite exposure. The results indicate that global DMT1 overexpression causes decreased superoxide generation upon stimulation in inflammatory cells, which presumably delayed the promotional stage of crocidolite-induced mesothelial carcinogenesis. DMT1Tg mice with low-stamina inflammatory cells may be helpful to evaluate the involvement of inflammation in various pathologies.     

Alternative Excision Repair Pathways

Alternative excision repair (AER) is a category of excision repair initiated by a single nick, made by an endonuclease, near the site of DNA damage, and followed by excision of the damaged DNA, repair synthesis, and ligation. The ultraviolet (UV) damage endonuclease in fungi and bacteria introduces a nick immediately 5′ to various types of UV damage and initiates its excision repair that is independent of nucleotide excision repair (NER). Endo IV-type apurinic/apyrimidinic (AP) endonucleases from Escherichia coli and yeast and human Exo III-type AP endonuclease APEX1 introduce a nick directly and immediately 5′ to various types of oxidative base damage besides the AP site, initiating excision repair. Another endonuclease, endonuclease V from bacteria to humans, binds deaminated bases and cleaves the phosphodiester bond located 1 nucleotide 3′ of the base, leading to excision repair. A single-strand break in DNA is one of the most frequent types of DNA damage within cells and is repaired efficiently. AER makes use of such repair capability of single-strand breaks, removes DNA damage, and has an important role in complementing BER and NER.NER and base excision repair (BER) are the major excision repair pathways present in almost all organisms. In NER, dual incisions are introduced, the damaged DNA between the incised sites is then removed, and DNA synthesis fills the single-stranded gap, followed by ligation. In BER, an AP site, formed by depurination or created by a base damage-specific DNA glycosylase, is recognized by an AP endonuclease that introduces a nick immediately 5′ to the AP site, followed by repair synthesis, removal of the AP site, and final ligation. Besides these two fundamental excision repair systems, investigators have found another category of excision repair—AER—an example of which is the excision repair of UV damage, initiated by an endonuclease called UV damage endonuclease (UVDE). UVDE introduces a single nick immediately 5′ to various types of UV lesions as well as other types of base damage, and this nick leads to the removal of the lesions by an AER process designated as UVDE-mediated excision repair (UVER or UVDR). Genetic analysis in Schizosaccharomyces pombe indicates that UVER provides cells with an extremely rapid removal of UV lesions, which is important for cells exposed to UV in their growing phase.Endo IV–type AP endonucleases from Escherichia coli and budding yeast and the Exo III–type human AP endonuclease APEX1 are able to introduce a nick at various types of oxidative base damage and initiate a form of excision repair that has been designated as nucleotide incision repair (NIR). Endonuclease V (ENDOV) from bacteria to humans recognizes deaminated bases, introduces a nick 1 nucleotide 3′ of the base, and leads to excision repair initiated by the nick. These endonucleases introduce a single nick near the DNA-damage site, leaving 3′-OH termini, and initiate repair of both the DNA damage and the nick. The mechanisms of AER may be similar to those of single-strand break (SSB) repair or BER except for the initial nicking process. However, how DNA damage is recognized determines the repair process within the cell. This article discusses the mechanisms and functional roles of AER. We begin with AER of UV damage, because genetic analysis has shown functional differences between this AER and NER in S. pombe.     

Inspection of frass ejection could decrease the risk of white-spotted longicorn beetle Anoplophora malasiaca (Coleoptera: Cerambycidae) infestation of Japanese pine bonsais to negligible levels

Applied Entomology and Zoology - To evaluate the infection risk of Anoplophora malasiaca (Thomson) (Coleoptera: Cerambycidae) in two species of Japanese pine bonsais (Japanese black pine and...     

Bacterial and Archaeal 16S rRNA Genes in Late Pleistocene to Holocene Muddy Sediments from the Kanto Plain of Japan

Microbial communities in ancient marine sediments composed of clay and silt obtained from the terrestrial subsurface were phylogenetically analyzed based on their 16S rRNA gene sequences. Chloroflexi and Miscellaneous Crenarchaeotic Group were predominant in bacterial and archaeal clone libraries, respectively. Of 44 operational taxonomic units (OTUs) that had close relatives in the database, 30 were close to sequences obtained from marine environments. Some sequences belonged to the candidate groups JS1, ANME-I, and Marine Benthic Group-C, which are typically found in marine sediments. Low chloride concentrations in the sediments suggest that these marine-affiliated sequences may not reflect currently active microbial communities. Our results indicate the existence of long-term preserved DNA or descendants of ancient oceanic microbial components in subsurface muddy sediments in a temperate region, which may reflect indigenous population of paleoenvironments.     

Substrate specificity and gene expression of two Penicillium chrysogenum α-l-arabinofuranosidases (AFQ1 and AFS1) belonging to glycoside hydrolase families 51 and 54

We previously isolated two α-l-arabinofuranosidases (ABFs), termed AFQ1 and AFS1, from the culture filtrate of Penicillium chrysogenum 31B. afq1 and afs1 complementary DNAs encoding AFQ1 and AFS1 were isolated by in vitro cloning. The deduced amino acid sequences of AFQ1 and AFS1 are highly similar to those of Penicillium purpurogenum ABF 2 and ABF 1, respectively, which belong to glycoside hydrolase (GH) families 51 and 54, respectively. Pfam analysis revealed an “Alpha-L-AF_C” domain in AFQ1 and “ArabFuran-catal” and “AbfB” domains in AFS1. Semi-quantitative RT-PCR analysis indicated that the afq1 gene was constitutively expressed in P. chrysogenum 31B at a low level, although the expression was slightly induced with arabinose, arabinitol, arabinan, and arabinoxylan. In contrast, expression of the afs1 gene was strongly expressed by the above four carbohydrates and less strongly induced by galactan. Recombinant enzymes (rAFQ1 and rAFS1) expressed in Escherichia coli were active against both p-nitrophenyl α-l-arabinofuranoside and polysaccharides with different specificities. ¹H-NMR analysis revealed that rAFS1 degraded arabinofuranosyl side chains that were both singly and doubly linked to the backbones of arabinoxylan and l-arabinan. On the other hand, rAFQ1 preferentially released arabinose linked to C-3 of single-substituted xylose or arabinose residues in the two polysaccharides.