Increased Renal Iron Accumulation in Hypertensive Nephropathy of Salt-Loaded Hypertensive Rats

Although iron is reported to be associated with the pathogenesis of chronic kidney disease, it is unknown whether iron participates in the pathophysiology of nephrosclerosis. Here, we investigate whether iron is involved in the development of hypertensive nephropathy and the effects of iron restriction on nephrosclerosis in salt- loaded stroke-prone spontaneously hypertensive rats (SHRSP). SHRSP were given either a normal or high-salt diet for 8 weeks. Another subset of SHRSP were fed a high-salt with iron-restricted diet. SHRSP given a high-salt diet developed severe hypertension and nephrosclerosis. As a result, survival rate was decreased after 8 weeks diet. Importantly, massive iron accumulation and increased iron content were observed in the kidneys of salt-loaded SHRSP, along with increased superoxide production, urinary 8-Hydroxy-2′-deoxyguanosine excretion, and urinary iron excretion; however, these changes were markedly attenuated by iron restriction. Of interest, expression of cellular iron transport proteins, transferrin receptor 1 and divalent metal transporter 1, was increased in the tubules of salt-loaded SHRSP. Notably, iron restriction attenuated the development of severe hypertension and nephrosclerosis, thereby improving survival rate in salt-loaded SHRSP. Taken together, these results suggest a novel mechanism by which iron plays a role in the development of hypertensive nephropathy and establish the effects of iron restriction on salt-induced nephrosclerosis.     

Dam Methylation Participates in the Regulation of PmrA/PmrB and RcsC/RcsD/RcsB Two Component Regulatory Systems in Salmonella enterica Serovar Enteritidis

The absence of Dam in Salmonella enterica serovar Enteritidis causes a defect in lipopolysaccharide (LPS) pattern associated to a reduced expression of wzz gene. Wzz is the chain length regulator of the LPS O-antigen. Here we investigated whether Dam regulates wzz gene expression through its two known regulators, PmrA and RcsB. Thus, the expression of rcsB and pmrA was monitored by quantitative real-time RT-PCR and Western blotting using fusions with 3×FLAG tag in wild type (wt) and dam strains of S. Enteritidis. Dam regulated the expression of both rcsB and pmrA genes; nevertheless, the defect in LPS pattern was only related to a diminished expression of RcsB. Interestingly, regulation of wzz in serovar Enteritidis differed from that reported earlier for serovar Typhimurium; RcsB induces wzz expression in both serovars, whereas PmrA induces wzz in S. Typhimurium but represses it in serovar Enteritidis. Moreover, we found that in S. Enteritidis there is an interaction between both wzz regulators: RcsB stimulates the expression of pmrA and PmrA represses the expression of rcsB. Our results would be an example of differential regulation of orthologous genes expression, providing differences in phenotypic traits between closely related bacterial serovars.     

Therapeutic Efficacy of C-Kit-Targeted Radioimmunotherapy Using 90Y-Labeled Anti-C-Kit Antibodies in a Mouse Model of Small Cell Lung Cancer

Small cell lung cancer (SCLC) is an aggressive tumor and prognosis remains poor. Therefore, the development of more effective therapy is needed. We previously reported that high levels of an anti-c-kit antibody (12A8) accumulated in SCLC xenografts. In the present study, we evaluated the efficacy of two antibodies (12A8 and 67A2) for radioimmunotherapy (RIT) of an SCLC mouse model by labeling with the ⁹⁰Y isotope.

Methods

¹¹¹In- or ¹²⁵I-labeled antibodies were evaluated in vitro by cell binding, competitive inhibition and cellular internalization assays in c-kit-expressing SY cells and in vivo by biodistribution in SY-bearing mice. Therapeutic efficacy of ⁹⁰Y-labeled antibodies was evaluated in SY-bearing mice upto day 28 and histological analysis was conducted at day 7.

Results

[¹¹¹In]12A8 and [¹¹¹In]67A2 specifically bound to SY cells with high affinity (8.0 and 1.9 nM, respectively). 67A2 was internalized similar to 12A8. High levels of [¹¹¹In]12A8 and [¹¹¹In]67A2 accumulated in tumors, but not in major organs. [¹¹¹In]67A2 uptake by the tumor was 1.7 times higher than for [¹¹¹In]12A8. [⁹⁰Y]12A8, but not [⁹⁰Y]67A2, suppressed tumor growth in a dose-dependent manner. Tumors treated with 3.7 MBq of [⁹⁰Y]12A8, and 1.85 and 3.7 MBq of [⁹⁰Y]67A2 (absorbed doses were 21.0, 18.0 and 35.9 Gy, respectively) almost completely disappeared approximately 2 weeks after injection, and regrowth was not observed except for in one mouse treated with 1.85 MBq [⁹⁰Y]67A2. The area of necrosis and fibrosis increased depending on the RIT effect. Apoptotic cell numbers increased with increased doses of [⁹⁰Y]12A8, whereas no dose-dependent increase was observed following [⁹⁰Y]67A2 treatment. Body weight was temporarily reduced but all mice tolerated the RIT experiments well.

Conclusion

Treatment with [⁹⁰Y]12A8 and [⁹⁰Y]67A2 achieved a complete therapeutic response when SY tumors received an absorbed dose greater than 18 Gy and thus are promising RIT agents for metastatic SCLC cells at distant sites.     

Interference in DNA Replication Can Cause Mitotic Chromosomal Breakage Unassociated with Double-Strand Breaks

Morphological analysis of mitotic chromosomes is used to detect mutagenic chemical compounds and to estimate the dose of ionizing radiation to be administered. It has long been believed that chromosomal breaks are always associated with double-strand breaks (DSBs). We here provide compelling evidence against this canonical theory. We employed a genetic approach using two cell lines, chicken DT40 and human Nalm-6. We measured the number of chromosomal breaks induced by three replication-blocking agents (aphidicolin, 5-fluorouracil, and hydroxyurea) in DSB-repair-proficient wild-type cells and cells deficient in both homologous recombination and nonhomologous end-joining (the two major DSB-repair pathways). Exposure of cells to the three replication-blocking agents for at least two cell cycles resulted in comparable numbers of chromosomal breaks for RAD54^−/−/KU70^−/− DT40 clones and wild-type cells. Likewise, the numbers of chromosomal breaks induced in RAD54^−/−/LIG4^−/− Nalm-6 clones and wild-type cells were also comparable. These data indicate that the replication-blocking agents can cause chromosomal breaks unassociated with DSBs. In contrast with DSB-repair-deficient cells, chicken DT40 cells deficient in PIF1 or ATRIP, which molecules contribute to the completion of DNA replication, displayed higher numbers of mitotic chromosomal breaks induced by aphidicolin than did wild-type cells, suggesting that single-strand gaps left unreplicated may result in mitotic chromosomal breaks.     

Meningitis and bacteremia by nonhemolytic Group B Streptococcus strain: A whole genome analysis

Group B streptococcus (GBS) is a leading cause of neonatal infections. Most isolates are β-hemolytic, and their activity is considered to be pivotal for GBS pathogenicity. We report a case of a neonate with meningitis caused by nonhemolytic GBS. The patient developed meningitis 3 days after birth. Genotyping was performed and the characteristics of the strain (GCMC97051) identified by whole genome sequence using next generation sequencing. GCMC97051 possesses genetic alterations such as disruption of cylA by IS1381A insertion and a frameshift mutation in cylE, resulting in a lack of hemolysis. Thus, nonhemolytic GBS can retain the potential to cause invasive infections.     

Development of Biotin-Prototrophic and -Hyperauxotrophic Corynebacterium glutamicum Strains

To develop the infrastructure for biotin production through naturally biotin-auxotrophic Corynebacterium glutamicum, we attempted to engineer the organism into a biotin prototroph and a biotin hyperauxotroph. To confer biotin prototrophy on the organism, the cotranscribed bioBF genes of Escherichia coli were introduced into the C. glutamicum genome, which originally lacked the bioF gene. The resulting strain still required biotin for growth, but it could be replaced by exogenous pimelic acid, a source of the biotin precursor pimelate thioester linked to either coenzyme A (CoA) or acyl carrier protein (ACP). To bridge the gap between the pimelate thioester and its dedicated precursor acyl-CoA (or -ACP), the bioI gene of Bacillus subtilis, which encoded a P450 protein that cleaves a carbon-carbon bond of an acyl-ACP to generate pimeloyl-ACP, was further expressed in the engineered strain by using a plasmid system. This resulted in a biotin prototroph that is capable of the de novo synthesis of biotin. On the other hand, the bioY gene responsible for biotin uptake was disrupted in wild-type C. glutamicum. Whereas the wild-type strain required approximately 1 μg of biotin per liter for normal growth, the bioY disruptant (ΔbioY) required approximately 1 mg of biotin per liter, almost 3 orders of magnitude higher than the wild-type level. The ΔbioY strain showed a similar high requirement for the precursor dethiobiotin, a substrate for bioB-encoded biotin synthase. To eliminate the dependency on dethiobiotin, the bioB gene was further disrupted in both the wild-type strain and the ΔbioY strain. By selectively using the resulting two strains (ΔbioB and ΔbioBY) as indicator strains, we developed a practical biotin bioassay system that can quantify biotin in the seven-digit range, from approximately 0.1 μg to 1 g per liter. This bioassay proved that the engineered biotin prototroph of C. glutamicum produced biotin directly from glucose, albeit at a marginally detectable level (approximately 0.3 μg per liter).     

Intermolecular Forces Involved in the Gelation and Gel Stability of Sesame 13S Globulin

The effect of various reagents on the formation and stability of heat-induced gels of sesame 13S globulins were investigated. Electrostatic interaction, the hydrophobic bond and the disulfide bond were important for forming the network structure of gels, and the hydrogen bond also had an influence on the formation of the gel. Hydrophobic bonds mainly contributed to the stability of the gel. Subunit analyses of the proteins solubilized from the gels showed the presence of a free acidic subunit (AS) and basic subunit (BS), a polymer of AS, a dimer of BS and the dimer of a fragment from AS or BS. From the results, sulfhydryl-disulfide exchange reactions during gelation are suggested.