High-resolution structure and biochemical properties of the LH1-RC photocomplex from the model purple sulfur bacterium, Allochromatium vinosum.

The mesophilic purple sulfur phototrophic bacterium Allochromatium (Alc.) vinosum (bacterial family Chromatiaceae) has been a favored model for studies of bacterial photosynthesis and sulfur metabolism, and its core light-harvesting (LH1) complex has been a focus of numerous studies of photosynthetic light reactions. However, despite intense efforts, no high-resolution structure and thorough biochemical analysis of the Alc. vinosum LH1 complex have been reported. Here we present cryo-EM structures of the Alc. vinosum LH1 complex associated with reaction center (RC) at 2.24 Å resolution. The overall structure of the Alc. vinosum LH1 resembles that of its moderately thermophilic relative Alc. tepidum in that it contains multiple pigment-binding α- and ß-polypeptides. Unexpectedly, however, six Ca ions were identified in the Alc. vinosum LH1 bound to certain α1/ß1- or α1/ß3-polypeptides through a different Ca2+-binding motif from that seen in Alc. tepidum and other Chromatiaceae that contain Ca2+-bound LH1 complexes. Two water molecules were identified as additional Ca2+-coordinating ligands. Based on these results, we reexamined biochemical and spectroscopic properties of the Alc. vinosum LH1-RC. While modest but distinct effects of Ca2+ were detected in the absorption spectrum of the Alc. vinosum LH1 complex, a marked decrease in thermostability of its LH1-RC complex was observed upon removal of Ca2+. The presence of Ca2+ in the photocomplex of Alc. vinosum suggests that Ca2+-binding to LH1 complexes may be a common adaptation in species of Chromatiaceae for conferring spectral and thermal flexibility on this key component of their photosynthetic machinery.

Structural Determination of the Nanocomplex of Borate with Styrene-Maleic Acid Copolymer-Conjugated Glucosamine Used as a Multifunctional Anticancer Drug.

The development of effective anticancer drugs is essential for chemotherapy that specifically targets cancer tissues. We recently synthesized a multifunctional water-soluble anticancer polymer drug consisting of styrene-maleic acid copolymer (SMA) conjugated with glucosamine and boric acid (BA) (SGB complex). It demonstrated about 10 times higher tumor-selective accumulation compared with accumulation in normal tissues because of the enhanced permeability and retention effect, and it inhibited tumor growth via glycolysis inhibition, mitochondrial damage, and thermal neutron irradiation. Gaining insight into the anticancer effects of this SGB complex requires a determination of its structure. We therefore investigated the chemical structure of the SGB complex by means of nuclear magnetic resonance, infrared (IR) spectroscopy, and liquid chromatography-mass spectrometry. To establish the chemical structure of the SGB complex, we synthesized a simple model compound─maleic acid-glucosamine (MAG) conjugate─by using a maleic anhydride (MA) monomer unit instead of the SMA polymer. We obtained two MAG-BA complexes (MAGB) with molecular weights of 325 and 343 after the MAG reaction with BA. We confirmed, by using IR spectroscopy, that MAGB formed a stable complex via an amide bond between MA and glucosamine and that BA bound to glucosamine via a diol bond. As a result of this chemical design, identified via analysis of MAGB, the SGB complex can release BA and demonstrate toxicity to cancer cells through inhibition of lactate secretion in mild hypoxia that mimics the tumor microenvironment. For clinical application of the SGB complex, we confirmed that this complex is stable in the presence of serum. These findings confirm that our design of the SGB complex has various advantages in targeting solid cancers and exerting therapeutic effects when combined with neutron irradiation.

Ectopic PTH-producing parathyroid cyst inside the thymus: a case report.

BACKGROUND: The hallmark of hyperparathyroidism is hypersecretion of parathyroid hormone (PTH) which results in hypercalcemia and hypophosphatemia. While hypercalcemia due to malignancy is often brought about by PTH-related protein in adults, PTH-producing tumors are quite rare in clinical practice. Additionally, from the point of embryology, it is very difficult to examine ectopic PTH-producing tissue such as ectopic parathyroid glands. Furthermore, clear histopathological criteria are not present. CASE PRESENTATION: A 57-year-old woman was referred to our hospital for hypercalcemia. Her parathyroid hormone (PTH) level was elevated, but there were no enlarged parathyroid glands. Although 99mTc-MIBI confirmed a localized and slightly hyperfunctioning parathyroid tissue in the anterior mediastinum, it was not typical as hyperfunctioning parathyroid. We finally diagnosed her as ectopic PTH-producing cyst-like tumor with venous sampling of PTH. She underwent anterosuperior mediastinal ectopic PTH-producing cyst-like tumor resection. It is noted that intact-PTH concentration of the fluid in the cyst was very high (19,960,000 pg/mL). Based on histopathological findings, we finally diagnosed her as ectopic PTH-producing parathyroid cyst inside the thymus. After resection of anterosuperior mediastinal thymus including ectopic PTH-producing parathyroid cyst, calcium and intact-PTH levels were decreased, and this patient was discharged without any sequelae. CONCLUSIONS: We should know the possibility of superior mediastinal ectopic PTH-producing parathyroid cyst inside the thymus among subjects with ectopic PTH-producing parathyroid glands. Particularly when the cyst is present in the superior mediastinum, it is necessary to do careful diagnosis based on not only positive but also negative findings in 99mTc-MIBI. It is noted that the patient's bloody fluid in the cyst contained 19,960,000 pg/mL of intact-PTH, and its overflow into blood stream resulted in hyperparathyroidism and hypercalcemia. Moreover, in such cases, the diagnosis is usually confirmed after through histological examination of ectopic PTH-producing parathyroid glands. We think that it is very meaningful to let clinicians know this case.

EPR-Effect Enhancers Strongly Potentiate Tumor-Targeted Delivery of Nanomedicines to Advanced Cancers: Further Extension to Enhancement of the Therapeutic Effect.

For more than three decades, enhanced permeability and retention (EPR)-effect-based nanomedicines have received considerable attention for tumor-selective treatment of solid tumors. However, treatment of advanced cancers remains a huge challenge in clinical situations because of occluded or embolized tumor blood vessels, which lead to so-called heterogeneity of the EPR effect. We previously developed a method to restore impaired blood flow in blood vessels by using nitric oxide donors and other agents called EPR-effect enhancers. Here, we show that two novel EPR-effect enhancers-isosorbide dinitrate (ISDN, Nitrol®) and sildenafil citrate-strongly potentiated delivery of three macromolecular drugs to tumors: a complex of poly(styrene-co-maleic acid) (SMA) and cisplatin, named Smaplatin® (chemotherapy); poly(N-(2-hydroxypropyl)methacrylamide) polymer-conjugated zinc protoporphyrin (photodynamic therapy and imaging); and SMA glucosamine-conjugated boric acid complex (boron neutron capture therapy). We tested these nanodrugs in mice with advanced C26 tumors. When these nanomedicines were administered together with ISDN or sildenafil, tumor delivery and thus positive therapeutic results increased two- to four-fold in tumors with diameters of 15 mm or more. These results confirmed the rationale for using EPR-effect enhancers to restore tumor blood flow. In conclusion, all EPR-effect enhancers tested showed great potential for application in cancer therapy.

Polymer-conjugated glucosamine complexed with boric acid shows tumor-selective accumulation and simultaneous inhibition of glycolysis.

We synthesized unique water-soluble synthetic-polymer, styrene-maleic acid copolymer (SMA) conjugated glucosamine (SG); which formed a stable complex with boric acid (BA). This complex had a mean particle size of 15 nm by light scattering, and single peak in gel permeation chromatography. The particles were taken up by tumor cells five times faster than free BA in vitro and liberated BA at acidic tumor pH (5-7). Liberated BA inhibited glycolysis and resulted in tumor suppression in vivo. Intravenously injected SGB-complex did bind with albumin, and plasma half-life was about 8 h in mice, and accumulated to tumor tissues about 10 times more than in normal organs. IC50 of SGB-complex for HeLa cells under pO2 of 6-9% was about 20 µg/ml (free BA equivalent), 150 times more potent than free BA. Neutron irradiation of human oral cancer cells with SGB-complex resulted in 16 times greater cell-killing than that without SGB-complex. In vivo antitumor effect was evaluated after neutron irradiation only once in SCC VII tumor bearing mice and significant tumor suppression was confirmed. These results indicate that SGB-complex is a unique multifunctional anticancer agent with much more potent activity under low pO2 conditions as in large advanced cancers.

GZD824 Inhibits GCN2 and Sensitizes Cancer Cells to Amino Acid Starvation Stress.

Eukaryotic initiation factor 2α (eIF2α) kinase general control nonderepressible 2 (GCN2) drives cellular adaptation to amino acid limitation by activating the integrated stress response that induces activating transcription factor 4 (ATF4). Here, we found that a multikinase inhibitor, GZD824, which we identified using a cell-based assay with ATF4 immunostaining, inhibited the GCN2 pathway in cancer cells. Indeed, GZD824 suppressed GCN2 activation, eIF2α phosphorylation, and ATF4 induction during amino acid starvation stress. However, at lower nonsuppressive concentrations, GZD824 paradoxically stimulated eIF2α phosphorylation and ATF4 expression in a GCN2-dependent manner under unstressed conditions. Such dual properties conceivably arose from a direct effect on GCN2, as also observed in a cell-free GCN2 kinase assay and shared by a selective GCN2 inhibitor. Consistent with the GCN2 pathway inhibition, GZD824 sensitized certain cancer cells to amino acid starvation stress similarly to ATF4 knockdown. These results establish GZD824 as a multikinase GCN2 inhibitor and may enhance its utility as a drug under development. SIGNIFICANCE STATEMENT: GZD824, as a direct general control nonderepressible 2 (GCN2) inhibitor, suppresses activation of the integrated stress response during amino acid limitation, whereas it paradoxically stimulates this stress-signaling pathway at lower nonsuppressive concentrations. The pharmacological activity we identify herein will provide the basis for the use of GZD824 to elucidate the regulatory mechanisms of GCN2 and to evaluate the potential of the GCN2-activating transcription factor 4 pathway as a target for cancer therapy.

Preparation of Biodegradable PLGA-Nanoparticles Used for pH-Sensitive Intracellular Delivery of an Anti-inflammatory Bacterial Toxin to Macrophages.

Poly(D,L-lactide-co-glycolic) acid (PLGA) is a synthetic copolymer that has been used to design micro/nanoparticles as a carrier for macromolecules, such as protein and nucleic acids, that can be internalized by the endocytosis pathway. However, it is difficult to control the intracellular delivery to target organelles. Here we report an intracellular delivery system of nanoparticles modified with bacterial cytotoxins to the endoplasmic reticulum (ER) and anti-inflammatory activity of the nanoparticles. Subtilase cytotoxin (SubAB) is a bacterial toxin in certain enterohemorrhagic Escherichia coli (EHEC) strains that cleaves the host ER chaperone BiP and suppresses nuclear factor-kappaB (NF-κB) activation and nitric oxide (NO) generation in macrophages at sub-lethal concentration. PLGA-nanoparticles were modified with oligo histidine-tagged (6 × His-tagged) recombinant SubAB (SubAB-PLGA) through a pH-sensitive linkage, and their translocation to the ER in macrophage cell line J774.1 cells, effects on inducible NO synthase (iNOS), and levels of tumor necrosis factor (TNF)-α cytokine induced by lipopolysaccharide (LPS) were examined. Compared with free SubAB, SubAB-PLGA was significantly effective in BiP cleavage and the induction of the ER stress marker C/EBP homologous protein (CHOP) in J774.1 cells. Furthermore, SubAB-PLGA attenuated LPS-stimulated induction of iNOS and TNF-α. Our findings provide useful information for protein delivery to macrophages and may encourage therapeutic applications of nanoparticles to the treatment of inflammatory diseases.