An anti-HIV-1 V3 loop antibody fully protects cross-clade and elicits T-cell immunity in macaques mucosally challenged with an R5 clade C SHIV

Neutralizing antibodies have been shown to protect macaques against SHIV challenge. However, genetically diverse HIV-1 clades have evolved, and a key question left unanswered is whether neutralizing antibodies can confer cross-clade protection in vivo. The novel human monoclonal antibody HGN194 was isolated from an individual infected with an HIV-1 clade AG recombinant circulating recombinant form (CRF). HGN194 targets an epitope in the third hypervariable loop (V3) of HIV-1 gp120 and neutralizes a range of relatively neutralization-sensitive and resistant viruses. We evaluated the potential of HGN194 to protect infant rhesus monkeys against a SHIV encoding a primary CCR5-tropic HIV-1 clade C envelope. After high-dose mucosal challenge, all untreated controls became highly viremic while all HGN194-treated animals (50 mg/kg) were completely protected. When HGN194 was given at 1 mg/kg, one out of two monkeys remained aviremic, whereas the other had delayed, lower peak viremia. Interestingly, all protected monkeys given high-dose HGN194 developed Gag-specific proliferative responses of both CD4+ and CD8+ T cells. To test whether generation of the latter involved cryptic infection, we ablated CD8+ cells after HGN194 clearance. No viremia was detected in any protected monkeys, thus ruling out virus reservoirs. Thus, induction of CD8 T-cell immunity may have resulted from transient "Hit and Run" infection or cross priming via Ag-Ab-mediated cross-presentation. Together, our data identified the HGN194 epitope as protective and provide proof-of-concept that this anti-V3 loop mAb can prevent infection with sterilizing immunity after challenge with virus of a different clade, implying that V3 is a potential vaccine target.     

ULTRASTRUCTURAL STUDY ON MICROSPORE WALL MORPHOGENESIS IN ISOETES JAPONICA (ISOETACEAE)

Spore wall morphogenesis of the microspore of Isoetes japonica was studied by transmission electron microscopy. The microspore wall consists of four layers: the perispore, outer exospore, inner exospore, and endospore. The perispore consists of electron-dense materials. The exospore is divided into outer and inner sections, with a large gap between the two. The outer exospore appears as an undulating plate consisting of tripartite lamellae with homogeneous sporopollenin. The inner exospore consists of an accumulation of tripartite lamellae on the microspore cell membrane. Immediately after meiosis, the tripartite lamellae of the outer exospore forms around the microspore. The lamellated inner exospore forms next, which adheres to the cell membrane of the microspore. The deposition of homogeneous sporopollenin material on the tripartite lamellae causes the plates of the outer exospore to thicken. Some homogeneous material may also be deposited on the inner exospore. Lastly, the electron-dense perispore is deposited on the outer exospore, and the electron-lucent endospore forms beneath the inner exospore. We conclude that the lamellae of the outer exospore, inner exospore, and endospore are formed and derived, in that order, from the gametophytic microspore cytoplasm. The homogeneous sporopollenin material of the outer exospore and perispore may be derived from the sporophytic tapetal cytoplasm.     

Requirement of the conserved, hydrophobic C-terminus region for the activation of heparanase

Heparanase is an endo-β-d-glucuronidase responsible for the cleavage of heparan sulfate, participating in extracellular matrix degradation and remodeling. Heparanase activity is well correlated with the potential for metastasis and angiogenesis in a large number of tumor-derived cell types, directly implicating the involvement of heparanase in tumor progression. Here, we provide the first evidence that the hydrophobic C-terminus region of heparanase has specific roles in intracellular trafficking, secretion, activation, and heparanase-mediated tumor cell migration. Furthermore, partial deletion of this hydrophobic C-terminus region, substitution within the hydrophobic C-terminus region to hydrophilic amino acids, and experiments of single amino acid mutations further point out the importance of the hydrophobic C-terminus region. Therefore, our findings suggest that the hydrophobic C-terminus region of heparanase is a determinant for its intracellular trafficking to the Golgi apparatus, followed by secretion, activation, and tumor cell migration.     

Rational design and synthesis of novel heparan sulfate mimetic compounds as antiadhesive agents

A biological evaluation of the antiadhesive activity of novel heparan sulfate glycosaminoglycans mimetic compounds (KI-compounds) is described. In an adhesion assay, KI-111 [2-(4-fluoro-3-nitrobenzoyl)benzoic acetic anhydride] was found to exert potent inhibitory activities against the adhesion of human fibrosarcoma HT1080 cells and HeLa cells to fibronectin. Cell growth, migration, and invasion of HT1080 cells were also inhibited by KI-111 at almost equal concentrations.     

An ultrastructural study of the polyp and strobila of Atorella japonica (Cnidaria,Coronatae) with respect to muscles and nerves

Atorella japonica were observed by TEM to examine the nerve plexus in the capitulum of the polyp and the cross-striated muscle cells of the strobila. The nerve plexus included a number of neuromuscular junctions and many interneural synapses. Neuromuscular junctions contained two types of synaptic vesicle: clear and small (ca 75 nm diam.), and dense cored and large (ca 120 nm diam.). The first type of vesicle always appeared near the presynaptic membrane and the second type was distributed behind the former. In interneural synapses, two types of vesicle which were similar to neuromuscular synaptic vesicles were recognized. They were distributed in a pattern similar to that of the neuromuscular synaptic vesicles, but these vesicles were found on both sides of the two synaptic membranes.     

Transmembrane domain of Bcl-2 is required for inhibition of ceramide synthesis,but not cytochrome c release in the pathway of inostamycin-induced apoptosis

Bcl-2 protein plays important roles in the regulation of apoptosis. However, the exact mechanism by which Bcl-2 blocks apoptosis is still unclear. In the present study, we found that overexpression of Bcl-2 in human small cell lung carcinoma Ms-1 cells inhibited not only the release of cytochrome c from mitochondria into cytosol but also de novo ceramide synthesis induced by inostamycin, a phosphatidylinositol turnover inhibitor. To investigate the correlation between the structure of Bcl-2 and its inhibitory function in inostamycin-induced apoptosis, Ms-1 cells that stably overexpress domain-deletional mutants of Bcl-2 were established. Transmembrane domain-deleted Bcl-2 failed to inhibit inostamycin-induced de novo ceramide synthesis, whereas it inhibited inostamycin-induced cytochrome c release, indicating that anchoring of Bcl-2 to membrane was a requirement for its inhibitory effect on inostamycin-induced ceramide synthesis, but not cytochrome c release. Thus, the deletion mutant of tarnsmembrane domain of Bcl-2 can suppress inostamycin-induced apoptosis by inhibiting cytochrome c release, a downstream event of ceramide synthesis in the pathway of inostamycin-induced apoptosis. We also found that the BH3 and BH4 domains of Bcl-2 were necessary for inhibition of inostamycin-induced apoptosis, and deletion of BH1 or BH2 did not affect the inhibitory effect of Bcl-2 to inostamycin-induced apoptotic events.     

Proteomics of beta2-microglobulin amyloid fibrils

Knowledge on the chemical structure of beta2-microglobulin in natural amyloid fibrils is quite limited because of the difficulty in obtaining tissue samples suitable for biochemical studies. We have reviewed the available information on the chemical modifications and we present new data of beta2-microglobulin extracted from non-osteotendinous tissues. beta2-microglobulin can accumulate in these compartments after long-term haemodialysis but rarely forms amyloid deposits. We confirm that truncation at the N-terminus is an event specific to beta2-microglobulin derived from fibrils but is not observed in the beta2-microglobulin from plasma or from the insoluble non-fibrillar material deposited in the heart and spleen. We also confirm the partial deamidation of Asn 17 and Asn 42, as well as the oxidation of Met 99 in fibrillar beta2-microglobulin. Other previously reported chemical modifications cannot be excluded, but should involve less than 1-2% of the intact molecule.     

The Oxyradical-Scavenging Activity of Azelaic Acid in Biological Systems

We have previously shown that azelaic acid, a C₉ dicarboxylic acid, as disodium salt (C₉2Na) is capable of inhibiting significantly the hydroxylation of aromatic compounds and the peroxidation of arachidonic acid due to reactive hydroxyl radicals (HO'). In this paper we have investigated the ability of C₉2Na to inhibit the oxyradical induced toxicity towards two tumoral cell lines (Raji and IRE 1) and normal human fibroblasts (HF). Oxyradicals were generated either by the addition of polyphenols to the medium, or by direct irradiation of phosphate buffered-saline in which cells were incubated from 15min prior to incubation in normal medium. The effects of C₉2Na were compared with those obtained by mannitol (MAN), superoxide dismutase (SOD) and catalase (CAT). C₉2Na, MAN, SOD and CAT significantly decreased the polyphenol toxicity towards cell lines cultured up to 24 h. After 48 h of incubation the above compounds lost the capability of protecting cells from polyphenol toxicity. This suggests that the toxic role of oxyradicals (O₂^-, H₂O₂, HO^.) persists for about 24h and, subsequently other toxic mechanisms must be involved, which are not affected by oxyradical scavengers. SOD and CAT did not show any protective effect on UV induced cytotoxicity, while both C₉2Na and MAN were capable of reducing significantly the UV damage towards cell lines, even after 48 h incubation. This can be explained by the fact that UV cytotoxicity depends mainly on the generation of HO', that can be “scavenged” by C₉2Na or MAN, but not by SOD or CAT. C₉2Na and MAN were not significantly degraded in the period during which they afford protection against HO^..     

Synthesis and evaluation of biological activities of vibsanin A analogs

Vibsanin A is an 11-membered vibsane diterpenoid and is reported to induce myeloid cell differentiation via activation of protein kinase C (PKC) without tumor-promoting activity. Therefore, vibsanin A is thought to be an attractive compound for acute myeloid leukemia (AML) therapy. In this study, we synthesized vibsanin A analogs and compared the activity of these compounds for PKC activation and myeloid cell differentiation. We found that the hydroxymethyl group in vibsanin A is an important substituent to induce differentiation of AML cells. Collectively, our results showed the biochemical features of vibsanin A and provided new insights into the development of new antileukemic drugs.     

Cell-dependent regulation of vasculogenic mimicry by carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1)

Vasculogenic mimicry (VM) promotes tumor migration, metastasis, and invasion in various types of cancer, but the relationship between VM and these phenotypes remains undefined. In this study, we examined carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) as a novel target of VM. We found that ectopic expression of CEACAM1 in HT1080 human fibrosarcoma cells suppressed the formation of a VM-like network. Further, cell migration and proliferation were abated by the introduction of CEACAM1 into HT1080 cells. Conversely, knockout (KO) of the CEACAM1 gene in SK-MEL-28 melanoma cells, which normally express high levels of CEACAM1, inhibited formation of a VM-like network, which was covered on reintroduction of CEACAM1. These results suggest that CEACAM1 differentially regulates formation of the VM-like network between cancer cell types and implicate CEACAM1 as a novel therapeutic target in malignant cancer.