Comprehensive Antigenic Map of a Cleaved Soluble HIV-1 Envelope Trimer

The trimeric envelope (Env) spike is the focus of vaccine design efforts aimed at generating broadly neutralizing antibodies (bNAbs) to protect against HIV-1 infection. Three recent developments have facilitated a thorough investigation of the antigenic structure of the Env trimer: 1) the isolation of many bNAbs against multiple different epitopes; 2) the generation of a soluble trimer mimic, BG505 SOSIP.664 gp140, that expresses most bNAb epitopes; 3) facile binding assays involving the oriented immobilization of tagged trimers. Using these tools, we generated an antigenic map of the trimer by antibody cross-competition. Our analysis delineates three well-defined epitope clusters (CD4 binding site, quaternary V1V2 and Asn332-centered oligomannose patch) and new epitopes at the gp120-gp41 interface. It also identifies the relationships among these clusters. In addition to epitope overlap, we defined three more ways in which antibodies can cross-compete: steric competition from binding to proximal but non-overlapping epitopes (e.g., PGT151 inhibition of 8ANC195 binding); allosteric inhibition (e.g., PGT145 inhibition of 1NC9, 8ANC195, PGT151 and CD4 binding); and competition by reorientation of glycans (e.g., PGT135 inhibition of CD4bs bNAbs, and CD4bs bNAb inhibition of 8ANC195). We further demonstrate that bNAb binding can be complex, often affecting several other areas of the trimer surface beyond the epitope. This extensive analysis of the antigenic structure and the epitope interrelationships of the Env trimer should aid in design of both bNAb-based therapies and vaccines intended to induce bNAbs.     

Identification of a novel substitution in the constant region of a gene coding for an amyloidogenic kappa1 light chain.

Current concepts regarding the association between immunoglobulin (Ig) light chain structure and AL amyloidosis (AL) emphasize Ig variable region amino acid substitutions because the majority of light chain amyloid fibrils that have been sequenced contain amino termini of the variable region with only small amounts of the constant region. In this report, we describe a patient with rapidly progressive AL whose amyloid deposits contained primarily monoclonal kappa light chain constant region fragments. We sequenced and analyzed this AL protein, determining that it was an O18-O8 kappa1 variant and that the constant region possessed an unusual Ser-->Asn substitution at position 177. Using pre-mortem bone marrow cells, we cloned and sequenced the cDNA for this AL protein (HCAK1) and, using DNA from post-mortem somatic tissue, we cloned and sequenced the patient's kappa germline O18-O8 donor and kappa constant region (Ckappa) gene segments. The cDNA that coded for HCAK1 contained a variable region that was derived from O18-O8, showing 96.1% homology to germline, and a Ckappa that had a nucleotide substitution (AGC to AAC), resulting in the 177Ser-->Asn replacement. Two Ckappa genes were cloned from somatic tissue DNA, one identical to a known Ckappa sequence and another containing this substitution which likely is a new Ckappa allotype. Our findings indicate that further investigation is warranted into the contributions genetic polymorphisms and light chain constant regions may make to amyloidogenesis.     

Bcl-2 protects against FCCP-induced apoptosis and mitochondrial membrane potential depolarization in PC12 cells.

This report addresses the relation between Bcl-2 and mitochondrial membrane potential (DeltaPsi(m)) in apoptotic cell death. Rat pheochromocytoma (PC12) cells are differentiated into neuron-like cells with nerve growth factor (NGF). It is known that Bcl-2 can attenuate apoptosis induced by deprivation of neurotrophic factor. The protective effect of Bcl-2 has been correlated with preservation of DeltaPsi(m). Protonophores, such as carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), collapse the proton gradient across the mitochondrial inner membrane, resulting in a complete abolition of the mitochondrial membrane potential. Based on the analysis of morphology, of phosphatidylserine exposure and of nuclear fragmentation we conclude that FCCP induces apoptosis in PC12 cells, which can be prevented by overexpression of Bcl-2. To determine whether the cytoprotective effect of Bcl-2 is due to stabilization of DeltaPsi(m), we investigated the effect of Bcl-2 on changes in DeltaPsi(m), induced by FCCP in PC12 cells. We showed that treatment with FCCP induced a reduction in DeltaPsi(m), as assessed with the lipophilic cationic membrane potential-sensitive dye JC-1, and that Bcl-2 protects against FCCP-induced changes in NGF differentiated PC12 cells. Our data indicate that Bcl-2 protects against FCCP-induced cell death by stabilizing DeltaPsi(m).     

Complexes of disubstituted benzene positional isomers with α-cyclodextrin

Stability constants for complex formation between α-cyclodextrin and some ortho-, meta-, and para-disubstituted benzenes were measured in aqueous solution at 25°C by potentiometry, spectrophotometry, competitive spectrophotometry, and solubility. All systems form 1:1 complexes, some para substrates form 1:2 complexes (one substrate to two cyclodextrins), but no meta substrates form 1:2 complexes. Ortho substrates form weak complexes. These observations are accounted for in terms of a binding site molecular model. On the average over many systems, K₁₁ (para) and K₁₁ (meta) are approximately equal. Major discrepancies (greater than a factor of two) are diagnostic of significantly different electronic or steric effects in the complexing abilities of the isomeric substrates.     

High mortality,fluctuation in numbers,and heavy subterranean insect herbivory in bush lupine,Lupinus arboreus

Sporadic patchy die-off of bush lupine, Lupinus arboreus, has long been known. We describe in detail a series of these incidents on the central California coast, based upon observational and comparative evidence. Stands of thousands of plants die, while nearby mature plants live on. In some sites, repeated die-off followed by regeneration from the seed bank has led to the cover and density of this woody, perennial plant fluctuating widely over the 40 year period for which records exist. Root damage by caterpillars of the ghost moth or swift Hepialus californicus (Lepidoptera, Hepialidae) is a major cause of individual bush death and a probable cause of die-off of stands of lupine. Hidden from view underground, a few of these insects readily kill a juvenile or young mature plant by girdling and reaming-out roots. The mass mortality of L. arboreus that we observed involved heavy root damage by these caterpillars in evenaged stands of plants in their first (1.5-year-old) or second (2.5-year-old) flowering season. The injured plants set seed before dying. Older, larger bush lupines better withstood root damage. In plants aged 3 or more years, damage and mortality were correlated with the intensity of ghost moth caterpillars in the roots. At the highest intensity (mean = 37.5, maximum = 62 caterpillars/root), a stand of large, old L. arboreus suffered 41% mortality; 45% of root cambium (median value) was destroyed by feeding caterpillars. Mass death of mature L. arboreus was not correlated with folivory, and leaf damage ranged from nil to moderate in instances of die-off. The western tussock moth, Orgyia vetusta, accounted for the highest levels of folivory, but this insect was rare when die-offs occurred. The lowest lupine mortality rates in our study occurred where tussock caterpillar intensities were high and where plants were repeatedly defoliated by this insect. However, experimental defoliation by high, but realistic, intensities of tussock moth caterpillars resulted in some mortality of mature bushes, and the combined effects of leaf and root herbivory have yet to be assessed. In its natural range on the California coast, bush lupine has several additional species of insect herbivores that can be locally abundant and injurious to the plant, although none is associated with die-off. Subterranean natural enemies of ghost moth caterpillars may play a role in the patchy waxing and waning of this shrub. Locally, a new species of entomophagous nematode (Heterorhabditis sp.) cause high mortality in the soil, before ghost moth caterpillars have entered the root. This natural enemy may thus afford lupines protection from heavy underground herbivory.     

Microbial Decomposition of Synthetic 14C-Labeled Lignins in Nature: Lignin Biodegradation in a Variety of Natural Materials

Lignin biodegradation in a variety of natural materials was examined using specifically labeled synthetic ¹⁴C-lignins. Natural materials included soils, sediments, silage, steer bedding, and rumen contents. Both aerobic and anaerobic incubations were used. No ¹⁴C-labeled lignin biodegradation to labeled gaseous products under anaerobic conditions was observed. Aerobic ¹⁴C-labeled lignin mineralization varied with respect to type of natural material used, site, soil type and horizon, and temperature. The greatest observed degradation occurred in a soil from Yellowstone National Park and amounted to over 42% conversion of total radioactivity to ¹⁴CO₂ during 78 days of incubation. Amounts of ¹⁴C-labeled lignin mineralization in Wisconsin soils and sediments were significantly correlated with organic carbon, organic nitrogen, nitrate nitrogen, exchangeable calcium, and exchangeable potassium.     

Naphthalene plasmids in pseudomonads.

A rapid method beginning with the direct lysis of bacteria in alkaline sodium dodecyl sulfate was used to detect naphthalene plasmids in pseudomonads. The strains NCIB 9816, PG, ATCC 17483, and ATCC 17484, which can grow on naphthalene as the sole source of carbon and energy, were examined. All except ATCC 17483 contained more than one plasmid. ATCC 17483 did not contain any plasmids. The largest pair of plasmids found in each of NCIB 9816 and PG(NAH2 and NAH3, respectively) determined naphthalene metabolism and could be transferred by conjugation. This also transferred the unusually regulated meta pathway enzymes for catechol metabolism. NAH2 determines the constitutive production of low concentrations of catechol 2,3-dioxygenase and 2-hydroxymuconic acid semialdehyde dehydrogenase, and NAH3 determines the constitutive production of high concentration of these. NAH2 and NAH3 gave identical fragments on digestion with BamHI or HindIII, but these were quite different from those of NAH. Nonetheless, NAH2 and NAH3 hybridized with NAH.     

Aspergillus terreus and its toxic metabolites as a food contaminant in some Egyptian Bakery products and grains

A. terreus isolates isolated from some bakery products, corn and rice were found to be able to produce territrems. 90% of theA. terreus isolated from bakery products were able to produce territrem A, with a mean of 0.09 ppm, while 80% ofA. terreus isolates produce territrem B with a mean of 0.24 ppm. On the other hand 31.8% of the isolates ofA. terreus from corn were able to produce territrem A with a mean of 0.44 ppm. ConcerningA. terreus isolates from rice, 66.7% were found to produce territrem A, with a mean of 5.28 ppm, and 77.8% of the isolates produced territrem B with a mean of 1.79 ppm.