Cross-reactive human immunodeficiency virus type 1-neutralizing human monoclonal antibody that recognizes a novel conformational epitope on gp41 and lacks reactivity against self-antigens

Broadly cross-reactive human immunodeficiency virus (HIV)-neutralizing antibodies are infrequently elicited in infected humans. The two best-characterized gp41-specific cross-reactive neutralizing human monoclonal antibodies, 4E10 and 2F5, target linear epitopes in the membrane-proximal external region (MPER) and bind to cardiolipin and several other autoantigens. It has been hypothesized that, because of such reactivity to self-antigens, elicitation of 2F5 and 4E10 and similar antibodies by vaccine immunogens based on the MPER could be affected by tolerance mechanisms. Here, we report the identification and characterization of a novel anti-gp41 monoclonal antibody, designated m44, which neutralized most of the 22 HIV type 1 (HIV-1) primary isolates from different clades tested in assays based on infection of peripheral blood mononuclear cells by replication-competent virus but did not bind to cardiolipin and phosphatidylserine in an enzyme-linked immunosorbent assay and a Biacore assay nor to any protein or DNA autoantigens tested in Luminex assays. m44 bound to membrane-associated HIV-1 envelope glycoproteins (Envs), to recombinant Envs lacking the transmembrane domain and cytoplasmic tail (gp140s), and to gp41 structures containing five-helix bundles and six-helix bundles, but not to N-heptad repeat trimers, suggesting that the C-heptad repeat is involved in m44 binding. In contrast to 2F5, 4E10, and Z13, m44 did not bind to any significant degree to denatured gp140 and linear peptides derived from gp41, suggesting a conformational nature of the epitope. This is the first report of a gp41-specific cross-reactive HIV-1-neutralizing human antibody that does not have detectable reactivity to autoantigens. Its novel conserved conformational epitope on gp41 could be helpful in the design of vaccine immunogens and as a target for therapeutics.     

Oxidation of diiron and triiron sulfurdithiolato complexes: mimics for the active site of [FeFe]-hydrogenase

The oxidation of the hexacarbonyl(1,3-dithiolato-S,S')diiron complexes 4a-4c with varying amounts of dimethyldioxirane (DMD) was systematically studied. The chemoselectivity of the oxidation products depended upon the substituent R (R=H, Me, 1/2 (CH2)(5)). For R=H, four oxidation products, 6a-6d, have been obtained. In the case of R=Me, three products, 7a-7c, were formed, and for R=1/2 (CH2)(5), only complex 8 was observed. These observations are due to steric and electronic effects caused by the substituent R. Additionally, oxidation of the triiron complex 5 with DMD was performed to yield the products 9a and 9b. X-Ray diffraction analyses were performed for 6a-6d, 7a, and 7c, as well as for 9a and 9b. The electronic properties were determined by density-functional theory (DFT) calculations.     

Functional role of CAAT box element of the nopaline synthase (nos) promoter

The nopaline synthase (nos) promoter is active in a wide range of plant tissues and regulated by various environmental stimuli. It was previously found that the CAAT box region is important for nos promoter activity. In the present study, the location of the CAAT box element was determined by site specific mutation analysis. Point mutations within the conserved CAAT box element significantly reduced the promoter response in transgenic tobacco plants and calli to wounding, H₂O₂, methyl jasmonate, and 2,4-D, but not to salicylic acid. However, mutations immediately upstream from the CAAT box did not affect these responses. These results suggest that the CAAT box element is important in responding to certain stimuli.     

A UNIQUELY CALCIFIED BROWN ALGA FROM HAWAII: NEWHOUSIA IMBRICATA GEN. ET SP. NOV. (DICTYOTALES,PHAEOPHYCEAE)1

An encrusting brown alga from subtidal habitats around the island of Oahu (Hawaiian Islands) represents only the second genus of the class Phaeophyceae to form calcium carbonate, which it deposits primarily as both extracellular and intracellular aragonite, admixed with small (3.3%) amounts of calcite. Plants form expanses 15–100+ cm in extent consisting of horizontally aligned imbricating tiers of distromatic blades 1–4 mm in diameter that are separated from one another by cementing layers of extracellular aragonite, the tiers forming stacks of dozens of laminae and anchored to coral substrata by a basement layer that adheres tightly without haptera or rhizoids. The hypodermal layer of each blade consists of lightly pigmented rectilinear cells bearing either one or two smaller deeply pigmented epidermal cells in cross‐sectional profiles and three or four in long‐sectional profiles, the cells of both layers becoming encased in rigid carbonate skeletons laid down in their outer wall matrices. The successive tiers become stacked by either overgrowing marginal proliferations or new blade primordia that arise from the hypodermal layer of surface laminae and initially spread centrifugally by means of continuous marginal meristems. Neither plurilocular nor unilocular reproductive structures are known. The alga is described as the new genus and species Newhousia imbricata Kraft, G.W. Saunders, Abbott et Haroun and is assigned on the basis of small subunit rDNA gene sequence analyses to the order Dictyotales, family Dictyotaceae, within a strongly supported monophyletic clade that includes Distromium, Lobophora, and Zonaria.     

Hidden keys to survival: the type,density, pattern and functional role of emperor penguin body feathers

Antarctic penguins survive some of the harshest conditions on the planet. Emperor penguins breed on the sea ice where temperatures drop below −40°C and forage in −1.8°C waters. Their ability to maintain 38°C body temperature in these conditions is due in large part to their feathered coat. Penguins have been reported to have the highest contour feather density of any bird, and both filoplumes and plumules (downy feathers) are reported absent in penguins. In studies modelling the heat transfer properties and the potential biomimetic applications of penguin plumage design, the insulative properties of penguin plumage have been attributed to the single afterfeather attached to contour feathers. This attribution of the afterfeather as the sole insulation component has been repeated in subsequent studies. Our results demonstrate the presence of both plumules and filoplumes in the penguin body plumage. The downy plumules are four times denser than afterfeathers and play a key, previously overlooked role in penguin survival. Our study also does not support the report that emperor penguins have the highest contour feather density.     

Cloning, expression and characterization of the murine orthologue of SBF2, the gene mutated in Charcot-Marie-Tooth disease type 4B2

Autosomal recessive hereditary motor and sensory neuropathy (HMSN) or Charcot-Marie-Tooth disease (CMT) is a clinically and genetically heterogeneous disorder of the peripheral nervous system. The clinical picture includes progressive distal weakness and atrophy, foot deformities, and distal sensory loss. For autosomal recessive CMT type 4B2 one locus was mapped to chromosome 11p15. Recently, mutations in SET binding factor 2 (SBF2), were identified as cause of CMT4B2. SBF2 is a member of the pseudo-phosphatase branch of myotubularins and all disease-associated mutations known to date lead to shortened or truncated proteins, also implicating loss-of-function. Here, we describe the molecular cloning and the expression pattern of Sbf2. The mRNA spans around 8 kb, and the protein shares high amino acid identity compared to the human protein suggesting a conserved function. Sbf2 is encoded by 40 exons on murine chromosome 7. In situ hybridization, Northern blots and RT-analysis revealed a very broad pattern of Sbf2 expression. Overexpressed epitope tagged Sbf2 showed cytoplasmic distribution. Taken together, this study provides information about the mRNA expression and subcellular localization of Sbf2 and as such helps in further understanding its function in development and disease.     

Charge-charge interactions are key determinants of the pK values of ionizable groups in ribonuclease Sa (pI=3.5) and a basic variant (pI=10.2)

The pK values of the titratable groups in ribonuclease Sa (RNase Sa) (pI=3.5), and a charge-reversed variant with five carboxyl to lysine substitutions, 5K RNase Sa (pI=10.2), have been determined by NMR at 20 degrees C in 0.1M NaCl. In RNase Sa, 18 pK values and in 5K, 11 pK values were measured. The carboxyl group of Asp33, which is buried and forms three intramolecular hydrogen bonds in RNase Sa, has the lowest pK (2.4), whereas Asp79, which is also buried but does not form hydrogen bonds, has the most elevated pK (7.4). These results highlight the importance of desolvation and charge-dipole interactions in perturbing pK values of buried groups. Alkaline titration revealed that the terminal amine of RNase Sa and all eight tyrosine residues have significantly increased pK values relative to model compounds.A primary objective in this study was to investigate the influence of charge-charge interactions on the pK values by comparing results from RNase Sa with those from the 5K variant. The solution structures of the two proteins are very similar as revealed by NMR and other spectroscopic data, with only small changes at the N terminus and in the alpha-helix. Consequently, the ionizable groups will have similar environments in the two variants and desolvation and charge-dipole interactions will have comparable effects on the pK values of both. Their pK differences, therefore, are expected to be chiefly due to the different charge-charge interactions. As anticipated from its higher net charge, all measured pK values in 5K RNase are lowered relative to wild-type RNase Sa, with the largest decrease being 2.2 pH units for Glu14. The pK differences (pK(Sa)-pK(5K)) calculated using a simple model based on Coulomb's Law and a dielectric constant of 45 agree well with the experimental values. This demonstrates that the pK differences between wild-type and 5K RNase Sa are mainly due to changes in the electrostatic interactions between the ionizable groups. pK values calculated using Coulomb's Law also showed a good correlation (R=0.83) with experimental values. The more complex model based on a finite-difference solution to the Poisson-Boltzmann equation, which considers desolvation and charge-dipole interactions in addition to charge-charge interactions, was also used to calculate pK values. Surprisingly, these values are more poorly correlated (R=0.65) with the values from experiment. Taken together, the results are evidence that charge-charge interactions are the chief perturbant of the pK values of ionizable groups on the protein surface, which is where the majority of the ionizable groups are positioned in proteins.