Association of a murine 53,000-dalton phosphoprotein with simian virus 40 large-T antigen in transformed cells.

Serum raised against a mouse 53,000-dalton (53K) phosphoprotein precipitates both the 53K immunogen and simian virus 40 large-T from lysates of simian virus 40-transformed 3T3 cells. This serum, designated F5, does not recognize antigenic determinants on native or denatured large-T and precipitates large-T because the 53K phosphoprotein forms a stable complex with large-T. This complex sediments at 23S on sucrose density gradients, corresponding to a molecular weight of 600K to 1,000K, and appears to contain only 53K and large-T as major components. It is held together by noncovalent bonds and is located in the cell nucleus. All the 53K immunoprecipitated from cell lysates by F5 is present in the high-molecular-weight complex, but large-T can be separated into a complexed and a free form on sucrose density gradients. The complexed form of large-T is more readily phosphorylated than the free form. We have been unable to detect an association of large-T with comparable host cell proteins during productive infections with simian virus 40.     

In-depth qualitative and quantitative analysis of composite glycosylation profiles and other micro-heterogeneity on intact monoclonal antibodies by high-resolution native mass spectrometry using a modified Orbitrap

Here, we describe a fast, easy-to-use, and sensitive method to profile in-depth structural micro-heterogeneity, including intricate N-glycosylation profiles, of monoclonal antibodies at the native intact protein level by means of mass spectrometry using a recently introduced modified Orbitrap Exactive Plus mass spectrometer. We demonstrate the versatility of our method to probe structural micro-heterogeneity by describing the analysis of three types of molecules: (1) a non-covalently bound IgG4 hinge deleted full-antibody in equilibrium with its half-antibody, (2) IgG4 mutants exhibiting highly complex glycosylation profiles, and (3) antibody-drug conjugates. Using the modified instrument, we obtain baseline separation and accurate mass determination of all different proteoforms that may be induced, for example, by glycosylation, drug loading and partial peptide backbone-truncation. We show that our method can handle highly complex glycosylation profiles, identifying more than 20 different glycoforms per monoclonal antibody preparation and more than 30 proteoforms on a single highly purified antibody. In analyzing antibody-drug conjugates, our method also easily identifies and quantifies more than 15 structurally different proteoforms that may result from the collective differences in drug loading and glycosylation. The method presented here will aid in the comprehensive analytical and functional characterization of protein micro-heterogeneity, which is crucial for successful development and manufacturing of therapeutic antibodies     

UNCLOS III and conflict management in straits

Abstract

The legal regime articulated for international straits in the United Nations Convention of 1982 is a significant milestone in the reconciliation of competing interests attendant upon navigation in international straits. However, the author also submits that the refinement of respective rights and obligations of states in international straits can only be harmonized through the process of claim and counterclaim, a slow and somewhat tedious process. Extremely difficult, yet highly important questions deserve thoughtful analysis in the context of the 1982 Convention's territorial sea and straits provisions.     

Overexpression of Bacillus subtilis phosphoribosylpyrophosphate synthetase and crystallization and preliminary X-ray characterization of the free enzyme and its substrate–effector complexes

Bacillus subtilis phosphoribosylpyrophosphate (PRPP) synthetase has been expressed to high levels in an Escherichia coli host strain devoid of endogenous PRPP synthetase. A rapid and efficient purification protocol has been developed allowing production of enzyme preparations with purity conforming to the stringent criteria required for crystallization. Crystallization experiments, in combination with dynamic light scattering studies, have led to the production of three crystal forms of the enzyme. These forms include the free enzyme, the enzyme in a binary complex with the substrate adenosine triphosphate (ATP), and the enzyme in a quaternary complex with the substrate analog α, β-methylene adenosine triphosphate (mATP), the substrate ribose-5-phosphate (Rib-5-P), and the allosteric inhibitor adenosine diphosphate (ADP). Diffraction data showed that all three crystal forms are suitable for structure determination. They crystallize in the same hexagonal space group, P6₃, with virtually identical unit cell dimensions of a = b = 115.6 Å c = 107.8 Å, and with two molecules in the asymmetric unit. The self-rotation function showed the existence of a non-crystallographic twofold axis perpendicular to the c axis. The availability of the different complexes should allow questions regarding the molecular mechanisms of catalysis and allostery in PRPP synthetase to be addressed.     

Methods for generating high-resolution structural models from electron microscope tomography data

Reconstructed volumes generated by tilt-image electron-microscope tomography offer the best spatial resolution currently available for studying cell structures in situ. Analysis is often accomplished by creating surface models that delineate grayscale contrast boundaries. Here, we introduce a specialized and convenient sequence of segmentation operations for making such models that greatly improves their reliability and spatial resolution as compared to current approaches, providing a basis for making accurate measurements. To assess the reliability of the surface models, we introduce a spatial uncertainty measurement based on grayscale gradient scale length. The model generation and measurement methods are validated by applying them to synthetic data, and their utility is demonstrated by using them to characterize macromolecular architecture of active zone material at the frog's neuromuscular junction.     

Enhanced detection of human immunodeficiency virus type 1-specific T-cell responses to highly variable regions by using peptides based on autologous virus sequences

The antigenic diversity of human immunodeficiency virus type 1 (HIV-1) represents a significant challenge for vaccine design as well as the comprehensive assessment of HIV-1-specific immune responses in infected persons. In this study we assessed the impact of antigen variability on the characterization of HIV-1-specific T-cell responses by using an HIV-1 database to determine the sequence variability at each position in all expressed HIV-1 proteins and a comprehensive data set of CD8 T-cell responses to a reference strain of HIV-1 in infected persons. Gamma interferon Elispot analysis of HIV-1 clade B-specific T-cell responses to 504 overlapping peptides spanning the entire expressed HIV-1 genome derived from 57 infected subjects demonstrated that the average amino acid variability within a peptide (entropy) was inversely correlated to the measured frequency at which the peptide was recognized (P = 6 x 10(-7)). Subsequent studies in six persons to assess T-cell responses against p24 Gag, Tat, and Vpr peptides based on autologous virus sequences demonstrated that 29% (12 of 42) of targeted peptides were only detected with peptides representing the autologous virus strain compared to the HIV-1 clade B consensus sequence. The use of autologous peptides also allowed the detection of significantly stronger HIV-1-specific T-cell responses in the more variable regulatory and accessory HIV-1 proteins Tat and Vpr (P = 0.007). Taken together, these data indicate that accurate assessment of T-cell responses directed against the more variable regulatory and accessory HIV-1 proteins requires reagents based on autologous virus sequences. They also demonstrate that CD8 T-cell responses to the variable HIV-1 proteins are more common than previously reported.     

The diageotropica mutation alters auxin induction of a subset of the Aux/IAA gene family in tomato

The diageotropica (dgt) mutation has been proposed to affect either auxin perception or responsiveness in tomato plants. It has previously been demonstrated that the expression of one member of the Aux/IAA family of auxin-regulated genes is reduced in dgt plants. Here, we report the cloning of ten new members of the tomato Aux/IAA family by PCR amplification based on conserved protein domains. All of the gene family members except one (LeIAA7) are expressed in etiolated tomato seedlings, although they demonstrate tissue specificity (e.g. increased expression in hypocotyls vs. roots) within the seedling. The wild-type auxin-response characteristics of the expression of these tomato LeIAA genes are similar to those previously described for Aux/IAA family members in Arabidopsis. In dgt seedlings, auxin stimulation of gene expression was reduced in only a subset of LeIAA genes (LeIAA5, 8, 10, and 11), with the greatest reduction associated with those genes with the strongest wild-type response to auxin. The remaining LeIAA genes tested exhibited essentially the same induction levels in response to the hormone in both dgt and wild-type hypocotyls. These results confirm that dgt plants can perceive auxin and suggest that a specific step in early auxin signal transduction is disrupted by the dgt mutation.     

Biochemical isolation and purification of ovulation-inducing factor (OIF) in seminal plasma of llamas

Background  

The objective of the present study was to isolate and purify the protein fraction(s) of llama seminal plasma responsible for the ovulation-inducing effect of the ejaculate.