Identification of the principal neutralizing determinant of human immunodeficiency virus type 1 as a fusion domain.

The V3 loop, located near the middle of the surface envelope glycoprotein gp120, is the major neutralizing domain of human immunodeficiency virus type 1 (HIV-1). Although the majority of the V3 loop is highly variable between different strains of HIV-1, a Gly-Pro-Gly-Arg motif at the tip of the loop is highly conserved. To determine whether this region plays a role in fusion mediated by the HIV-1 envelope glycoproteins, we introduced seven single-amino-acid changes in the V3 loop. The mutant envelope glycoproteins were expressed from an HIV-1 envelope expression vector and analyzed for their ability to induce cell fusion in the absence of virus replication. Our results indicated that single-amino-acid changes in the V3 loop were capable of completely abolishing or greatly reducing the ability of the HIV-1 envelope glycoproteins to induce cell fusion, thereby identifying the V3 loop as a fusion domain of HIV-1. Mutations in the highly conserved tip of the loop or in a nonconserved region flanking the highly conserved tip had no effect on envelope glycoprotein synthesis, processing, transport, or binding to the CD4 receptor molecule. Mutation of the putative disulfide bridge-forming Cys at residue 336 blocked gp160 cleavage and CD4 binding.     

Brain grafts and Parkinson's disease

In animal models, grafts derived from several different tissues, principally fetal substantia nigra and adrenal medulla from young adults, have been found to be effective in alleviating some of the manifestations of lesions of the substantia nigra. It has been suggested that these grafts function by diffusely secreting dopamine, by exerting trophic effects on the host brain, or by producing a new innervation of the host corpus striatum. Evidence for each of these modes of action is briefly reviewed. Several brain tissue transplantation techniques have been described. Each of these techniques has significant limitations in animal models. The significance of these limitations for human application is described, and possibilities for improving the efficacy of brain tissue transplantation in animal models and for human application are discussed.     

Bipartite signal sequence mediates nuclear translocation of the plant potyviral NIa protein.

The NIa protein of certain plant potyviruses localizes to the nucleus of infected cells. Previous studies have shown that linkage of NIa to reporter protein beta-glucuronidase (GUS) is sufficient to direct GUS to the nucleus in transfected protoplasts and in cells of transgenic plants. In this study, we mapped sequences in NIa that confer karyophilic properties. A quantitative transport assay using transfected protoplasts, as well as in situ localization technique using epidermal cells from transgenic plants, were employed. Two domains within NIa, one between amino acid residues 1 to 11 (signal domain I) and the other between residues 43 to 72 (signal domain II), were found to function additively for efficient localization of fusion proteins to the nucleus, although either region independently could facilitate a low level of translocation. Like signals from animal cells, both nuclear transport domains of NIa contain a high concentration of basic (arginine and lysine) residues. Nuclear transport signal domain II overlaps or is very near Tyr62, which is the residue that mediates covalent attachment of a subset of NIa molecules to the 5' terminus of viral RNA within infected cells. The nature of the NIa nuclear transport signal and the possibility for regulation of NIa translocation are discussed.     

Measurement of Serotonin Turnover Rate in Rat Dorsal Raphe Nucleus by In Vivo Electrochemistry

5-Hydroxytryptamine (5-HT; serotonin) turnover rate in dorsal raphe nucleus of the urethane-anesthetized rat was estimated by using the in vivo electrochemical detector to measure the decay of extraneuronal 5-hydroxyindole acetic acid (5-HIAA) after monoamine oxidase inhibition. Carbon paste electrodes were scanned by semiderivative voltammetry and revealed two peaks: one at +0.15 V and the other at +0.25 V. The higher potential peak is composed primarily of the 5-HT metabolite 5-HIAA. After administration of pargyline, 75 mg/kg i.p., this peak declined exponentially. Regression analysis of these data by an exponential decay model yielded the fractional rate constant 0.82 +/- 0.06 h-1 (mean +/- SEM). This rate constant of 5-HIAA disappearance measured by in vivo electrochemistry is identical to the rate constant found by others measuring 5-HIAA disappearance by direct tissue assay methods. In animals not treated with pargyline, tissue 5-HIAA concentrations in the dorsal raphe nucleus were measured by HPLC with electrochemical detection. The average 5-HT turnover rate calculated as the product of the fractional rate constant and steady-state tissue 5-HIAA concentration was 12.6 nmol/g/h. These results demonstrate that electrochemical detection of extraneuronal 5-HIAA combined with monoamine oxidase inhibition can be used to measure neurotransmitter turnover in vivo in a discrete brain region.     

Regional mapping of unique DNA sequences from human chromosome 3 derived from a flow-sorted chromosome library

Eight single-copy DNA probes specific for human chromosome 3 were isolated by screening a human chromosome 3-derived genomic library. Southern blot analyses of DNAs isolated from a panel of somatic cell hybrids allowed us to regionally assign all probes to subregions on chromosome 3. Three clones were localized to the short arm of chromosome 3 (3p21----pter), two to the long arm (3q21----qter), and three to the 3q21----3p21 subregion. Six of these DNA sequences map to regions overlapping a segment of chromosome 3 (3p14----p23) frequently deleted in small cell lung cancer cells. Restriction fragment length polymorphism analyses indicate that at least three of the eight single-copy probes studies show MspI or BglII polymorphisms. This library is a useful source of chromosome 3-specific probes.     

The asymmetric function of Dph1–Dph2 heterodimer in diphthamide biosynthesis

JBIC Journal of Biological Inorganic Chemistry - Diphthamide, the target of diphtheria toxin, is a post-translationally modified histidine residue found in archaeal and eukaryotic translation...     

Strategies of Bringing Drug Product Marketing Applications to Meet Current Regulatory Standards

In the past decade, many guidance documents have been issued through collaboration of global organizations and regulatory authorities. Most of these are applicable to new products, but there is a risk that currently marketed products will not meet the new compliance standards during audits and inspections while companies continue to make changes through the product life cycle for continuous improvement or market demands. This discussion presents different strategies to bringing drug product marketing applications to meet current and emerging standards. It also discusses stability and method designs to meet process validation and global development efforts.At the 2014 American Association of Pharmaceutical Scientists (AAPS) annual meeting in San Diego, CA, Yan Wu (Merck) and Anita Freed (Pfizer) led a symposium entitled “Bringing Drug Product Marketing Applications to Current Regulatory Standards: Trials and Tribulations.” This symposium was very timely as this topic is a growing industry concern, evidenced by over 300 attendees, and considering the new guidances (1–8) that have been established over the past decade. While most of these quality standards are applicable to new drug products, there is a risk that currently marketed products, known as legacy products, will not meet the new compliance standards during audits and inspections. Companies also need to continuously make process or method changes for in-line products as part of product life cycle management efforts or to meet different market needs. If legacy (or in-line) products undergo a change, the question is how much extra effort is needed to have these products meet current standards to support the associated submission. This symposium addressed these issues and offered modeling tools using existing data or other approaches and case studies to effectively manage post-approval changes. Presentations included the following:

• Modeling historical data to support process and method stability changes
• Food and Drug Administration (FDA) perspectives on application of International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) Q8 to legacy products
• Assessment of impact on stability with manufacturing, packaging, and/or method changes
• Applying Association of Southeast Asian Nations (ASEAN) stability requirements to legacy products and managing specifications across climatic zones

This paper provides an overview of the presentations and highlights strategies and points of consideration when bringing marketing applications of the legacy drug products to current and emerging standards.     

Modeling large regions in proteins: applications to loops, termini, and folding

Template-based methods for predicting protein structure provide models for a significant portion of the protein but often contain insertions or chain ends (InsEnds) of indeterminate conformation. The local structure prediction "problem" entails modeling the InsEnds onto the rest of the protein. A well-known limit involves predicting loops of ≤12 residues in crystal structures. However, InsEnds may contain as many as ~50 amino acids, and the template-based model of the protein itself may be imperfect. To address these challenges, we present a free modeling method for predicting the local structure of loops and large InsEnds in both crystal structures and template-based models. The approach uses single amino acid torsional angle "pivot" moves of the protein backbone with a C(β) level representation. Nevertheless, our accuracy for loops is comparable to existing methods. We also apply a more stringent test, the blind structure prediction and refinement categories of the CASP9 tournament, where we improve the quality of several homology based models by modeling InsEnds as long as 45 amino acids, sizes generally inaccessible to existing loop prediction methods. Our approach ranks as one of the best in the CASP9 refinement category that involves improving template-based models so that they can function as molecular replacement models to solve the phase problem for crystallographic structure determination.     

Plasma polyunsaturated fatty acids and regional cerebral glucose metabolism in major depression

Deficiencies in polyunsaturated essential fatty acids (PUFA) are implicated in mood disorders, although mechanisms of action and regional specificity in the brain are unknown. We hypothesized that plasma phospholipid PUFA levels are correlated with regionally specific relative cerebral metabolic rates of glucose (rCMRglu). Medication-free depressed subjects (N=29) were studied using [¹⁸F]-fluoro-2-deoxyglucose positron emission tomography. Docosahexaenoic acid (22:6n-3), arachidonic acid (20:4n-6), and eicosapentaenoic acid (20:5n-3) were assessed as a percentage of total phospholipid PUFA (DHA%, AA%, and EPA%, respectively). DHA% and AA% correlated positively with rCMRglu in temporoparietal cortex. In addition, DHA% correlated negatively with rCMRglu in prefrontal cortex and anterior cingulate. No correlations were seen with EPA%. Thus, under conditions of low plasma DHA, rCMRglu was higher in temporoparietal cortex and lower in anterior cingulate/prefrontal cortex. Opposing effects of DHA on these regions is a hypothesis that could be addressed in future prospective studies with n-3 supplementation. This pilot study is the first to demonstrate fatty acid and regionally specific correlations in the brain between plasma PUFA and rCMRglu in humans.