Domain flexibility in retroviral proteases: structural implications for drug resistant mutations

Rigid body rotation of five domains and movements within their interfacial joints provide a rational context for understanding why HIV protease mutations that arise in drug resistant strains are often spatially removed from the drug or substrate binding sites. Domain motions associated with substrate binding in the retroviral HIV-1 and SIV proteases are identified and characterized. These motions are in addition to closure of the flaps and result from rotations of approximately 6-7 degrees at primarily hydrophobic interfaces. A crystal structure of unliganded SIV protease (incorporating the point mutation Ser 4 His to stabilize the protease against autolysis) was determined to 2.0 A resolution in a new space group, P3221. The structure is in the most "open" conformation of any retroviral protease so far examined, with six residues of the flaps disordered. Comparison of this and unliganded HIV structures, with their respective liganded structures by difference distance matrixes identifies five domains of the protease dimer that move as rigid bodies against one another: one terminal domain encompassing the N- and C-terminal beta sheet of the dimer, two core domains containing the catalytic aspartic acids, and two flap domains. The two core domains rotate toward each other on substrate binding, reshaping the binding pocket. We therefore show that, for enzymes, mutations at interdomain interfaces that favor the unliganded form of the target active site will increase the off-rate of the inhibitor, allowing the substrate greater access for catalysis. This offers a mechanism of resistance to competitive inhibitors, especially when the forward enzymatic reaction rate exceeds the rate of substrate dissociation.     

All that you can be: stereotyping of self and others in a military context

The authors tested the shifting standards model (M. Biernat, M. Manis, & T. E. Nelson, 1991) as it applies to sex- and race-based stereotyping of self and others in the military. U. S. Army officers attending a leadership training course made judgments of their own and their groupmates' leadership competence at 3 time points over a 9-week period. We examined the effects of officer sex and race on both subjective (rating) and objective/common-rule (ranking/Q-sort) evaluations. Stereotyping generally increased with time, and in accordance with the shifting standards model, pro-male judgment bias was more evident in rankings than in ratings, particularly for White targets. Self-judgments were also affected by sex-based shifting standards, particularly in workgroups containing a single ("solo") woman. Differential standard use on the basis of race was less apparent, a finding attributed to the Army's explicit invocation against the use of differential race-based standards.     

Analogues of capsaicin with agonist activity as novel analgesic agents: structure-activity studies. 4. Potent, orally active analgesics

Structural features of three regions of the capsaicin molecule necessary for agonist properties were delineated by a previously reported modular approach. These in vitro agonist effects were shown to correlate with analgesic potency in rodent models. Combination of optimal structural features from each of these regions of the capsaicin molecule have led to highly potent agonists (eg., 1b). Evaluation in vivo established that 1b had analgesic properties but poor oral activity, short duration of action, and excitatory side effects which precluded further development of this compound. Preliminary metabolism studies had shown that the phenol moiety of 1b was rapidly glucuronidated in vivo, providing a possible explanation for the poor pharmacokinetic profile. Subsequent specific modification of the phenol group led to compounds 2a-j, which retained in vitro potency. The in vivo profiles of two representatives of this series, 2a,h, were much improved over the "parent" phenol series, and they are candidates for development as analgesic agents.     

Roald Amundsen--a study of personality

Roald Amundsen is the most famous of the Norwegian polar explorers. His ancestors came from a group of islands south-east of the Oslofjord. From being fishermen and sailors, they progressed to becoming captains and shipowners in the course of two generations. Amundsen's father, Jens, stayed at sea until his ship went down with all the crew. Roald was 14 years of age at the time, the youngest of four competing brothers. Jens had left the close-knit local family community before that, and bought a flat in the capital, Oslo, so that his sons could get a better education. Roald's mother wanted him to study medicine. He did as she wished for a time, but was not at all interested. When his mother died, he abruptly left the university and went to sea, which had been the tradition in his family for decades. As a young boy he was an admirer of Sir John Franklin and his explorers of the Northwest Passage. Fridtjof Nansen became his ideal. The biographies about Roald Amundsen are very diverging--some hold him a hero, others reflect a strongly critical attitude. Here, the author tries to define his personality and places him firmly within the narcissistic domain. His tendency to seek the company of married women, but to take immediate flight when they really became interested reflects an Oedipus complex from before puberty. The tragic death of his father, the sea captain, may have been a supposition; puberty can be seen as a period of coping with ambivalence towards an earlier idealized father. His genius combined ambitious goals with a sharp eye for details as regards the equipment used in his expeditions. In his travels in the Arctic and the Antartic he was driven forward by the energy of the nation. His heroic death, trying to save his earlier "enemy", Nobile, was probably caused by an urge for self-destruction.     

CHL1 encodes a component of the low-affinity nitrate uptake system in Arabidopsis and shows cell type-specific expression in roots

The Arabidopsis CHL1 (AtNRT1) gene confers sensitivity to the herbicide chlorate and encodes a nitrate-regulated nitrate transporter. However, how CHL1 participates in nitrate uptake in plants is not yet clear. In this study, we examined the in vivo function of CHL1 with in vivo uptake measurements and in situ hybridization experiments. Under most conditions tested, the amount of nitrate uptake by a chl1 deletion mutant was found to be significantly less than that of the wild type. This uptake deficiency was reversed when a CHL1 cDNA clone driven by the cauliflower mosaic virus 35S promoter was expressed in transgenic chl1 plants. Furthermore, tissue-specific expression patterns showed that near the root tip, CHL1 mRNA is found primarily in the epidermis, but further from the root tip, the mRNA is found in the cortex or endodermis. These results are consistent with the involvement of CHL1 in nitrate uptake at different stages of root cell development. A functional analysis in Xenopus oocytes indicated that CHL1 is a low-affinity nitrate transporter with a K(m) value of approximately 8.5 mM for nitrate. This finding is consistent with the chlorate resistance phenotype of chl1 mutants. However, these results do not fit the current model of a single, constitutive component for the low-affinity uptake system. To reconcile this discrepancy and the complex uptake behavior observed, we propose a "two-gene" model for the low-affinity nitrate uptake system of Arabidopsis.     

Differential effects of elevated potassium ion concentration and of theophylline on growth hormone release by rat adenohypophyses in vitro

The effects of 27 mM K+ and of 6.7 mM theophylline on the release of growth hormone (GH) by rat hemipituitaries in vitro were investigated using bioassay (rat tibia test) and radioimmunoassay (RIA). Both agents markedly increased the release of immunoreactive GH. High K+ also promoted the release of bioactive GH but to a much lesser degree than RIA-GH. Theophylline did not consistently affect the release of bioassay-detectable GH. The results suggest that these agents promote massive release of a form of immunoreactive GH (possibly "immature") that has little or no activity in the bioassay. Theophylline is relatively more effective in this regard than is elevated K+.     

Environmental life-cycle inventory of detergent-grade surfactant sourcing and production

Life-cycle inventories were compiled to characterize natural resource requirements and environmental emissions associated with the sourcing and production of selected, detergent-grade surfactants and surfactant feedstocks. Petrochemical surfactant types examined were linear alkylbenzene sulfonate, alcohol sulfate (AS), alcohol ethoxylate (AE) and alcohol ethoxylate sulfate (AES). Oleochemical surfactants derived from palm oil, palm kernel oil and inedible tallow were AS, AE, AES and (for palm oil and tallow) methyl ester sulfonate. It was determined that natural resource requirements were primarily related to the source of feedstock and secondarily to surfactant type. Likewise, the composition and mass of atmospheric, aqueous and solid emissions were principally determined by feedstock source. Energy requirements varied as a function of both feedstock and surfactant type. The inventories do not support fundamental shifts in surfactant usage or feedstock sourcing on the basis of environmental concerns, as no single surfactant or feedstock was identified as superior across all resource and emissions criteria examined. The data provide baselines for evaluating opportunities for resource optimization, pollution prevention and waste minimization within each production technology surveyed.     

The in vitro and in vivo investigation of inhaled migraine therapies using a novel aerosol delivery system consisting of an air pressurized capsule device (APCD) in combination with a pMDI spacer for endotracheal dosing into beagle dogs

Abstract

Context: Aerosol delivery to animals in preclinical settings has historically been very challenging, requiring the use of techniques, such as intratracheal instillation and dry powder insufflation, that are somewhat invasive, inefficient and not representative of clinical inhalation.

Objective: The objective of this work is to develop a system to deliver dry powder to dogs in an efficient and effective manner for the study of new anti-migraine compounds in development.

Materials and methods: The new device uses a metered aliquot of a dry gas to force dry powder drug from a pre-filled HPMC capsule into an AeroChamber® spacer for subsequent inhalation by the animal.

Results: The delivery of two invesigational migraine drugs via the new device was assessed in vitro using abbreviated Andersen cascade impaction and showed the device is capable of generating a reproducible delivered dose of up to ～68% with more than 50% of the dose in the respirable range. In vivo studies have also been performed showing that this device effectively delivered the migraine drugs to spontaneously breathing dogs using a proprietary validated dog inhalation model.

Discussion: Results confirmed that the air pressurized capsule device (APCD) was effective in delivering the APIs to lungs of the animals. The in vivo data verified the advantages of inhaled delivery over oral delivery for this class of drugs and were used to establish the cardiopulmonary and respiratory side effect liability profile for these compounds.

Conclusions: This work has demonstrated the utility of this device for quick and accurate screening of prospective drug candidates, representing a significant improvement in ease of use and reprodicibility over current delivery methods.     

Multiple phases of expression and regulation of mouse Hoxc8 during early embryogenesis

Hox genes are expressed in dynamic patterns during embryogenesis consistent with their role in axial specifications. To study the distribution of mouse Hoxc8, a homeodomain containing protein, we raised monoclonal antibodies against the least conserved portion of Hoxc8. Using these antibodies, we have examined early and mid-gestation embryos for the distribution of the protein. At the end of gastrulation Hoxc8 is expressed in the caudal portion of the embryo. In the neural tube, an early phase when all cells express Hoxc8 is distinguished from a late phase with predominant expression in differentiating neurons. A comparison of this expression pattern with that of a reporter gene under the control of the early Hoxc8 enhancer demarcates three separate regulatory components: (1) initiation and establishment; (2) maintenance; and (3) downregulation. We propose that Hoxc8 expression during embryogenesis is established in multiple phases. Possible regulatory mechanisms involved in generating such a complex domain of Hox gene expression are discussed.     

Psychological characteristics of South African street children

Research has shown that no treatment program designed for street children can succeed unless the community is prepared to respect, protect, and provide opportunities for them (Tacon, cited in Schurink & Rip, 1993). Unfortunately, these children are abused in many parts of the world (Aptekar, 1994). This paper investigates why the general public, as well as those charged with enforcing the law, often treat street children with scorn and hostility.