Stable, stoichiometric delivery of diverse protein functions.

As contemporary "genomics" steadily reveals an increasing number of novel gene sequences, the need for efficient methodologies to functionally characterize these genes in vivo increases significantly. Reliable coupling of target gene expression to a variety of surrogate reporter functions is critical to properly assay novel gene function in complex cell populations. Ideally, independent target and reporter proteins would be derived from a single open reading frame creating a stoichiometric relationship without obscuring subcellular localization. We report here effective strategies for assaying gene function through the stable production of chimeric polyproteins, processed intracellularly by inclusion of an intervening 19-amino-acid sequence from the 2A region of the Foot and Mouth Disease virus. Using drug-resistance and flow cytometry-based assay systems, we demonstrate that diverse protein functions are effectively delivered to various cell types by retroviral constructs as single 2A-cleaved polyproteins. For example, cells infected with a retrovirus encoding a nuclear cell cycle regulator, linked via the 2A-motif to a marker membrane protein, showed a direct correlation between cell cycle arrest and surface marker level. This demonstrates the utility of this methodology for stable and stoichiometric delivery of distinctly localized protein functionalities. In particular, the ability to exploit multiple cellular functions will serve to accelerate the functional characterization of gene products and facilitate novel gene therapy approaches.

Retroviral delivery of peptide modulators of cellular functions.

Stable transduction of genetic material, in combination with sensitive methodologies for in vivo study of cell physiology, provides an opportunity to efficiently evaluate the functions of regulatory proteins. To dissect the minimal therapeutic function of such proteins, we have stably expressed protein microdomains as fusions, composed of short peptides, and detected specific subfunctions distinct from holoprotein function, using flow cytometry and other techniques. We demonstrate that retroviral delivery of the 24-amino-acid proliferating cell nuclear antigen-binding motif (p21C), derived from the C-terminus of the cell cycle inhibitor protein, p21, is sufficient to induce cell cycle arrest. Cells expressing this peptide motif reversibly execute both G1- and G2-checkpoint controls that are normally activated subsequent to interference with DNA synthesis. The p21C effect is distinct from results obtained with an intact p21 protein that also binds cyclin-CDK complexes and arrested cells exclusively at the G1/S transition. Thus, microdomains can exert unique biological effects compared to the parental molecules from which they were derived. To further evaluate the peptide delivery strategy, we analyzed the role of various kinases in IgE-mediated stimulation of mast cell exocytosis. Primary bone marrow-derived mast cells were transduced with retroviral constructs encoding short-kinase inhibitor motifs and analyzed by flow cytometry for effects on exocytosis. We found that a specific protein kinase A (PKA) inhibitor peptide suppressed IgE-mediated stimulation of mast cell exocytosis. This anti-exocytotic effect was mimicked by a small molecule inhibitor of PKA (KT5720). Thus, the ability to express protein microdomains can be a powerful means to subtly perturb cellular physiology in manners that reveal new paths for therapeutic intervention. We believe that such approaches might allow for new forms of gene therapy to become available.

Users' evaluation of the Navy Computer-Assisted Medical Diagnosis System.

U.S. Navy Independent Duty Corpsmen (IDCs) aboard small ships and submarines are responsible for all clinical and related health care duties while at sea. During deployment, life-threatening illnesses sometimes require evacuation to a shore-based treatment facility. At-sea evacuations are dangerous, expensive, and may compromise the mission of the vessel. Therefore, Group Medical Officers and IDCs were trained to use the Navy Computer-Assisted Medical Diagnosis (NCAMD) system during deployment. They were then surveyed to evaluate the NCAMD system. Their responses show that NCAMD is a cost-efficient, user-friendly package. It is easy to learn, and is especially valuable for training in the diagnosis of chest and abdominal complaints. However, the delivery of patient care at sea would significantly improve if computer hardware were upgraded to current industry standards. Also, adding various computer peripheral devices, structured forms, and reference materials to the at-sea clinician's resources could enhance shipboard patient care.

Comparisons of wartime and peacetime disease and non-battle injury rates aboard ships of the British Royal Navy.

Disease and non-battle injury rates were computed for ships of the British Royal Navy which were deployed during wartime and peacetime operations. The wartime sick list admission rates were lower aboard carriers, battleships, and cruisers when compared with their counterparts deployed in peacetime; rate differences for battleships and cruisers were statistically significant (p < 0.05). Several categories of disease also yielded significant differences in the wartime/peacetime contrasts. Infections and parasitic disorders aboard carriers, skin diseases aboard battleships, and skin diseases, injuries, and generative system disorders occurring on cruisers were all lower during wartime than on peacetime deployments. Illness rates also varied by ship type, with the lowest rates evidenced aboard carriers.

Subsistence economy of el paraiso, an early peruvian site.

Studies of food remains from the Preceramic monumental site of E1 Paraíso, Peru (1800 to 1500 B.C.), have shed new light on a debate regarding the relative importance of seafood versus terrestrial resources and the role of cultigens in subsistence economies during the early development of Peruvian civilization. Fish was the primary animal food at the site whereas plant foods consisted of a mixture of cultivated resources (squashes, beans, peppers, and jicama) with an additional reliance on fruits (guava, lucuma, and pacae). Wild plants, especially the roots of sedges and cat-tail, also may have accounted for a substantial part of the diet. Cotton was a chief crop, used in making fishing tackle and the textiles that served as clothing and items of high value and status. As an example of the beginnings of civilization, El Paraíso is a case in which impressive architecture was built on a relatively simple subsistence economy and energy was expended in the production of resources useful in local and regional exchange systems.

Solubilization of high affinity corticotropin-releasing factor receptors from rat brain: characterization of an active digitonin-solubilized receptor complex.

The binding characteristics of CRF receptors in rat frontal cerebral cortex membranes solubilized in 1% digitonin were determined. The binding of [125I]Tyro-ovine CRF ([125I]oCRF) to solubilized membrane proteins was dependent on incubation time, temperature, and protein concentration, was saturable and of high affinity, and was absent in boiled tissue. The solubilized receptors retained their high affinity for [125I] oCRF in the solubilized state, exhibiting a dissociation constant (KD) of approximately 200 pM, as determined by direct binding saturation isotherms. Solubilized CRF receptors maintained the rank order of potencies for various related and unrelated CRF peptides characteristic of the membrane CRF receptor: rat/human CRF congruent to ovine CRF congruent to Nle21,38-rat CRF greater than alpha-helical oCRF-(9-41) greater than oCRF-(7-41) much greater than vasoactive intestinal peptide, arginine vasopressin, or the substance-P antagonist. Furthermore, the absolute potencies (Ki values) for the various CRF-related peptides in solubilized receptors were almost identical to those observed in the membrane preparations, indicating that the CRF receptor retained its high affinity binding capacity in the digitonin-solubilized state. Chemical affinity cross-linking of digitonin-solubilized rat cortical membrane proteins revealed a specifically labeled protein with an apparent mol wt of 58,000 which was similar to the labeled protein in native membrane homogenates. Although solubilized CRF receptors retained their high affinity for agonists, their sensitivity for guanine nucleotide was lost. Size exclusion chromatography substantiated these results, demonstrating that in the presence or absence of guanine nucleotides, [125I]oCRF labeled the same size receptor complex. These data suggest that either the guanine nucleotide-binding protein (Ns) is tightly associated with the CRF receptor after solubilization and is insensitive to guanine nucleotides, or that high affinity binding for soluble CRF receptors is not dependent on the coupling of a guanine nucleotide-binding protein. The solubilization of CRF receptors from membranes in digitonin should allow for the more complete molecular and functional characterization of CRF-mediated events and purification of the receptor.

Beans for valdivia.

Carbonized remains of Canavalia beans were recovered from archaeological deposits excavated at Real Alto (OGCh-12), southwestern Ecuador. The identification, context and dating of the earliest beans from Real Alto demonstrate their use from the beginning of Valdivia (about 3300 B.C.) and the Early Formative in coastal Ecuador.

Phytolith analysis of archeological soils: evidence for maize cultivation in formative ecuador.

Soil samples from the archeological sites of Real Alto and OGCh-20, Santa Elena Peninsula, Ecuador, show the presence of cross-shaped silica bodies identifiable as maize (Zea mays L.) phytoliths by size comparison with known wild grass and maize phytoliths. These results support arguments for the cultivation of maize at 2450 B.C. in coastal Ecuador.