The NMR structure of the nucleocapsid protein from the mouse mammary tumor virus reveals unusual folding of the C-terminal zinc knuckle

The nucleocapsid protein (NC) from the mouse mammary tumor virus (MMTV) has been overexpressed in Escherichia coli and purified to homogeneity for structural studies by nuclear magnetic resonance (NMR) spectroscopy. The protein contains two copies of a conserved zinc-coordinating "CCHC array" or "zinc knuckle" motif common to the nucleocapsid proteins of nearly all known retroviruses. The residues comprising and adjacent to the zinc knuckles were assigned by standard two-dimensional (1)H and three-dimensional (1)H-(15)N NMR methods; the rotational dynamic properties of the protein were determined from (15)N relaxation experiments, and distance restraints derived from the nuclear Overhauser effect (NOE) data were used to calculate the three-dimensional structure. The (1)H-(1)H NOE and (15)N relaxation data indicate that the two zinc knuckles do not interact with each other, but instead behave as independently folded domains connected by a flexible 13-residue linker segment. The proximal zinc knuckle folds in a manner that is essentially identical to that observed previously for the two zinc knuckles of the human immunodeficiency virus type 1 nucleocapsid protein and for the moloney murine leukemia virus nucleocapsid zinc knuckle domain. However, the distal zinc knuckle of MMTV NC exhibits a rare three-dimensional fold that includes an additional C-terminal beta-hairpin. A similar C-terminal reverse turn-like structure was observed recently in the distal zinc knuckle of the Mason-Pfizer monkey virus nucleocapsid protein [Gao, Y., et al. (1998) Protein Sci. 7, 2265-2280]. However, despite a high degree of sequence homology, the conformation and orientation of the beta-hairpin in MMTV NC is significantly different from that of the reverse turn in MPMV NC. The results support the conclusion that structural features of NC zinc knuckle domains can vary significantly among the different genera of retroviridae, and are discussed in terms of the recent and surprising discovery that MMTV NC can facilitate packaging of the HIV-1 genome in chimeric MMTV mutants.     

Quantitative Circulatory Physiology: an integrative mathematical model of human physiology for medical education

We have developed Quantitative Circulatory Physiology (QCP), a mathematical model of integrative human physiology containing over 4,000 variables of biological interactions. This model provides a teaching environment that mimics clinical problems encountered in the practice of medicine. The model structure is based on documented physiological responses within peer-reviewed literature and serves as a dynamic compendium of physiological knowledge. The model is solved using a desktop, Windows-based program, allowing students to calculate time-dependent solutions and interactively alter over 750 parameters that modify physiological function. The model can be used to understand proposed mechanisms of physiological function and the interactions among physiological variables that may not be otherwise intuitively evident. In addition to open-ended or unstructured simulations, we have developed 30 physiological simulations, including heart failure, anemia, diabetes, and hemorrhage. Additional stimulations include 29 patients in which students are challenged to diagnose the pathophysiology based on their understanding of integrative physiology. In summary, QCP allows students to examine, integrate, and understand a host of physiological factors without causing harm to patients. This model is available as a free download for Windows computers at http://physiology.umc.edu/themodelingworkshop.     

Production of a recombinant form of early pregnancy factor that can prolong allogeneic skin graft survival time in rats

Early pregnancy factor (EPF), an extracellular chaperonin 10 homologue, has immunosuppressive and growth factor properties. In order to carry out more extensive studies on the in vivo characteristics of EPF, a recombinant form of the molecule has been prepared. Recombinant human EPF (rEPF) was expressed in Escherichia coli using the plasmid pGEX-2T expression system. Potency of rEPF in vitro in the rosette inhibition test, the bioassay for EPF, was equivalent to that of native EPF (nEPF), purified from human platelets, and synthetic EPF (sEPF). However, the half-life of activity (50% decrease in the log value) in serum, following i.p. injection, was significantly decreased (3.2 h, compared with nEPF 6.2 days, sEPF 5.8 days). This was thought to be due to modification of the N-terminus of the recombinant molecule inhibiting binding to serum carrier proteins. Because EPF can modify Th1 responses, the ability of the recombinant molecule to suppress allogeneic graft rejection was investigated. Following skin grafts from Lewis rats to DA rats and vice versa, rEPF was delivered locally at the graft site and the effect on survival time of the allografts noted. Results demonstrated that rEPF treatment significantly prolonged skin graft survival time by as much as 55% in stringent models of transplantation across major histocompatibility barriers.     

Enzymes of Choline Synthesis in Spinach (Response of Phospho-Base N-Methyltransferase Activities to Light and Salinity)

In spinach (Spinacia oleracea L.), choline is synthesized by the sequential N-methylation of phosphoethanolamine -> phosphomono- -> phosphodi- -> phosphotrimethylethanolamine (i.e. phosphocholine) followed by hydrolysis to release choline. Differential centrifugation of spinach leaf extracts shows that enzymes catalyzing the three N-methylations are cytosolic. These enzymes were assayed in leaf extracts prepared from plants growing under various light/dark periods. Under a diurnal, 8-h light/16-h dark photoperiod, the activity of the enzyme catalyzing the N-methylation of phosphoethanolamine is highest at the end of the light period and lowest following the dark period. Prolonged dark periods (exceeding 16 h) lead to a further reduction in the activity of this enzyme, although activity is restored when plants are reexposed to light. In contrast, the activity of the enzyme(s) catalyzing the N-methylations of phosphomono- and phosphodimethylethanolamine does not undergo comparable changes in response to light/dark treatments. Salt shock of plants with 200 mM NaCl results in a 2-fold increase in all three N-methylation activities relative to nonsalinized controls but only in plants exposed to light. Thus, light is required for the salt-responsive up-regulation of choline synthesis in spinach.     

Dissecting the serotonergic food signal stimulating sensory-mediated aversive behavior in C. elegans

Nutritional state often modulates olfaction and in Caenorhabditis elegans food stimulates aversive responses mediated by the nociceptive ASH sensory neurons. In the present study, we have characterized the role of key serotonergic neurons that differentially modulate aversive behavior in response to changing nutritional status. The serotonergic NSM and ADF neurons play antagonistic roles in food stimulation. NSM 5-HT activates SER-5 on the ASHs and SER-1 on the RIA interneurons and stimulates aversive responses, suggesting that food-dependent serotonergic stimulation involves local changes in 5-HT levels mediated by extrasynaptic 5-HT receptors. In contrast, ADF 5-HT activates SER-1 on the octopaminergic RIC interneurons to inhibit food-stimulation, suggesting neuron-specific stimulatory and inhibitory roles for SER-1 signaling. Both the NSMs and ADFs express INS-1, an insulin-like peptide, that appears to cell autonomously inhibit serotonergic signaling. Food also modulates directional decisions after reversal is complete, through the same serotonergic neurons and receptors involved in the initiation of reversal, and the decision to continue forward or change direction after reversal is dictated entirely by nutritional state. These results highlight the complexity of the "food signal" and serotonergic signaling in the modulation of sensory-mediated aversive behaviors.     

Cytokinetic properties of asynchronous and cytosine arabinoside perturbed murine tumors measured by simultaneous bromodeoxyuridine/DNA analyses

This paper describes the determination of cytokinetic properties of asynchronous and cytosine arabinoside- (Ara-C) treated KHT tumors growing in vivo using the bromodeoxyuridine (BrdUrd)/DNA analysis technique. The cytokinetic properties of asynchronously growing tumors were estimated by computer analysis of sequential BrdUrd/DNA distributions measured at 2- to 3-h intervals after administration of a single i.p. injection of BrdUrd. The cytokinetic properties of the Ara-C-treated tumors were estimated by computer analysis of BrdUrd/DNA distributions measured at 2- to 3-h intervals after Ara-C treatment. BrdUrd was injected 30 min prior to tumor harvest. The cytokinetic properties of the cells rendered nonclonogenic by Ara-C were followed in BrdUrd/DNA distributions measured at 2- to 3-h intervals after Ara-C treatment of tumors that were labeled with BrdUrd 30 min prior to Ara C injection. The G1-, S-, and G2M-phase durations were estimated to be 7.6, 10.9, and 2.0 h prior to Ara-C; decreasing to 1.2, 4.1, and 1.4 after Ara-C. The growth fraction was estimated to be 0.8 prior to Ara-C. Complete recruitment of the normally noncycling subpopulation was observed after Ara-C treatment. Ara-C-killed cells were removed from the tumor within 24 h following Ara-C injection. These cytokinetic properties were similar to those reported in other studies.