Antiserum inhibition of propagating viruses

The design and implementation of controlled environments to continuously culture and evolve viruses provides a means to track how their populations respond to natural and designed anti-viral agents. We have previously demonstrated how the growth of viruses in spreading plaques enables detection and characterization of their evolutionary dynamics. Using plaques of phage T7 growing on E. coli as a model system, we observe here that velocities of propagation can be readily controlled by the level of anti-viral antiserum incorporated into the propagation medium. Further, we develop a simple analytic expression for the radial velocity of propagation in terms of the microscopic rates of viral amplification, Fickian diffusion of the virions and their neutralization by antiserum. Our analysis captures the essential dependence of propagation velocity on antiserum concentration. This study provides an ex vivo foundation for exploring how medically relevant viruses escape suppression by the immune system. (c) 1997 John Wiley & Son, Inc. Biotechnol Bioeng 55: 542-546, 1997.     

Design and development of a micro-thermocouple sensor for determining temperature and relative humidity patterns within an airstream

This paper describes the production and calibration of a miniature psychrometer treated with a specially developed porous coating. The investigation was conducted to determine localized patterns of rapidly changing temperature and relative humidity in dynamic flowing gas environments (e.g., with particular attention to future applications to the human respiratory system). The technique involved the use of dry miniature thermocouples and wetted miniature thermocouples coated with boron nitride to act as a wicking material. A precision humidity generator was developed for calibrating the psychrometer. It was found that, in most cases, the measured and expected (i.e., theoretically predicted) relative humidity agreed to within 0.5 to 1.0 percent relative humidity. Procedures that would decrease this discrepancy even further were pinpointed, and advantages of using the miniature psychrometer were assessed.     

The escape strategy of green lacewings from orb webs

When green lacewings (Neuroptera: Chrysopidae) fly into spider orb webs, they often simply reverse their flight direction and pull away (Table I). If a lacewing is trapped, it uses a specialized escape behavior. It first cuts away the sticky strands entangling head, feet, and antennae. If an antenna cannot be freed by tugging, it uses an antenna climb (Fig. 5A). After its body is free, the lacewing remains suspended by its hair-covered wings, which are held in a characteristic cruciform position (Fig. 5B). Orb web sticky strands adhere poorly to the hairy wings (Fig. 7), so the chrysopid may just wait until the strands slide off and it falls free. If placed in an orb web when the spider is at the web hub and ready to attack, a lacewing usually does not have time to escape (Fig. 1). When the spider is at the hub but eating, the chances of escape improve, and when the spider is away from the hub attacking other prey, nearly all lacewings in our experiment were able to escape. This finding emphasizes the importance of the spider's activity in its capture success.Paper No. 88 of the series Defense Mechanisms of Arthropods.     

Pyrrolizidine alkaloid deters ant predators of Utetheisa ornatrix eggs: effects of alkaloid concentration,oxidation state,and prior exposure of ants to alkaloid-laden prey

To examine the chemical defense of lepidopteran eggs towards ant predators, eggs of the arctiid moth Utetheisa ornatrix were offered to laboratory colonies of the ant Leptothorax longispinosus. The ants rapidly devoured Utetheisa eggs produced by parents reared on an alkaloid-free diet, but left eggs that had been endowed with parental alkaloid largely unmolested. That defense can be attributed directly to the presence of a pyrrolizidine alkaloid since topical application of monocrotaline in either its free base or N-oxide state conferred protection on otherwise palatable eggs. The N-oxide and free base were not equally effective in conferring protection however; free-base treated eggs were subject to greater predation than N-oxide treated eggs after 24 h. The emergence of differential alkaloid effectiveness only after prolonged exposure is consistent with the notion that chemical protection is seldom absolute. That difference was resolved only when the otherwise food-deprived ants exceeded a certain threshold of hunger. The efficacy of applied monocrotaline as a predator deterrent increased with increasing concentration of N-oxide, but was lowest for eggs treated with an intermediate concentration of free base. The latter concentration effect likely represents an artefact of the behaviour of the ant colonies offered eggs treated with an intermediate concentration, as those colonies were the most voracious predators in reference tests with palatable eggs. In addition to the immediate deterrent value of pyrrolizidine alkaloid, ant colonies that had been exposed to alkaloid-laden eggs subsequently avoided even palatable, unprotected eggs offered 33 days later. Our data provide the first demonstration of such long-term avoidance of chemically protected lepidopteran prey by an invertebrate. The discovery of a mechanism promoting learned avoidance on the part of ant predators has important ramifications to egg-laying strategies of female Utetheisa, both in terms of the dispersion of eggs, and the extent to which eggs are provisioned with alkaloid.     

Higher urine desmosine levels are associated with mortality in patients with acute lung injury

Desmosine is a stable breakdown product of elastin that can be reliably measured in urine samples. We tested the hypothesis that higher baseline urine desmosine would be associated with higher mortality in 579 of 861 patients included in the recent Acute Respiratory Distress Syndrome Network trial of lower tidal volume ventilation (1). We also correlated urine desmosine levels with indexes of disease severity. Finally, we assessed whether urine desmosine was lower in patients who received lower tidal volumes. Desmosine was measured by radioimmunoassay in urine samples from days 0, 1, and 3 of the study. The data were expressed as a ratio of urine desmosine to urine creatinine to control for renal dilution. The results show that higher baseline (day 0) urine desmosine-to-creatinine concentration was associated with a higher risk of death on adjusted analysis (odds ratio 1.36, 95% confidence interval 1.02-1.82, P=0.03). Urine desmosine increased in both ventilator groups from day 0 to day 3, but the average rise was higher in the 12-ml/kg predicted body weight group compared with the 6-ml/kg predicted body weight group (P=0.053, repeated-measures model). In conclusion, patients with acute lung injury ventilated with lower tidal volumes have lower urine desmosine levels, a finding that may reflect reduced extracellular matrix breakdown. These results illustrate the value of evaluating urinary biological markers that may have prognostic and pathogenetic significance in acute lung injury.     

Scanning mutagenesis of the alpha repeats and of the transmembrane acidic residues of the human retinal cone Na/Ca-K exchanger

The Na/Ca-K exchanger (NCKX) utilizes the inward sodium gradient and outward potassium gradient for Ca(2+) extrusion; two distinct NCKX isoforms are expressed in the outer segments of retinal rod (NCKX1) and cone (NCKX2) photoreceptors, respectively, where NCKX extrudes Ca(2+) that enters photoreceptors via the cGMP-gated channels. We carried out the first systematic NCKX mutagenesis study in which 96 residues were mutated in the human cone NCKX2 cDNA, and functional consequences of these mutations were measured; the residues selected for mutagenesis are conserved between rod and cone NCKX, the large majority are also conserved in NCKX paralogs found in lower organisms, and finally, they include the few residues conserved between members of the NCKX and members of the NCX (potassium-independent Na/Ca exchange) gene families. Twenty-five residues were identified for which mutagenesis reduced NCKX function to <20% of wild-type cone NCKX2 activity, while protein expression and plasma membrane targeting were not affected. Three classes of residues were found to be most sensitive toward mutagenesis: acidic (glutamate/aspartate) residues, polar (serines/threonine) residues, and glycine residues. These results are discussed with respect to residues that may contribute to the NCKX cation binding site(s).     

Aldose reductase induced by hyperosmotic stress mediates cardiomyocyte apoptosis: differential effects of sorbitol and mannitol

Cells adapt to hyperosmotic conditions by several mechanisms, including accumulation of sorbitol via induction of the polyol pathway. Failure to adapt to osmotic stress can result in apoptotic cell death. In the present study, we assessed the role of aldose reductase, the key enzyme of the polyol pathway, in cardiac myocyte apoptosis. Hyperosmotic stress, elicited by exposure of cultured rat cardiac myocytes to the nonpermeant solutes sorbitol and mannitol, caused identical cell shrinkage and adaptive hexose uptake stimulation. In contrast, only sorbitol induced the polyol pathway and triggered stress pathways as well as apoptosis-related signaling events. Sorbitol resulted in activation of the extracellular signal-regulated kinase (ERK), p54 c-Jun N-terminal kinase (JNK), and protein kinase B. Furthermore, sorbitol treatment resulting in induction and activation of aldose reductase, decreased expression of the antiapoptotic protein Bcl-xL, increased DNA fragmentation, and glutathione depletion. Apoptosis was attenuated by aldose reductase inhibition with zopolrestat and also by glutathione replenishment with N-acetylcysteine. In conclusion, our data show that hypertonic shrinkage of cardiac myocytes alone is not sufficient to induce cardiac myocyte apoptosis. Hyperosmolarity-induced cell death is sensitive to the nature of the osmolyte and requires induction of aldose reductase as well as a decrease in intracellular glutathione levels.     

Ligand crowding at a nascent signal sequence

We have systematically analyzed the molecular environment of the signal sequence of a growing secretory protein from Escherichia coli using a stage- and site-specific cross-linking approach. Immediately after emerging from the ribosome, the signal sequence of pOmpA is accessible to Ffh, the protein component of the bacterial signal recognition particle, and to SecA, but it remains attached to the surface of the ribosome via protein L23. These contacts are lost upon further growth of the nascent chain, which brings the signal sequence into sole proximity to the chaperone Trigger factor (TF). In its absence, nascent pOmpA shows extended contacts with L23, and even long chains interact in these conditions proficiently with Ffh. Our results suggest that upon emergence from the ribosome, the signal sequence of an E. coli secretory protein gradually becomes sequestered by TF. Although TF thereby might control the accessibility of pOmpA's signal sequence to Ffh and SecA, it does not influence interaction of pOmpA with SecB.     

X-ray absorption fine-structure spectroscopy studies of Fe sites in natural human neuromelanin and synthetic analogues.

X-ray absorption fine-structure spectroscopy is used to study the local environment of the iron site in natural (human) neuromelanin extracted from substantia nigra tissue and in various synthetic neuromelanins. All the materials show Fe centered in a nearest neighbor sixfold (distorted) oxygen octahedron; the Fe-O distances, while slightly different in the natural and synthetic neuromelanin, are both approximately 2.0 A. Appreciable differences arise, however, in the second (and higher) coordination shells. In this case the synthetic melanin has the four planar oxygens bound to carbon rings with Fe-C distances of approximately 2.82 and 4.13 A; the human sample does not show the 2.82 A link but instead indicates a double shell at approximately 3.45 and 3.78 A.