Amino-acid-type selective isotope labeling of proteins expressed in Baculovirus-infected insect cells useful for NMR studies

Culture conditions for successful amino–acid-type selective isotope labeling of proteins expressed in Baculovirus-infected insect cells are described. The method was applied to the selective labeling of the catalytic domain of c-Abl kinase with ¹⁵N-phenylalanine, ¹⁵N-glycine, ¹⁵N-tyrosine or ¹⁵N-valine. For the essential amino acids phenylalanine, tyrosine and valine high ¹⁵N-label incorporation rates of 90% and approximately the expected number of resonances in the HSQC spectra were observed, which was not the case for the non-essential amino acid glycine. The method should be applicable to amino-acid-type selective isotope labeling of other recombinant proteins which have not been amenable to NMR analysis.     

Directed walk designs for dose-response problems with competing failure modes

We examine adaptive allocation designs for the problem of determining the optimal therapeutic dose for subjects in early-phase clinical trials. A subject can fail due to lack of efficacy or due to a toxic reaction. Successful subjects will have both a positive response and no toxic side effects. Thus, we seek to maximize the product of the nontoxicity and efficacy dose-response curves. We are interested in sampling rules that perform well along several criteria, including the ethical criterion that, as often as possible, experimental subjects be treated at or close to the maximum in question. Statistically, we wish to identify the optimum dose with high probability at the close of the experiment. Here, we propose designs that combine new allocation policies, directed walks, with new smoothed shape-constrained curve-fitting techniques. These are compared with a variety of other curve-fitting techniques and with up-and-down and equal allocation rules.     

Xanthelasma palpebrarum: a review and current management principles

LEARNING OBJECTIVES: After studying this article, the participant should be able to: 1. Describe the differential diagnosis of xanthelasma palpebrarum. 2. Discuss the various nonsurgical and surgical treatment options and their advantages and disadvantages. 3. Describe the circumstances in which recurrence is most likely after treatment. Xanthelasma palpebrarum is the most common cutaneous xanthoma. It typically presents in middle-aged and older adults, most often around the eyelids. The diagnosis can often be made on clinical grounds alone. For the plastic surgeon, it is important to apply an algorithmic approach to the treatment of these lesions. Depending on the size and location, several different methods can be used to address this problem, ranging from simple excision, to laser treatment, to chemical peeling. This article addresses the underlying pathophysiology of xanthelasma and the currently accepted modes of treatment.     

The ecology and genetics of a host shift: microbotryum as a model system

The need to prevent and cure emerging diseases often precludes their continuing study in situ. We present studies on the process of disease emergence by host shifts using the model system of anther-smut disease (Microbotryum violaceum) on the plant genus Silene (Caryophyllaceae). This system has little direct social impact, and it is readily amenable to experimental manipulation. Our microevolutionary studies have focused on the host shift of Microbotryum from Silene alba (=latifolia; white campion) onto Silene vulgaris (bladder campion) in a population in Virginia. Karyotypic variation shows that the host shift is recent and originates from the disease on sympatric S. alba. Analysis of the spatial pattern of disease shows that the host shift has been contingent on the co-occurrence of the two species at a local scale. Cross-inoculation studies show that families of the new host differ greatly in their susceptibility to the pathogen, indicating the potential for rapid evolution of resistance. Disease expression on the new host is frequently abnormal, suggesting that the pathogen is imperfectly adapted to its new host. In experimental populations, disease transmission within populations of the old host is greater than within populations of the new host. However, there is also a high transmission rate of the disease from the new host back to the old host, suggesting a feedback effect that increases disease prevalence in the community as a whole. Continuing studies of these populations are designed to determine whether this new host-pathogen system is likely to be self-sustaining and to quantify evolutionary changes in both the host and the pathogen.     

Potential Contributions of Viral Envelope and Host Genetic Factors in a Human Immunodeficiency Virus Type 1-Infected Long-Term Survivor

The lack of clinical progression in some individuals despite prolonged human immunodeficiency virus type 1 (HIV-1) infection may result from infection with less-pathogenic viral strains. To address this question, we examined the HIV-1 envelope protein from a donor with a low viral burden, stable CD4⁺ T-lymphocyte counts, and little evidence of CD8⁺ T-cell expansion, activation, or immune activity. To avoid potential changes in envelope function resulting from selection in vitro, envelope clones were constructed by using viral RNA isolated from uncultured peripheral blood mononuclear cells (PBMC). The data showed that recombinant viruses containing envelope sequences derived from RNA isolated from patient PBMC replicated poorly in primary CD4⁺ T cells but demonstrated efficient growth in macrophages. The unusual phenotype of these viruses could not be explained solely by differential utilization of coreceptors since the chimeric viruses, as well as an uncloned isolate obtained from the same visit date, can utilize CCR5. In addition, the donor’s own cells appeared resistant to infection with chimeric viruses containing autologous envelope sequences. Genotype analysis revealed that the donor was heterozygous for the previously described 32-bp deletion in CCR5 which may be linked with prolonged survival in HIV-1-infected individuals. These data suggest that the changes in envelope sequences confer properties of viral attenuation, which together with the CCR5 +/Δ32 genotype could account for the long-term survival of this patient.     

Were there mammalian pursuit predators in the tertiary? Dances with wolf avatars

Fast-running, long-legged pursuit carnivores are familiar members of the present-day ecosystem, and it has been assumed that extinct large predators took similar ecomorphological roles (i.e., were wolf avatars) in past faunas. While these fossil taxa may also have been meat-specialists, we present evidence from limb morphology to show that there was no modern type of pursuit predator until the latest Tertiary. In contrast, ungulates evolved longer legs similar to those of present-day cursorial taxa by the middle Tertiary, some 20 million years earlier. These data suggest the need for the reevaluation of many classical evolutionary stories, not only about assignation of fossil taxa to a wolf-like mode of predatory behavior, but also to issues such as the coevolution of long legs and fast running speeds between predator and prey, and even the implicit assumption that cursorial morphologies are primarily an adaptation for speed. We conclude that evolutionary change in ungulate limb morphologies represents an adaptation to decrease transport costs in association with Tertiary climatic changes and that the present-day predation mode of long distance pursuit is a Plio-Pleistocene phenomenon, related to the development of colder and more arid climates.     

Effects of gene dosage and sequence modification on the frequency and timing of transposition of the maize element Activator (Ac) in tobacco

The effect of Ac copy number on the frequency and timing of germinal transposition in tobacco was investigated using the streptomycin phosphotransferase gene (SPT) as an excision marker. The activity of one and two copies of the element was compared by selecting heterozygous and homozygous progeny of transformants carrying single SPT::Ac inserts. It was observed that increasing gene copy not only increases the transposition frequency, but also occasionally alters the timing of transposition such that earlier events are obtained. The result is that some homozygous plants generate multiple streptomycin resistant progeny carrying the same transposed Ac (trAc) element. We have also investigated the effect of modification of the sequence in the region around 82 bp downstream of the polyadenylation site and 177 bp from the 3 end of the element on germinal excision frequencies. Alteration of three bases to create a BglII site at this location caused a minor decrease in germinal excision events, but insertion of four bases to create a Cla I site caused a 10-fold decrease in the transposition activity of the Ac element.     

Influence of interganglionic interactions on steroid-mediated dendritic reorganization and death of proleg motor neurons during metamorphosis in Manduca sexta

In Manduca sexta, the larval abdominal prolegs and their muscles degenerate at pupation. The proleg motor neurons undergo a period of dendritic regression, after which a specific subset of them dies. The surviving motor neurons undergo dendritic outgrowth during pupal-adult development, and most die after adult emergence. All of these events are regulated hormonally by ecdysteroids and juvenile hormone, but interactions of the motor neurons with other cells may potentially contribute as well. To investigate the possible influence of interganglionic neural interactions, we chronically isolated individual abdominal ganglia by severing the adjacent rostral and caudal connectives in the larval stage. Subsequent metamorphic changes in proleg motor neurons were examined in the isolated ganglia and ganglia adjacent to the isolated ganglia. Two abnormalities were observed: (1) some imprecision in the timing of motor neuron death, both at pupation and after adult emergence, and (2) the growth of ectopic neurites outside the neuropil boundaries during pupal-adult development (in ganglia with or without neuromas caused by connective transections). Other aspects of proleg motor neuron metamorphosis, including the segment-specific death of motor neurons at pupation, were the same as that in intact and sham-operated insects. Thus, interganglionic interactions appear to play a relatively minor role in the steroid-mediated metamorphic transformation of proleg motor neurons. © 1994 John Wiley & Sons, Inc.     

Heavy-Metal and Antibiotic Resistance in the Bacterial Flora of Sediments of New York Bight

The New York Bight extends seaward some 80 to 100 miles (ca. 129 to 161 km) from the Long Island and New Jersey shorelines to the edge of the continental shelf. Over 14 × 10⁶ m³ of sewage sludge, dredge spoils, acid wastes, and cellar dirt are discharged into this area each year. Large populations of Bacillus sp. resistant to 20 μg of mercury per ml were observed in Bight sediments contaminated by these wastes. Resistant Bacillus populations were much greater in sediments containing high concentrations of Hg and other heavy metals than in sediments from areas further offshore where dumping has never been practiced and where heavy-metal concentrations were found to be low. Ampicillin resistance due mainly to β-lactamase production was significantly (P < 0.001) more frequent in Bacillus strains from sediments near the sewage sludge dump site than in similar Bacillus populations from control sediments. Bacillus strains with combined ampicillin and Hg resistances were almost six times as frequent at the sludge dump site as in control sediments. This observation suggests that genes for Hg resistance and β-lactamase production are simultaneously selected for in Bacillus and that heavy-metal contamination of an ecosystem can result in a selection pressure for antibiotic resistance in bacteria in that system. Also, Hg resistance was frequently linked with other heavy-metal resistances and, in a substantial proportion of Bacillus strains, involved reduction to volatile metallic Hg (Hg°).