Investigation of the role of tyrosine-114 in the activity of human O6-alkylguanine-DNA alkyltranferase

Tyrosine-114 is one of 13 totally conserved amino acids in all known sequences of O6-alkylguanine-DNA alkyltransferase (AGT). The importance of this amino acid in repair of alkylated DNA by AGT was studied by changing it to phenylalanine (F), alanine (A), threonine (T), or glutamic acid (E) in human AGT. The activities of the mutant proteins were then compared to those of the wild type with regard to abilities to do the following: (a) protect Escherichia coli from the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG); (b) repair methylated DNA in vitro; (c) bind to oligodeoxynucleotides containing O6-methylguanine; and (d) react with the low molecular weight pseudosubstrate, O6-benzylguanine. When expressed at high levels in E. coli strain GWR109, lacking endogenous AGT, the wild type and the Y114F mutant were highly effective in reducing mutations and cell killing by MNNG. The Y114A mutant had a much smaller protective effect, and mutants Y114T and Y114E were inactive. Purified preparations of all four AGT mutants showed an approximately similar degree (74-120-fold) of reduction in the rate of reaction with O6-benzylguanine. In contrast, the degree of reduction in activity toward methylated DNA substrates in vitro varied according to the mutation with the more conservative Y114F producing only a 30-fold reduction and the most drastic change of Y114E abolishing activity completely. Alteration Y114A produced a 1000-fold reduction whereas Y114T reduced activity by 10000-fold. All of the mutations affected the binding of AGT to single- or double-stranded oligodeoxynucleotides containing O6-methylguanine. The extent of increase in the Kd varied according to the amino acid with 2-5-fold (F), 7-11-fold (A), 167-200-fold (T), and 600-1000-fold (E) increases. These results are consistent with tyrosine-114 playing a role both in the binding of AGT to its DNA substrate and in facilitating the transfer of the alkyl group. It is probable that AGT resembles other DNA repair proteins in bringing about a "flipping out" of the target base from the DNA helix. Tyrosine-114 is therefore an excellent candidate for a key role in the interaction with the flipped O6-methylguanine. The results also show that when large amounts of AGT are produced in the cell, substantial decreases in the efficiency with which AGT can repair methylated DNA do not prevent the ability to protect E. coli from toxic alkylating agents. Mutant Y114F, whose activity was reduced by 30-fold, was equal to wild-type AGT in bringing about this protection.     

Gene therapy in autoimmune diseases

The influence of "shinrin-yoku" (forest-air bathing and walking) on blood glucose levels in diabetic patients was examined. Eighty-seven (29 male and 58 female) non-insulin-dependent diabetic patients [61 (SEM 1) years old] participated in the present study. Shinrin-yoku was performed nine times over a period of 6 years. The patients were divided into two parties. They then walked in the forest for 3 km or 6 km according to their physical ability and/or the existence of diabetic complications. The mean blood glucose level after forest walking changed from 179 (SEM 4) mg.100 ml-1 to 108 (SEM 2) mg.100 ml-1 (P < 0.0001). The level of glycated haemoglobin A1c also decreased from 6.9 (SEM 0.2)% (before the first shinrin-yoku) to 6.5 (SEM 0.1)% (after the last shinrin-yoku; P < 0.05). Blood glucose values declined by 74 (SEM 9) mg.100 ml-1 and 70 (SEM 4) mg.100 ml-1 after short- and long-distance walking respectively. There was no significant difference between these values. Since the forest environment causes changes in hormonal secretion and autonomic nervous functions, it is presumed that, in addition to the increased calorie consumption and improved insulin sensitivity, walking in a forest environment has other beneficial effects in decreasing blood glucose levels.     

A reactive coordination scheme for a many-robot system

This paper presents a novel approach for coordinating a homogeneous system of mobile robots using implicit communication in the form of broadcasts. The broadcast-based coordination scheme was developed for the Army Ant swarm-a system of small, relatively inexpensive mobile robots that can accomplish complex tasks by cooperating as a team. The primary drawback, however, of the Army Ant system is that the absence of a central supervisor poses difficulty in the coordination and control of the agents. Our coordination scheme provides a global "group dynamic" that controls the actions of each robot using only local interactions. Coordination of the swarm is achieved with signals we call "heartbeats". Each agent broadcasts a unique heartbeat and responds to the collective behavior of all other heartbeats. We generate heartbeats with van der Pol oscillators. In this application, we use the known properties of coupled van der Pol oscillators to create predictable group behavior. Some of the properties and behaviors of coupled van der Pol oscillators are discussed in detail. We emphasize the use of this scheme to allow agents to simultaneously perform an action such as lifting, steering, or changing speed. The results of experiments performed on three actual heartbeat circuits are presented and the behavior of the realized system is compared to simulated results. We also demonstrate the application of the coordination scheme to global speed control.     

Spectroscopic determination of pressure-induced shifts in inclusion complexation equilibria

The effect of modest hydrostatic pressure (<350 bar) on condensed-phase equilibrium processes has been largely overlooked, due in large part to the small compressibility of these phases relative to gases or supercritical fluids. Although the bulk properties of condensed phases are not significantly modified by pressure in this modest regime, the solvation processes driving inclusion complexation may be appreciably affected. In this paper, we examine this hypothesis using steady-state fluorescence spectroscopy to determine the pressure dependence of association constants. The widely used host molecule, β-cyclodextrin, provides an incompressible hydrophobic cavity into which structurally analogous fluorescent probes are encapsulated. By comparing the unique pressure dependencies of these equilibria, the importance of local site solvation and rim interactions in influencing the pressure dependence is demonstrated. The structurally analogous complexes chosen for these studies are expected to have similar pressure-dependent behavior based on comparable solvation structures. However, pressure-induced changes in the association constant for these two analogs are quite distinct, with differences in K(c) ranging from clearly pressure dependent (-14%) to pressure independent over 338 bar. Additional solvation perturbations are observed in the pressure dependence of the quantum efficiency for both complexes (-7.3% and -9.4%). Thus, pressure-induced perturbation in the fluorescence properties of the complex need not be accompanied by simultaneous changes in the complexation equilibrium. Finally, these pressure-induced changes in complexation selectivity are important for all measurements conducted under variable pressure conditions, including liquid chromatography and process monitoring.     

Effect of inflammation on the proliferation of human gingival epithelial cells in vitro

The adaptive or pathologic responses of epithelial cells to inflammation are poorly characterized. The purpose of this study was to determine if epithelial cells cultured from clinically healthy and inflamed human gingival tissues express differences in proliferation rate and viability. Briefly, the inflammation status of individual donor sites from 101 patients was visually assessed at the time of periodontal surgery and categorized as either non-to-slightly inflamed, moderately inflamed, or severely inflamed. Discarded gingival tissues were then processed to obtain primary cell cultures, for which proliferation rates were determined by calculating the ratio of mean population doublings to the number of days required for cultures to become confluent. In general, the cells in the minimally inflamed group exhibited characteristics different than cells in the moderately and severely inflamed groups. Specifically, the cells obtained from clinical sites which exhibited no-to-slight inflammation had a significantly higher mean proliferation rate than cells in either the moderate inflammation group or the severe inflammation group. Based on trypan blue exclusion, the cells obtained from clinical sites which exhibited no-to-slight inflammation also were more viable than cells obtained from sites with moderate inflammation or severe inflammation. Microscopic evaluation showed morphological changes associated with increased inflammation. Cell cycle analysis by fluorescent-activated cell sorting (FACS) revealed a directly proportional relationship between the degree of inflammation and apoptosis, and a strong inversely proportional trend between the degree of inflammation and the numbers of cells undergoing mitosis. Taken together, these data suggest that epithelial cell proliferation and viability are inversely associated with the degree of gingival inflammation, once a putative "adaptive threshold" is exceeded. Elucidation of the underlying mechanisms will likely lead to improvements in clinical diagnosis and treatment.     

The geriatric gravida: multifetal pregnancy reduction, donor eggs, and aggressive infertility treatments

OBJECTIVE: Recent technologic advances and societal acceptance have dramatically increased the use of donor eggs for infertile couples who require assisted reproductive technologies. Now many "older" couples can access assisted reproductive technologies to achieve pregnancies. We sought to evaluate the changing pattern of patients referred for multifetal pregnancy reduction as a result of donor eggs and age factors in aggressive infertility treatment. STUDY DESIGN: Patients undergoing multifetal pregnancy reduction from 1986 to 1996 were included and categorized by year groupings, age, and the use of donor eggs. RESULTS: A total of 523 patients were referred for and underwent multifetal pregnancy reduction. Before 1994, only 4 of 226 (1.8%) had received donor eggs, whereas in 1994 to 1996, 29 of 297 (9.8%) had received donor eggs (chi 2 = 12.6, p < 0.001). Eight of 9 patients aged > or = 45 years undergoing multifetal pregnancy reduction received donor eggs. There were no patients aged > or = 45 years before 1994 but 9 in 1994 to 1996. Four of 9 patients aged > or = 45 years with multifetal pregnancies chose reduction to singleton gestation. The proportions of patients aged > or = 40 years have increased from 0% to 11% in the last 8 years. CONCLUSIONS: The availability of donor eggs has dramatically increased the use of assisted reproductive technologies and subsequent use of multifetal pregnancy reduction in older patients. Older patients are more inclined to want reduction to singleton gestation; they cite parental demands, financial issues, and their ability to parent in their 60s and 70s as reasons for reduction to singleton gestation.     

Permeable packings and perfusion chromatography in protein separation

The use of permeable packings in perfusion chromatography for protein separation is reviewed. Mass transport mechanisms in large-pore materials include forced convection in addition to diffusive transport. The key concept in perfusion chromatography is the "augmented" diffusivity by convection which explains the improved efficiency of perfusive packings compared with conventional supports. An extended Van Deemter equation has to be applied when calculating the height equivalent to a theoretical plate (HETP) of chromatographic columns with flow-through particles. It is shown that the effect of forced convective flow in pores is to drive the separation performance between diffusion-controlled and equilibrium limits. A methodology to understand mass transfer mechanisms in permeable packings is proposed. Experimental results for protein separation by high-performance liquid chromatography in new packing media are discussed. Simulated moving bed technology is addressed.     

Initial genetic characterization of the 1918 "Spanish" influenza virus

The "Spanish" influenza pandemic killed at least 20 million people in 1918-1919, making it the worst infectious pandemic in history. Understanding the origins of the 1918 virus and the basis for its exceptional virulence may aid in the prediction of future influenza pandemics. RNA from a victim of the 1918 pandemic was isolated from a formalin-fixed, paraffin-embedded, lung tissue sample. Nine fragments of viral RNA were sequenced from the coding regions of hemagglutinin, neuraminidase, nucleoprotein, matrix protein 1, and matrix protein 2. The sequences are consistent with a novel H1N1 influenza A virus that belongs to the subgroup of strains that infect humans and swine, not the avian subgroup.     

Sotahexal pharmacodynamics in patients with ischemic heart disease

Therapeutic and prophylactic antiarrhythmic efficacy of sotalol hydrochloride (Sotahexal, "Hexal", Germany) and its effects in intracardiac hemodynamics and ECG parameters were evaluated in 95 patients with ischemic heart disease (IHD). The highest response to the drug was observed in ventricular extrasystoles, arterial flutter and fibrillation. Acute episodes of arrhythmia are managed by bolus administration of Sotahexal [correction of Hexal]. In this case greater risk of side effects exists. It is desirable to decide on the drug dose, mode of administration on the individual basis with due consideration of the risks and dangers which could be avoided in case of adequate instrumental control.