Biomedical subjects
Publications and source records attributed to J M Weber.
We probe the repulsive Coulomb barrier of the doubly charged anion PtBr(4) (2-) by photodetachment spectroscopy. The results are discussed in terms of models for the photoemission process, the excitation spectrum of PtBr(4) (2-), and calculations of the energy-dependent tunneling probability for various model potentials.
The (CO2)n- clusters are thought to accommodate the excess electron by forming a localized molecular anion, or "core ion", solvated by the remaining, largely neutral CO2 molecules. Earlier studies interpreted discontinuities in the (CO2)n- photoelectron spectra to indicate that both the CO2- and C2O4- species were present in a size-dependent fashion. Here we use vibrational predissociation spectroscopy to unambiguously establish the molecular structures of the core ions in the 2 < or = n < or = 17 size range. Spectra are reported in the 2300-3800 cm(-1) region, which allows us to independently monitor the contribution of each ion through its characteristic overtone and combination bands. These signature bands are observed to be essentially intact in the larger clusters, establishing that the CO2- and C2O4- molecular ions are indeed the only electron accommodation modes at play. The size dependence of the core ion suggested in earlier analyses of the photoelectron spectra is largely confirmed, although both species are present over a range of clusters near the expected critical cluster sizes, as opposed to the prompt changes inferred earlier. Perturbations in the bands associated with the nominally neutral CO2 "solvent" molecules are correlated with the changes in the molecular structure of the core ion. These observations are discussed in the context of a diabatic model for electron delocalization over the CO2 dimer. In this picture, the driving force leading to the transient formation of the monomer ion is traced to the solvent asymmetry inherent in an incomplete coordination shell.
We examined 267 red foxes (Vulpes vulpes) from the canton of Geneva, Switzerland, for intestinal infections with Echinococcus multilocularis. This region is situated in the core area of the endemic range of this zoonotic cestode in Central Europe. Several factors were taken into account and urbanisation level appeared to be the most explicative to describe observed differences. The prevalence decreased significantly from rural and residential areas (prevalence of 52%, CI 43-62%, and 49%, CI 38-59 %, respectively) to the urban area (prevalence of 31%, CI 19-42%). A few juvenile foxes harboured very high burdens up to more than 120,000 worms and were significantly more heavily infected than adults. The intensity of infection decreased from rural and residential areas to the city, suggesting a lower contamination of the urban environment.
Parathyroid hormone (PTH)-related protein (PTH-rP) is an important autocrine/paracrine attenuator of programmed cell differentiation whose expression is restricted to the epithelial layer in tooth development. The PTH/PTHrP receptor (PPR) mRNA in contrast is detected in the dental papilla, suggesting that PTHrP and the PPR may modulate epithelial-mesenchymal interactions. To explore the possible interactions, we studied the previously described transgenic mice in which a constitutively active PPR is targeted to osteoblastic cells. These transgenic mice have a vivid postnatal bone and tooth phenotype, with normal tooth eruption but abnormal, widened crowns. Transgene mRNA expression was first detected at birth in the dental papilla and, at 1 week postnatally, in odontoblasts. There was no transgene expression in ameloblasts or in other epithelial structures. Prenatally, transgenic molars and incisors revealed no remarkable change. By the age of 1 week, the dental papilla was widened, with disorganization of the odontoblastic layer and decreased dentin matrix. In addition, the number of cusps was abnormally increased, the ameloblastic layer disorganized, and enamel matrix decreased. Odontoblastic and, surprisingly, ameloblastic cytodifferentiation was impaired, as shown by in situ hybridization and electron microscopy. Interestingly, ameloblastic expression of Sonic Hedgehog, a major determinant of ameloblastic cytodifferentiation, was dramatically altered in the transgenic molars. These data suggest that odontoblastic activation of the PPR may play an important role in terminal odontoblastic and, indirectly, ameloblastic cytodifferentiation, and describe a useful model to study how this novel action of the PPR may modulate mesenchymal/epithelial interactions at later stages of tooth morphogenesis and development.
Stem cell fate is influenced by specialized microenvironments that remain poorly defined in mammals. To explore the possibility that haematopoietic stem cells derive regulatory information from bone, accounting for the localization of haematopoiesis in bone marrow, we assessed mice that were genetically altered to produce osteoblast-specific, activated PTH/PTHrP receptors (PPRs). Here we show that PPR-stimulated osteoblastic cells that are increased in number produce high levels of the Notch ligand jagged 1 and support an increase in the number of haematopoietic stem cells with evidence of Notch1 activation in vivo. Furthermore, ligand-dependent activation of PPR with parathyroid hormone (PTH) increased the number of osteoblasts in stromal cultures, and augmented ex vivo primitive haematopoietic cell growth that was abrogated by gamma-secretase inhibition of Notch activation. An increase in the number of stem cells was observed in wild-type animals after PTH injection, and survival after bone marrow transplantation was markedly improved. Therefore, osteoblastic cells are a regulatory component of the haematopoietic stem cell niche in vivo that influences stem cell function through Notch activation. Niche constituent cells or signalling pathways provide pharmacological targets with therapeutic potential for stem-cell-based therapies.
BACKGROUND: Carboxymethylcellulose is a carbohydrate widely used as additive in tablets, cosmetics, some injectable hormone formulations, food (as E466) and as active principle in hydrocolloid dressings. Anaphylaxis to carboxymethylcellulose in parenteral corticosteroid preparations has previously been reported. Typically, skin tests were positive in such cases, occasionally specific IgE or histamine release have been demonstrated. CASE REPORT: We report on 3 patients who suffered from anaphylactic symptoms after local injection of corticosteroid preparations. Intracutaneous skin tests with carboxymethylcellulose were positive; in 2, sulfidoleukotriene release could be measured in the cellular antigen stimulation test (CAST). Specific IgE could not be identified. Oral provocation tests with typical doses of carboxymethylcellulose as found in food and tablets were negative. CONCLUSION: In patients with anaphylaxis to parenteral administration of carboxymethylcellulose, small amounts are tolerated by the oral route. Skin tests and CAST are useful diagnostic tools.
Subsequent to the discovery of 4-[(2,4-dichlorophenyl)amino]-6,7-dimethoxy-3-quinolinecarbonitrile (1a) as an inhibitor of Src kinase activity (IC(50) = 30 nM), several additional analogues were prepared. Optimization of the C-4 anilino group of 1a led to 1c, which contains a 2,4-dichloro-5-methoxy-substituted aniline. Replacement of the methoxy group at C-7 of 1c with a 3-(morpholin-4-yl)propoxy group provided 2c, resulting in increased inhibition of both Src kinase activity and Src-mediated cell proliferation. Analogues of 2c with other trisubstituted anilines at C-4 were also potent Src inhibitors, and the propoxy group of 2c was preferred over ethoxy, butoxy, or pentoxy. Replacement of the morpholine group of 2c with a 4-methylpiperazine group provided 31a, which had an IC(50) of 1.2 nM in the Src enzymatic assay, an IC(50) of 100 nM for the inhibition of Src-dependent cell proliferation and was selective for Src over non-Src family kinases. Compound 31a, which had higher 1 and 4 h plasma levels than 2c, effectively inhibited tumor growth in xenograft models.
Screening of a directed compound library in a yeast-based assay identified 4-[(2,4-dichlorophenyl)amino]-6,7-dimethoxy-3-quinolinecarbonitrile (2a) as a Src inhibitor. An enzymatic assay established that 2a was an ATP-competitive inhibitor of the kinase activity of Src. We present here SAR data for 2a which shows that the aniline group at C-4, the carbonitrile group at C-3, and the alkoxy groups at C-6 and C-7 of the quinoline are crucial for optimal activity. Increasing the size of the C-2 substituent of the aniline at C-4 of 2a from chloro to bromo to iodo resulted in a corresponding increase in Src inhibition. Furthermore, replacement of the 7-methoxy group of 2a with various 3-heteroalkylaminopropoxy groups provided increased inhibition of both Src enzymatic and cellular activity. Compound 25, which contains a 3-morpholinopropoxy group, had an IC(50) of 3.8 nM in the Src enzymatic assay and an IC(50) of 940 nM for the inhibition of Src-dependent cell proliferation.
Over 100 serotypes of adenoviruses have been implicated in a variety of human and domesticated animal pathologies and some serotypes are widely used as gene transfer vectors. Aside from the limited use of vaccines for specific serotypes, little effort has been expended in the development of antivirals. The objective here was to study the effect of cystatins from human saliva (CS) and tears (CT), two points of viral entry, on adenain, the adenovirus type 2 encoded proteinase, which is absolutely required for infectivity. Two molecular weight species (13 and 14.5 kDa) were purified from both fluids at a yield of 5 mg/l. In vitro adenain activity was inhibited to 50% at a molar ratio of 5 CS:1 adenain and 3 CT:1 adenain. By comparison, papain was inhibited to 50% at a molar ratio of 2 CS:1 papain and 1.5 CT:1 papain. Adenain differed from papain in response to CS and chicken egg white (CEW) cystatin in being stimulated at low concentrations, and in being inhibited only at very high concentrations of cystatins. The presence of cleavage consensus sites specific to adenain in the human cystatins could drive the adenain-cystatin interaction predominantly in the substrate pathway direction. However, we found that the cystatins could only be digested after denaturation and by highly active fresh enzyme preparations. Our experiments designed to test the nature of the interaction between adenain and cystatins suggest a docking model for the adenain-human cystatin interaction, similar to that proposed for papain and CEW. At equilibrium the dissociation constant, K(d), between adenain and CT was 1.2 nM. The kinetic parameters determined here suggest a simple reversible mechanism for the inhibition of adenain by human cystatins. We conclude that the cystatins present in tears and saliva are unlikely to play a significant role in inhibiting adenovirus infections.
BACKGROUND: Historically, group A beta-hemolytic streptococci (GAS) burn wound infection has been a major source of morbidity and mortality in burn patients and has prompted the prophylactic administration of antibiotics to children with burns. Wound monitoring, surveillance cultures, and early excision of deep wounds may have changed this. Our objective in this project was to determine the efficacy of routine antibiotic prophylaxis in the era of early excision and closure of deep burn wounds. METHODS: Two cohorts of burned children were compared: all children admitted during calendar years 1992 through 1994 (group 1) and during calendar years 1995 through 1997 (group 2). All group 1 children received routine GAS antibiotic prophylaxis. Only those group 2 children with documented positive admission or surveillance cultures for GAS were treated. RESULTS: There were 511 children in group 1 and 406 children in group 2. They were well matched for age (4.7 +/- 0.21 years vs. 5.3 +/- 0.26 years, p = 0.06) and burn size (11.0% +/- 0.7% vs. 12.4% +/- 0.8%, p = 0.18). GAS species were recovered at admission or during hospitalization from 11 (2.6%) of group 1 children and 18 (4.4%) of group 2 children (p = 0.05), indicating a marginally higher rate of carriage in group 2. Nevertheless, in group 1 there were three (0.6%) who developed GAS wound infection and in group 2 there were four (0.98%, p = 0.71). The incidence of GAS infection in those patients with positive admission cultures was three (27%) of group 1 and four (22%) of group 2. No child developed fulminant GAS infection. CONCLUSION: Routine antibiotic prophylaxis of burn wounds in children in not effective in further reducing a low baseline incidence of GAS wound infection if admission screening by culture is used to identify those children who carry the organism and early excision of deep burns is practiced.
High-altitude acclimation alters lipid metabolism during exercise, but it is unknown whether this involves changes in rates of lipolysis or reesterification, which form the triacylglycerol/fatty acid (TAG/FA) cycle. We combined indirect calorimetry with [2-(3)H]glycerol and [1-(14)C]palmitate infusions to simultaneously measure total lipid oxidation, lipolysis, and rate of appearance (R(a)) of nonesterified fatty acids (NEFA) in high-altitude-acclimated (HA) rats exercising at 60% maximal O(2) uptake (VO(2 max)). During exercise, relative total lipid oxidation (%VO(2)) equaled sea-level control (SL) values; however, acclimation greatly stimulated lipolysis (+75%) but had no effect on R(a) NEFA. As a result, TAG/FA cycling increased (+119%), due solely to an increase in recycling (+144%) within adipocytes. There was no change in either group in these variables with the transition from rest to exercise. We conclude that, in HA, 1) acclimation is a potent stimulator of lipolysis; 2) rats do not modify TAG/FA cycling with the transition to exercise; and 3) in normoxia, HA and SL derive the same fraction of their total energy from lipids and carbohydrates.
Src is a nonreceptor tyrosine kinase involved in signaling pathways that control proliferation, migration, and angiogenesis. Increased Src expression and activity are associated with an increase in tumor malignancy and poor prognosis. Several quinolines and quinazolines were identified as potent and selective inhibitors of Src kinase activity.
A new kind of versatile adenoviral vector (AdV) has been constructed, one that is completely replication disabled in the absence of Ad-E1 proteins but is capable of a single round of replication when Ad-E1 is present. This was made possible by deletion of the Ad protease gene (PS), which is essential for many steps of the Ad life cycle. The PS-deleted virus can be propagated in 293-derived cell lines engineered to express PS. In these new complementing cells, the PS gene was expressed from a tetracycline-inducible promoter in a dicistronic vector coexpressing the green fluorescent protein (GFP). When induced, the best 293-PS stable clones produced the PS in amounts greater than the level reached after Ad infection. Biological activity was first demonstrated by the ability of 293-PS cells to support the replication of Ad2ts1, a mutant expressing a functionally defective PS. While overexpression of the Ad PS slightly affected cell growth, moderate expression at levels sufficient to fully complement Ad2ts1 was well tolerated in 293 cells. Two PS-deleted mutants, deleted or not deleted for E1/E3, were then generated and characterized. Despite their complete loss of infectivity after a single round of replication in permissive cells, the PS-deleted mutants produced as much viral protein as wildtype Ad. These new vectors should thus be both safer and more efficient for applications in which enhancement of transgene expression is desirable, as in the case of vaccination, in situ therapy for tumors, protein production, or the large-scale production of other viral vectors such as adeno-associated virus (AAV).
Adenoviruses encode a cysteine protease, adenain, required for uncoating and virion maturation. Adenain activity is regulated by an 11-amino-acid peptide cofactor thiol-bonded distal to the active site. Structural and experimental data suggest that the peptide might stabilize adenain in an optimal conformation for enzyme activity by bridging two noncontiguous regions of the molecule. The sequence requirements for this mechanism were examined both in vitro and ex vivo by means of mutant peptides and databank analysis. The results of in vitro experiments suggested that activation is not an all or nothing mechanism. With the exception of the smallest peptide, the mutant peptides bound to adenain, activated it, and competed with the wild-type peptide, but all of this occurred with reduced efficiency. When added to the medium of infected cells, most of the peptides inhibited infectious virus production to varying degrees in a dose-dependent manner and in accordance with their in vitro activity on adenain. We interpret this inhibition to be due to unscheduled adenain activation. Examination of the activation peptide sequences from 19 adenovirus serotypes revealed a limited number of conserved sequence features. These features were in agreement with the experimental data. We conclude that binding and activation of adenain by pVIc may be reversible and this reversibility may be an integral aspect of the in vivo regulation of enzyme activity in the course of virus assembly. The peptide cofactor binding domain is therefore a potential target for the development of anti-adenoviral agents.
The serpins SCCA1 and SCCA2 are highly expressed in the epithelium of the conducting airways, a common site of infection by group C adenoviruses, such as human adenovirus type 2 (Ad2). Based on the common location we examined a possible interaction between them. In vitro experiments with recombinant proteins showed that SCCA1 inhibited the viral protease in a dose-dependent manner. Both serpins were cleaved in a manner consistent with hydrolysis within their reactive site loop, without the formation of an SDS-resistant complex, as in the case of papain. Infection of SCCA1-expressing cells did not result in the cleavage of SCCA1, nor was the yield of infectious virus affected as compared to SCCA1-negative parental cells. This may be due to differential localization, the serpin being cytoplasmic and viral protease being nuclear. Surprisingly, however, virus infection, which tends to inhibit host protein synthesis, caused a significant increase in SCCA1 expression well into the late phase of infection.
Many people harbor herpes simplex virus, often with a known history of "cold sores". During the relatively immunosuppressed state associated with a serious burn, recrudescence of such infections can occur. We report four adults and two children who developed severe herpetic ulceration, over the face and neck in five patients and in a partial thickness wound in one patient. Herpetic infection was diagnosed by culture and direct immunofluorescence testing and treatment was immediately instituted with systemic and topical Acylovir(R) (Zovirax, Glaxo Wellcome). Ulceration healed under treatment and did not leave visible scarring in any of the patients. Although these infections are rapidly progressive, they respond to prompt treatment with antiviral chemotherapy. Rapidly progressive vesicles and ulceration appearing on the face or in the wounds of burn patients should prompt immediate evaluation for herpetic infection.
A specially designed apparatus that can simulate the waveform of the dawn or dusk signal at any latitude and any day of the year has been shown to phase shift the circadian pacemaker in rodents and primates at a fraction of the illuminance previously used. Until recently, it was considered that rather high illuminances or rather long exposure episodes to room light were necessary to phase shift human circadian rhythms. This experiment shows that, under controlled conditions of a modified constant routine protocol, a single dawn signal is sufficient to phase advance the timing of the onset of secretion of the pineal hormone melatonin. The significant phase advance of salivary melatonin of 20 minutes, which is enhanced to 34 minutes after three consecutive dawn signals, is small, but appears to be of sufficient magnitude to entrain the human circadian pacemaker, which has an endogenous period of about 24.2h.
The rate of hepatic glucose production (R(a) glucose) of rainbow trout (Oncorhynchus mykiss) was measured in vivo by continuous infusion of [6-(3)H]glucose and in vitro on isolated hepatocytes to examine the role of epinephrine (Epi) in its regulation. By elevating Epi concentration and/or blocking beta-adrenoreceptors with propranolol (Prop), our goals were to investigate the mechanism for Epi-induced hyperglycemia to determine the possible role played by basal Epi concentration in maintaining resting R(a) glucose and to assess indirect effects of Epi in the intact animal. In vivo infusion of Epi caused hyperglycemia (3.75 +/- 0.16 to 8.75 +/- 0.54 mM) and a twofold increase in R(a) glucose (6.57 +/- 0.79 to 13.30 +/- 1.78 micromol. kg(-1). min(-1), n = 7), whereas Prop infusion decreased R(a) from 7.65 +/- 0.92 to 4.10 +/- 0.56 micromol. kg(-1). min(-1) (n = 10). Isolated hepatocytes increased glucose production when treated with Epi, and this response was abolished in the presence of Prop. We conclude that Epi-induced trout hyperglycemia is entirely caused by an increase in R(a) glucose, because the decrease in the rate of glucose disappearance normally seen in mammals does not occur in trout. Basal circulating levels of Epi are involved in maintaining resting R(a) glucose. Epi stimulates in vitro glucose production in a dose-dependent manner, and its effects are mainly mediated by beta-adrenoreceptors. Isolated trout hepatocytes produce glucose at one-half the basal rate measured in vivo, even when diet, temperature, and body size are standardized, and basal circulating Epi is responsible for part of this discrepancy. The relative increase in R(a) glucose after Epi stimulation is similar in vivo and in vitro, suggesting that indirect in vivo effects of Epi, such as changes in hepatic blood flow or in other circulating hormones, do not play an important role in the regulation of glucose production in trout.