Crystal structure of muconolactone isomerase at 3.3 A resolution

The crystal structure of muconolactone isomerase from Pseudomonas putida, a unique molecule with ten 96 amino acid subunits and 5-fold, and 2-fold symmetries, has been solved at 3.3 A resolution. The non-crystallographic symmetries were used to refine the initial single isomorphous replacement phases and produce an interpretable 10-fold averaged map. The backbone trace is complete and confirmed by the amino acid sequence fit. Each subunit is composed of a body with two alpha-helices and an antiparallel twisted beta-sheet of four strands, and an extended arm. The helices and the sheet fold to form a two-layered structure with an enclosed hydrophobic core and a partially formed putative active site pocket. The C-terminal arm of another subunit related by a local dyad symmetry extends over the core to complete this pocket. The decameric protein is almost spherical, with the helices forming the external coat. There is a large hydrophilic cavity in the center with open ends along the 5-fold axis. Molecular interactions between subunits are extensive. Each subunit contacts four neighbors and loses nearly 40% of its solvent contact area on oligomerization.     

Treatment of osteoporosis with human parathyroid peptide and observations on effect of sodium fluoride.

OBJECTIVE--To evaluate the need for a randomised study of treatment of spinal osteoporosis with human parathyroid peptide in the secondary prevention of crush fractures; to study the effect of human parathyroid hormone peptide 1-34 plus sex hormones on vertebral body cancellous bone; and, separately, to determine the effect of relatively low doses of sodium fluoride plus calcium on spinal bone mineral density. DESIGN--Open study of patients with primary or postmenopausal osteoporosis. All patients had serial bone densitometry of the spine by quantitative computed tomography and dual photon absorptiometry as well as serial densitometry of the radial midshaft (cortical) and radial distal (trabecular) bone by quantitative computed tomography. Changes in the spinal bone not forming the spongiosa of the vertebral bodies ("cortical" bone) were determined from the difference between the two axial measurements, after correction to the same units of measurement. SETTING--Northwick Park Hospital and Medical Research Council Clinical Research Centre. PATIENTS--24 Patients who fulfilled the conventional criteria for type 1 (vertebral) osteoporosis not secondary to recognised causes other than sex hormone deficiency and with at least one crush or wedge vertebral fracture and a spinal bone density (quantitative computed tomography) less than 80 mg/cm3 or two or more fractures. Twelve patients received human parathyroid peptide and 12 sodium fluoride; they were not randomised. MAIN OUTCOME MEASURES--Trends in axial and peripheral bone mass values determined by linear, time dependent regression analyses. RESULTS--The patients receiving the peptide showed a substantial increase in vertebral spongiosa (mean 25.6 mg/cm2 two years after the start of treatment). No significant changes were seen in spinal cortical or radial bone density. The patients receiving sodium fluoride showed roughly equal increases in cancellous and cortical bone over the same period (mean increase in vertebral spongiosa 16.1 mg/cm3). No significant changes were seen in radial bone. CONCLUSIONS--Treatment of postmenopausal women with human parathyroid peptide selectively increases spinal cancellous bone density by amounts that may prove useful in secondary prevention. Peptide treatment should now be tested in a randomised study in which the important end point is prevention of fractures as the usefulness of sodium fluoride in this context is doubtful.     

Hyperleptinemia and reduced TNF-alpha secretion cause resistance of db/db mice to endotoxin

Leptin deficiency in ob/ob mice increases susceptibility to endotoxic shock, whereas leptin pretreatment protects them against LPS-induced lethality. Lack of the long-form leptin receptor (Ob-Rb) in db/db mice causes resistance. We tested the effects of LPS in C57BL/6J db(3J)/db(3J) (BL/3J) mice, which express only the circulating leptin receptors, compared with C57BL/6J db/db (BL/6J) mice, which express all short-form and circulating isoforms of the leptin receptor. Intraperitoneal injections of LPS significantly decreased rectal temperature and increased leptin, corticosterone, and free TNF-alpha in fed and fasted BL/3J and BL/6J mice. TNF-alpha was increased three- and fourfold in BL/3J and BL/6J, respectively. LPS (100 microg) caused 50% mortality of fasted BL/6J mice but caused no mortality in fasted BL/3J mice. Pretreatment of fasted BL/3J mice with 30 microg leptin prevented the drop in rectal temperature, blunted the increase in corticosterone, but had no effect on TNF-alpha induced by 100 microg LPS. Taken together, these data provide evidence that fasted BL/3J mice are more resistant than BL/6J mice to LPS toxicity, presumably due to the absence of leptin receptors in BL/3J mice. This resistance may be due to high levels of free leptin cross-reacting with other cytokine receptors.     

Cooperative interactions between the contractile proteins of cardiac and skeletal muscle.

The calcium activation of the ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity of cardiac actomyosin reconstituted from bovine cardiac myosin and a complex of actin-tropomyosin-troponin extracted from bovine cardiac muscle at 37 degrees C was studied and compared with similar proteins from rabbit fast skeletal muscle. The proteins of the actin complex were identified by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Half-maximal activation of the cardiac actomyosin was seen at a calcium concentration of 1.2 +/- 0.002 (S.E. of mean) muM. A hybridized reconstituted actomyosin made with cardiac myosin and the actin-tropomyosin-troponin complex extracted from rabbit skeletal muscle was also activated by calcium but the half-maximal value was shifted to 0.65 +/- 0.02 (S.E. of mean) muM Ca2+. Homologous rabbit skeletal actomyosin showed half-maximal activation at 0.90 +/- 0.01 (S.E. of mean) muM Ca2+ and the value for a hybridized actomyosin made with rabbit skeletal myosin and the actin-complex from cardiac muscle was found at 1.4 +/- 0.03 (S.E. of mean) muM Ca2+ concentration. Kinetic analysis of the Ca2+ activated ATPase activity of reconstituted bovine cardiac actomyosin indicated some degree of cooperativity with respect to calcium. Double reciprocal plots of reconstituted actomyosins made with bovine cardiac actin complex were curvilinear and significantly different than those of reconstituted actomyosins made with the rabbit fast skeletal actin complex. The Ca2+-dependent cooperativity was of a mixed type as determined from Hill plots for homologous reconstituted bovine cardiac and rabbit fast skeletal actomyosin. The results show that cooperative interactions in reconstituted actomyosins were greater when the actin-tropomyosin-troponin complex was derived from cardiac than skeletal muscle.     

Axonal shortening and the mechanisms of axonal motility

Axons in tissue culture retract and shorten if their tips are detached from the substrate. The shortening reaction of the axon involves contractile forces that also arise during normal axonal motility, elongation, and retraction. We studied shortening in axonal segments isolated from their parent axons by transecting the axon between the growth cone and the most distal point of adhesion to the substrate. Within 15-20 minutes after transection, an isolated axonal segment shortened and pulled its tail end toward the growth cone. During the shortening process, long sinusoidal bends arose along the axon. The identical shortening reaction occurs without transection, when the axon tip is detached from the substrate. Pharmacological studies with inhibitors of glycolysis indicate that the shortening mechanisms utilize metabolic energy, presumably ATP. The rate of sinusoidal shortening is similar to both the rate of polymer translocation in the axon by slow axonal transport and the rate of normal axonal elongation. Taxol inhibits the shortening reaction with a similar dose dependence to its inhibition of axonal growth. Together, all these observations suggest that the same basic intracellular motility mechanisms are involved in normal axonal growth, in slow axonal transport, and in the shortening reaction: the intracellular dynamic system that utilizes ATP to generate longitudinal movements of polymers within the axon may be the same mechanism underlying both the retraction and the elongation of the axon.     

Materno-embryonally transferred antibodies precipitate autoimmune thyroiditis in obese strain (OS) chickens

In Obese strain (OS) chickens the role of maternal antibodies, passively transferred through the egg to the developing chick, was evaluated as a causative factor in the early development of spontaneous autoimmune thyroiditis (SAT). In the egg, passive antibody titers were highest in the yolk and lower in the allantoic fluid and sera of developing embryos. This passage of antibodies was documented by use of radiolabeled antibodies. In dams with high antibody titers, antibodies could be found in the sera of chicks at the time of hatch. Thyroglobulin was absent in the yolk of OS eggs during embryonal life, as compared with its detection in normal eggs. Immune complexes (thyroglobulin-autoantibody) detected in the thyroids of OS, but not CS, chicks at the time of hatch, or earlier, appear to reflect the presence of the maternally transferred antibodies. A pair of crosses between OS chickens, with thyroiditis, and the C strain (CS), without thyroiditis, was made to evaluate the role of transferred antibodies in the pathogenesis of autoimmune disease. When an OS chicken was the dam, maternal antibodies could be passively transferred; when a CS chicken was the dam, no maternal antibodies were present to be transferred. Nevertheless, both hybrids developed full-blown thyroiditis, demonstrating that binding of transferred maternal antibody to thyroglobulin is not a prerequisite for the induction of SAT. However, presence of maternal antibodies precipitated the onset of disease. Immune complexes formed in the embryonic thyroid are likely to participate in early autoimmune disease, although the development of full-blown thyroiditis may await the competency of the chick's immune system to provide the characteristic cellular infiltrate.     

Characterization of Environmentally Persistent Escherichia coli Isolates Leached from an Irish Soil

Soils are typically considered to be suboptimal environments for enteric organisms, but there is increasing evidence that Escherichia coli populations can become resident in soil under favorable conditions. Previous work reported the growth of autochthonous E. coli in a maritime temperate Luvic Stagnosol soil, and this study aimed to characterize, by molecular and physiological means, the genetic diversity and physiology of environmentally persistent E. coli isolates leached from the soil. Molecular analysis (16S rRNA sequencing, enterobacterial repetitive intergenic consensus PCR, pulsed-field gel electrophoresis, and a multiplex PCR method) established the genetic diversity of the isolates (n = 7), while physiological methods determined the metabolic capability and environmental fitness of the isolates, relative to those of laboratory strains, under the conditions tested. Genotypic analysis indicated that the leached isolates do not form a single genetic grouping but that multiple genotypic groups are capable of surviving and proliferating in this environment. In physiological studies, environmental isolates grew well across a broad range of temperatures and media, in comparison with the growth of laboratory strains. These findings suggest that certain E. coli strains may have the ability to colonize and adapt to soil conditions. The resulting lack of fecal specificity has implications for the use of E. coli as an indicator of fecal pollution in the environment.Escherichia coli is a well-established indicator of fecal contamination in the environment. The organism''s validity as an indicator of water pollution is dependent, among other factors, on its fecal specificity and its inability to multiply outside the primary host, the gastrointestinal tracts of humans and warm-blooded animals (9). While many pathogens and indicator organisms are considered to be poorly adapted for long-term survival, or proliferation, outside their primary hosts (24), there is increasing evidence that this view needs to be reconsidered with respect to E. coli (17, 38). In particular, questions remain about its fate and survival capacity in environmental matrices, such as soil. While the habitat within the primary host is characterized by constant warm temperature conditions and a ready availability of nutrients and carbon, that of soil is often characterized by oligotrophic and highly dynamic conditions, temperature and pH variation, predatory populations, and competition with environmentally adapted indigenous microflora (39). Soils are thus typically considered to be suboptimal environments for enteric organisms, and growth is thought to be negligible, with die-off of organisms at rates reported to be a function of the interaction of numerous factors, including the type and physiological state of the microorganism, the physical, chemical, and biological properties of the soil, atmospheric conditions (including sunlight, moisture, and temperature), and organism application method (10).In recent years, the growth of E. coli in soils, sediments, and water in tropical and subtropical regions has been widely documented, and the organism is considered to be an established part of the soil biota within these regions (4, 5, 7, 12, 14, 19, 25, 32). The integration of E. coli as a component of the indigenous microflora in soils of tropical and subtropical regions may be attributable to the nutrient-rich nature and warm temperatures of these habitats (21, 39), combined with the metabolic versatility of the organism and its simple nutritional requirements (21). In addition to tropical and subtropical regions, the presence of autochthonous E. coli populations in the cooler soils of temperate and northern temperate regions has also been reported (6, 20, 22, 37), with one report on an alpine soil (34) and, most recently, a report on a maritime temperate grassland soil (3). The growth of E. coli within soils can act as a reservoir for the further contamination of bodies of water (20, 31, 32), compromising the indicator status of E. coli within these regions. As such, an understanding of the ecological characteristics of E. coli in soil is critical to its validation as an indicator organism. With respect to the input of pathogenic E. coli into the environment, this knowledge becomes essential for assessing the potential health risk to human and animal hosts from agricultural activities such as landspreading of manures and slurries (24).It has been suggested that E. coli can sustain autochthonous populations within soils in temperate regions, wherever favorable conditions exist (21). The phenotypic traits of the organism (including its metabolic diversity and its ability to grow both aerobically and anaerobically in a broad temperature range) may assist the persistence, colonization, and growth of E. coli when conditions permit. The challenging nature of the soil environment and the disparity of conditions between the primary host and the secondary habitat raises the question of how these E. coli populations survive and compete for niche space among the highly competitive and diverse coexisting populations of the indigenous microflora (15, 21). There is some evidence that naturalized E. coli may form genetically distinct populations in the environment (17, 20, 34, 36). This suggests that autochthonous E. coli populations in soil may have increased environmental fitness, facilitating their residence in soil (20, 34, 38). Little is known, however, of the physiology of these organisms, and their capacity for survival in soil remains poorly understood (21).Previous work (3) recorded continuous low-level leaching of viable E. coli from lysimeters of a poorly drained Luvic Stagnosol soil type, more than 9 years after the last application of fecal material. This finding was indicative of the growth of E. coli within the soil and suggested the presence of autochthonous E. coli populations within the soil that could be leached subsequently. To our knowledge, prior to this report, naturalized autochthonous E. coli populations persisting under the relatively oligotrophic, low-temperature conditions of maritime temperate soil environments had not been described previously. Growth within this soil was attributed chiefly to favorable characteristics of the soil, which include high clay and moisture contents, nutrient retention, and the presence of anaerobic zones. The objective of this work was to characterize, by molecular and physiological means, the genetic diversity and physiology of environmentally persistent E. coli isolates leached. In particular, we were interested in determining if the isolates possessed phenotypic characteristics that may enhance their capacity to survive and occupy niche space within the soil. This study tested the hypothesis that E. coli clones persisting in lysimeters of this soil form a genetically distinct grouping and possess a physiology tailored to the soil environment.