Identification and characterization of Escherichia coli DNA helicase II mutants that exhibit increased unwinding efficiency

Ganglion cells with intraretinal axon collaterals have been described in monkey (Usai et al., 1991), cat (Dacey, 1985), and turtle (Gardiner & Dacey, 1988) retina. Using intracellular injection of horseradish peroxidase and Neurobiotin in in vitro whole-mount preparations of human retina, we filled over 1000 ganglion cells, 19 of which had intraretinal axon collaterals and wide-field, spiny dendritic trees stratifying in the inner half of the inner plexiform layer. The axons were smooth and thin (approximately 2 microm) and gave off thin (<1 microm), bouton-studded terminal collaterals that extended vertically to terminate in the outer half of the inner plexiform layer. Terminal collaterals were typically 3-300 microm in length, though sometimes as long as 700 microm, and were present in clusters, or as single branched or unbranched varicose processes with round or somewhat flattened lobular terminal boutons 1-2 microm in diameter. Some cells had a single axon whereas other cells had a primary axon that gave rise to 2-4 axon branches. Axons were located either in the optic fiber layer or just beneath it in the ganglion cell layer, or near the border of the ganglion cell layer and the inner plexiform layer. This study shows that in the human retina, intraretinal axon collaterals are associated with a morphologically distinct ganglion cell type. The synaptic connections and functional role of these cells are not yet known. Since distinct ganglion cell types with intraretinal axon collaterals have also been found in monkey, cat, and turtle, this cell type may be common to all vertebrate retinas.     

Combinatorial chemistry in insects: a library of defensive macrocyclic polyamines

The pupal defensive secretion of the coccinellid beetle Epilachna borealis is composed principally of a combinatorial library of macrocyclic polyamines. These compounds constitute a previously unrecognized family of natural products, characterized by extremely large-ring lactonic structures derived from a small set of (2-hydroxyethylamino)alkanoic acids. The combinatorial assembly of these simple building blocks generates a high degree of structural diversity, which is further increased by slow, spontaneous intramolecular rearrangement of the macrocycles.     

Nitric oxide synthase activities in human myometrium and villous trophoblast throughout pregnancy

OBJECTIVE: To study the changes in nitric oxide synthase activities in human myometrium and trophoblast throughout pregnancy and around delivery. METHODS: Samples of villous trophoblast were collected from women undergoing elective cesarean delivery at term (n = 12) or voluntary termination of pregnancy in the first (n = 27) or second (n = 11) trimesters of pregnancy. Myometrial samples were obtained from nonpregnant women undergoing hysterectomy (n = 5) and pregnant women both before (n = 7) and after (n = 7) the onset of spontaneous labor at term. Nitric oxide synthase activity was quantified for homogenized samples using the L-citrulline assay in the presence and absence of calcium. RESULTS: The highest levels of nitric oxide synthase activity were found in first-trimester villi (range 2-29 nmol L-citrulline/minute/g protein), with a significant fall in activity in the third trimester (range 2-10 nmol L-citrulline/minute/g protein; P < .001 for both calcium-dependent and calcium-independent activity). Myometrial activities were relatively low compared with those in the trophoblast (0-2 nmol L-citrulline/minute/g protein), with no significant differences in calcium-dependent activities between subgroups. Myometrial calcium-independent activities were lower in pregnant than in nonpregnant women (P = .007), with those in labor having levels higher than those not in labor (P = .048). CONCLUSION: Levels of nitric oxide synthase activity are relatively high in villous trophoblast, particularly during the first trimester. Although the contribution to total nitric oxide production in the uterus by myometrial nitric oxide synthase appears to be relatively small, nitric oxide produced by the trophoblast may play a role in maintaining uterine quiescence by a paracrine effect. Further work is needed to test this hypothesis and explore other possible roles for trophoblast-derived nitric oxide in early pregnancy.     

Systemic inflammatory response syndrome treatment by powdered sorbent pheresis: the BioLogic-Detoxification Plasma Filtration System

Systemic inflammatory response syndrome (SIRS) is one of the most common causes of death in intensive care unit patients. The detoxification plasma filtration (DTPF) system (HemoCleanse, Inc., West Lafayette, IN) combines the DT hemodiabsorption system in series with a push-pull pheresis PF system (a suspension of powdered sorbents surrounding 0.5 microm plasma filter membranes). Bidirectional plasma flow (at 80-100 ml/min) across the PF membranes provides direct contact between plasma proteins and powdered sorbents, as well as clearance of cytokines (tumor necrosis factor-alpha, interleukin-1beta, and interleukin-6) at a rate of 15-25 ml/min, without evidence of saturation for 90 minutes. In a U.S. Food and Drug Administration approved study we treated eight patients with SIRS and organ failure with a single DTPF treatment, using powdered charcoal as sorbent in four patients and powdered charcoal and silica in four patients. Treatments proceeded for 6 hours with proper heparin anticoagulation (activated clotting time 250-300 sec) and appeared safe. All patients improved during the treatments and each had increased blood pressure and decreased need for pressor agents. Plasma cytokine levels stabilized or decreased during treatment and were significantly lower the morning after treatment. Multiple organ dysfunction (MOD) and Acute Physiology Chronic Health Evaluation II scores and organ function gradually improved in most patients, and two patients survived for more than 28 days and two for more than 14 days. The DTPF System may prove beneficial in treatment of patients with sepsis.     

A number-needed-to-treat analysis of the use of respiratory syncytial virus immune globulin to prevent hospitalization

OBJECTIVES: To estimate how many infants in selected high-risk subgroups would require treatment with respiratory syncytial virus immune globulin (RSV-IG) to avoid 1 hospital admission and to determine whether this is economically justified. DESIGN: Cost-benefit analysis. Data from 3 randomized controlled trials of RSV-IG are used to estimate the number needed to treat to prevent 1 hospital admission for respiratory syncytial virus infection. The threshold number needed to treat is computed according to a formula incorporating costs and benefits of RSV-IG prophylaxis. Estimates of the willingness to pay were obtained from a sample of 39 health care providers (35 physicians and 4 nurses). MAIN OUTCOME MEASURES: The number needed to treat to prevent 1 hospital admission for respiratory syncytial virus infection. The threshold number needed to treat that would balance costs with benefits. RESULTS: More than 16 (95% confidence interval, 12.5-23.8) infants would need to be treated with RSV-IG to avoid 1 hospital admission for respiratory syncytial virus infection, ranging from 63 for premature infants without chronic lung disease to 12 (confidence interval, 6.3-100.0) for infants with bronchopulmonary dysplasia. A sensitivity analysis of the costs and values of hospital admission for respiratory syncytial virus infection and RSV-IG treatment resulted in a weak recommendation against the treatment of infants with bronchopulmonary dysplasia and strong recommendations that the costs and risks of RSV-IG treatment outweigh the benefits for the combined sample of infants and premature infants without lung disease. CONCLUSIONS: The number-needed-to-treat procedures offer a method to assess evidence of treatment effects and decision rules for whether to accept treatment recommendations. Under plausible assumptions, treatment with RSV-IG is not recommended for infants without lung disease. Institutions can examine cost and benefit assumptions that best fit their own practice setting.     

Sodium, potassium-activated adenosine triphosphatase activity is impaired in the guinea pig pancreatic duct system in streptozotocin-induced diabetes

In patients with type I diabetes mellitus, clinical studies have demonstrated decreased secretion of pancreatic juice by the pancreatic excretory duct system. The cause of this decrease is unknown, but could involve changes in initial signal transduction pathways or one or more of the electrolyte transport components that subserve regulated fluid secretion. We have compared responsiveness to secretin in pancreatic ducts isolated from healthy and diabetic Hartley guinea pigs and also have compared the expression of CFTR and Na+, K(+)-ATPase in these two groups, as the activities of these two proteins are essential for secretion of pancreatic juice. The increases in cyclic AMP levels evoked by exposure to either 0.1 nM or 0.1 microM secretin were not significantly different in pancreatic ducts isolated from healthy and diabetic guinea pigs nor were levels of CFTR or Na+, K(+)-ATPase expression. By contrast, Na+, K(+)-ATPase activity in pancreatic ducts isolated from diabetic guinea pigs was decreased by 70%, suggesting a change in the enzyme's catalytic properties in the diabetic tissues. The observed decrease would be expected to seriously compromise the production of pancreatic juice.     

Binding of 9-anthroylcholine monitors the interactions of adenosine cyclic 3',5'-phosphate-dependent protein kinase with MgATP, substrates, and regulatory subunits

The isolated catalytic subunit of cAMP-dependent protein kinase and smooth muscle myosin light chain kinase undergo interactions with the fluorescent dye 9-anthroylcholine (9AC) that are responsive to the two enzymes' associations with substrates and effectors. Additionally, the binding of 9AC is highly sensitive to subtle structural or functional differences among closely related protein kinases. Skeletal muscle myosin light chain kinase and the catalytically active chymotryptic fragment of the gamma-subunit of phosphorylase kinase do not associate with 9AC. The 1:1 fluorescent complex of the isolated catalytic subunit of cAMP-dependent protein kinase with 9AC exhibits a dissociation constant of 21 microM. The association of the catalytic subunit with either of the regulatory subunits, RI and RII, results in decreases in the observed 9AC fluorescence that are reversed upon the addition of cAMP. The effects of MgATP and of polypeptide substrates (Kemptide, troponin I, protamine) on the 9AC-catalytic subunit complex are consistent with a general noncompetitive model in which the interactions of 9AC and the other ligands with the enzyme are mutually antagonistic but not purely competitive.     

Aurally and visually guided visual search in a virtual environment

BACKGROUND: Atopic dermatitis (AD) is characterized by skin infiltrates of leukocytes, such as lymphocytes and eosinophils. OBJECTIVE: To describe the mechanisms determining this inflammatory process, we have analyzed expression of adhesion molecules and their regulation on skin endothelial cells (ECs). METHODS: Expression of adhesion molecules on ECs was analyzed by immunohistochemistry by using Ulex europaeus agglutin 1 as a pan-endothelial marker. RESULTS: Vascular cell adhesion molecule-1 (VCAM-1), E-selectin, and P-selectin were not found in skin of nonatopic individuals, whereas expression of these surface molecules was observed in nonlesional skin of patients with AD and was even more pronounced in lesional skin or after epicutaneous application of aeroallergen. Induction of adhesion molecule expression was examined on both macrovascular ECs from human umbilical cord vein (HUVECs) and human microvascular ECs (HMEC-1) from skin. TNF-alpha very potently upregulated adhesion molecule expression in vitro on both EC cell types. To verify the in vivo relevance of TNF-alpha, we performed TNF-alpha staining in the skin. TNF-alpha was observed in the dermis of nonatopic skin, both in chymase-containing mast cells and CD68+ macrophages. The increase in the number of TNF-alpha-containing cells was concomitant with the increase in adhesion molecule expression in the skin of patients with AD. IL-4 is supposed to be important in atopic diseases because of its IgE- and VCAM-1-inducing properties. However, IL-4 addition failed to induce VCAM-1 expression on HMEC-1, although in the same set of experiments, a clear induction of VCAM-1 expression by IL-4 on HUVECs was demonstrated. Flow cytometry revealed the absence of 11-4 receptor alpha-chains on HMEC-1 and their presence on HUVECs. Immunohistochemistry examination on skin sections showed no binding of the IL-4R alpha-chain antibodies to ECs. CONCLUSION: We conclude that adhesion molecule expression is increased in the skin of patients with AD. Most probably, this increased expression is not a (direct) effect of IL-4 on skin endothelium, but other cytokines, such as TNF-alpha, might be responsible for this increased adhesion molecule expression. Continuous adhesion molecule expression may facilitate T-cell extravasation in a nonantigen-specific manner, thus explaining the presence of increased T-cell numbers in nonlesional skin of patients with AD.