Measuring the Local Velocity along Transition Paths during the Folding of Single Biological Molecules.

Transition paths are the most interesting part of folding reactions but remain little studied. We measured the local velocity along transition paths in DNA hairpin folding using optical tweezers. The velocity distribution agreed well with diffusive theories, yielding the diffusion coefficient. We used the average velocity to calculate the transmission factor in transition-state theory (TST), finding observed rates that were ∼10^{5}-fold slower than predicted by TST. This work quantifies the importance of barrier recrossing events and highlights the effectiveness of the diffusive model of folding.

The mammalian Na+/H+ antiporters NHE-1, NHE-2, and NHE-3 are electroneutral and voltage independent, but can couple to an H+ conductance.

Na+/H+ exchange in vertebrates is thought to be electroneutral and insensitive to the membrane voltage. This basic concept has been challenged by recent reports of antiport-associated currents in the turtle colon epithelium (Post and Dawson, 1992, 1994). To determine the electrogenicity of mammalian antiporters, we used the whole-cell patch clamp technique combined with microfluorimetric measurements of intracellular pH (pHi). In murine macrophages, which were found by RT-PCR to express the NHE-1 isoform of the antiporter, reverse (intracellular Na(+)-driven) Na+/H+ exchange caused a cytosolic acidification and activated an outward current, whereas forward (extracellular Na(+)-driven) exchange produced a cytosolic alkalinization and reduced a basal outward current. The currents mirrored the changes in pHi, were strictly dependent on the presence of a Na+ gradient and were reversibly blocked by amiloride. However, the currents were seemingly not carried by the Na+/H+ exchanger itself, but were instead due to a shift in the voltage dependence of a preexisting H+ conductance. This was supported by measurements of the reversal potential (Erev) of tail currents, which identified H+ (equivalents) as the charge carrier. During Na+/H+ exchange, Erev changed along with the measured changes in pHi (by 60-69 mV/pH). Moreover, the current and Na+/H+ exchange could be dissociated. Zn2+, which inhibits the H+ conductance, reversibly blocked the currents without altering Na+/H+ exchange. In Chinese hamster ovary (CHO) cells, which lack the H+ conductance, Na+/H+ exchange produced pHi changes that were not accompanied by transmembrane currents. Similar results were obtained in CHO cells transfected with either the NHE-1, NHE-2, or NHE-3 isoforms of the antiporter, indicating that exchange through these isoforms is electroneutral. In all the isoforms tested, the amplitude and time-course of the antiport-induced pHi changes were independent of the holding voltage. We conclude that mammalian NHE-1, NHE-2, and NHE-3 are electroneutral and voltage independent. In cells endowed with a pH-sensitive H+ conductance, such as macrophages, activation of Na(+)-H+ exchange can modulate a transmembrane H+ current. The currents reported in turtle colon might be due to a similar "cross-talk" between the antiporter and a H+ conductance.

Prevention of endotoxin-induced mortality by antitissue factor immunization.

BACKGROUND: Microvascular thrombosis with intravascular fibrin deposition is a characteristic pathologic alteration during endotoxic shock. This effect is predominantly mediated by expression of the cellular procoagulant tissue factor by endothelial cells and cells of monocyte or macrophage lineage, resulting in acceleration of the coagulation cascade and fibrin deposition. OBJECTIVE: To determine whether modulation of this response by treatment with an antitissue factor antibody might have beneficial effects. DESIGN: A polyclonal antibody to murine tissue factor was prepared by injecting rabbits with a synthesized peptide sequence of murine tissue factor. To determine the activity of the antibody, elicited murine peritoneal macrophages were treated for 4 hours with 10-micrograms/mL lipopolysaccharide (LPS), and procoagulant activity was determined via a clotting assay (milliunits of activity per 10(6) macrophages). RESULTS: The tissue factor antibody abrogated LPS-induced macrophage procoagulant activity, confirming activity of the antibody (macrophages, 236 +/- 28 mU/10(6) macrophages; macrophages/LPS, 3801 +/- 190* mU/10(6) macrophages; macrophages/LPS/alpha-tissue factor, 753 +/- 92* mU/10(6) macrophages; n = 3; the asterisk indicates P < .05 by an analysis of variance). Additionally, antibody-protein affinity was confirmed by Western blot analysis. Having determined the activity of the antibody in vitro, we tested its efficacy in vivo in a lethal endotoxemia model. Mice were immunized with 200 microL of antiserum intraperitoneally 2 hours before injection with 250 micrograms of LPS intraperitoneally and 24 hours later. Control animals received 200 microL of saline solution. All animals initially exhibited lethargy and piloerection, characteristic of the predicted response to LPS. However, immunized animals had a significantly (P < .05) reduced mortality compared with control animals. Fibrinogen levels were significantly (P < .05) higher in the immunized mice, suggesting decreased consumption of coagulation factors, a finding consistent with an antitissue factor effect. Further, plasma tumor necrosis factor levels 90 minutes after LPS injection were similar in both groups, suggesting normal induction of the cytokine cascade. CONCLUSIONS: Modulation of microvascular fibrin deposition by abrogating tissue factor-mediated coagulation significantly (P < .05) improved survival in this model without attenuating the initiation of the cytokine cascade. These findings suggest a pathogenic role for coagulation in the induction of acute organ injury during sepsis.

Isolation and characterisation of a novel spirochaete from severe virulent ovine foot rot.

A novel spirochaete was isolated from a case of severe virulent ovine foot rot (SVOFR) by immunomagnetic separation with beads coated with polyclonal anti-treponemal antisera and prolonged anaerobic broth culture. The as yet unnamed treponeme differs considerably from the only other spirochaete isolated from ovine foot rot as regards morphology, enzymic profile and 16S rDNA sequence. On the basis of 16S rDNA, it was most closely related to another unnamed spirochaete isolated from cases of bovine digital dermatitis in the USA, raising the possibility of cross-species transmission. Further information is required to establish this novel ovine spirochaete as the cause of SVOFR.

Bilateral neonatal Sturge-Weber-Dimitri disease: CT and MR findings.

The CT and MR features of cortical calcification and meningeal angiomatosis are typical of Sturge-Weber-Dimitri disease but are unusual in children less than 1 year of age. This case describes a child presenting with both of these features bilaterally in the neonatal period and represents an unusual presentation of this disorder.

Assessment of genotoxic potential of ethylenediamine: in vitro and in vivo studies.

Ethylenediamine (EDA) was evaluated for potential genotoxic activity using a battery in vitro and in vivo mammalian tests. The tests employed were the Chinese hamster ovary (CHO) gene mutation assay, the sister-chromatid exchange (SCE) test with CHO cells, unscheduled DNA synthesis (UDS) assays with primary rat hepatocytes and a dominant lethal study with Fischer 344 rats. EDA did not produce a positive, dose-related, mutagenic effect in either the CHO mutation assay or in the SCE test when evaluated both with and without the addition of a rat-liver S9 activation system. With hepatocytes, no positive effects of EDA upon UDS values were noted in 2 separate studies using either a scintillation counting procedure or an autoradiographic method to determine UDS activity. In a dominant lethal study, male rats fed for 23 weeks with dietary levels of EDA X 2HCl of 0, 0.05, 0.15 or 0.50 g/kg/day, and mated with 1 virgin female/week for 3 consecutive weeks, showed no dose-related or statistically significant effects upon fertility, total number of implantations/female, or the number of living and dead implants per female; marked effects upon the incidence of dominant lethal mutations were noted in the positive control group injected intraperitoneally with one dose of 0.25 mg/kg triethylenemelamine. We conclude that EDA was not genotoxic in the in vitro and in vivo mammalian test systems employed.

Severity of medical and neurologic complications as a determinant of neurodevelopmental outcome at 6 and 12 months in very low birth weight infants.

Very low birth weight (n = 154) and term infants (n = 119) had neurologic and developmental assessment at 6 and 12 months of age. Preterm infants with severe neonatal complications were considered to be at high risk, and those with milder complications were considered to be at low risk, for neurodevelopmental abnormality. Compared to term infants, high- and low-risk infants had abnormalities at 6 months in total neurologic score, cranial nerves, motor tone, motor coordination, and reflexes (P < .001). At 12 months, all groups had improved. However, high-risk infants had persistent abnormalities in the same subcategories (P < .001), whereas low-risk infants differed from term infants only in motor tone (P < .001). Bayley developmental scores were different for all groups at 6 months (P < .001), but at 12 months only high-risk infants differed from term infants (P < .01). These results demonstrate improvement in neurologic and developmental scores over time in very low birth weight infants. The degree of neurodevelopmental abnormality and improvement over time is related to severity of neonatal complications in preterm infants.

Children of alcoholics: helping a vulnerable group.

There are 28 million children of alcoholics in the United States--1 of every 8 Americans. They are more likely than others to suffer from alcoholism and a wide range of physical, emotional, and mental health problems. It is probable that an inherited predisposition for the disease of alcoholism exists. Most children of alcoholics do not become alcoholic, but they are at increased risk for many other health problems. Records of the use of services provided by health maintenance organizations and of health insurance claims show that children of alcoholics use more medical and hospital services than other children. Children of alcoholics are more likely to have problems in school and to abuse alcohol and other drugs. Their mental and physical health problems persist into adulthood. Clinical findings show that life in an alcoholic family is often characterized by pain, guilt, fear, tension, and insecurity. Children do not know that alcoholism is a disease which they cannot cause, control, or cure. Because alcoholism is a family secret, children rarely seek help, even as adults. Because the children of alcoholics are in many medical and social service systems, greater awareness and understanding by health and human service professionals can lead to identification and help for this vulnerable group. It is critical for family physicians, obstetricians, pediatricians, nurses, social workers, hospital staff, and others to incorporate questions about family alcoholism in routine screening procedures for youth and adults. Recommendations and useful materials are discussed.

Prediction of 12-month neurodevelopmental outcome from a 6-month neurologic examination in premature infants.

This study examined whether a neurologic examination at 6 months of age is predictive of neurodevelopmental outcome at 12 months in very-low-birth-weight (VLBW) infants. A neurologic examination and the Bayley Scales of Infant Development were performed at 6 and 12 months with VLBW infants and full-term (FT) controls. VLBW infants were categorized based on early medical complications. High-risk (HR) infants had diagnoses of bronchopulmonary dysplasia, pulmonary immaturity, grade III or IV intraventricular hemorrhage, and/or periventricular leukomalacia. VLBW infants with other diagnoses were placed in the low-risk (LR) group. Total neurologic scores (NS) improved over time for all three groups but improved more for HR infants, who had more abnormal NS at both time points; NS at 6 months predicted neurologic and developmental scores at 12 months for all three groups, but the relation between 6- and 12-month outcomes was strongest for the HR infants. The neurologic examination may be helpful in assessing VLBW infants' need for referral to early childhood intervention programs.

Fostering supportive learning environments in long-term care: the case of WIN A STEP UP.

The education of direct care workers (DCWs) is key to improving job quality and the quality of care in long-term care (LTC). This paper describes the successful integration of a supervisory training program into a continuing education intervention (WIN A STEP UP) for DCWs, identifies the factors that appear to influence the integration of the learning into practice, and discusses the implications for educators. The WIN A STEP UP program achieved its strongest results when the DCW curriculum was paired with Coaching Supervision. Attention to pre-training, training and post-training conditions is necessary to successfully integrate learning into practice in LTC.

Medical costs of children of alcoholics--pay now or pay later.

Inpatient hospital utilization rates are compared for children of alcoholics (COAs) and other children. Data for the study are from hospital utilization statistics on 1.6 million insurance subscribers and their dependents. The findings indicate that COAs have higher rates of inpatient hospital admissions, spend more days in the hospital and incur greater hospital charges, and are more susceptible to specific illnesses than other children. COAs are also more susceptible than other children to specific illnesses such as mental illness, substance abuse, and injuries and poisonings. The findings demonstrate the need to train health care professionals about the relationship between parental alcoholism and childhood illnesses and the need for drug and alcohol prevention and intervention programs for children.

Sphingosylphosphorylcholine activates an amiloride-insensitive Na+-H+-exchange mechanism in GH4C1 cells.

The effect of sphingosylphosphorylcholine (SphPCho) on the intracellular pH (pHi) in GH4C1 cells was investigated. SphPCho evoked a very slow increase in basal pHi. In cells acidified with nigericin, SphPCho induced a rapid alkalinization of the cells. The effect was inhibited in a Na+-free buffer solution, but was insensitive to ethylisopropyl amiloride, a potent inhibitor of Na+-H+ exchangers (NHE). Reverse transcription and PCR showed that the predominant isoform of the antiport expressed in GH4C1 cells is NHE-1. The rate of alkalinization after stimulation with propionate, and after addition of Na+ to cells acidified with NH4Cl, was enhanced in cells treated with SphPCho. The initial rate of alkalinization after addition of Na+ to acidified cells treated with SphPCho gave an apparent Km value of 15 +/- 2 mM for Na+. The Vmax value was 9 +/- 2 mM H+/min. The effect was insensitive to ouabain, staurosporine and bafilomycin A. However, the SphPCho-evoked alkalinization was abolished in cells treated with 2-deoxy-D-glucose. The effect was not due to the charge of the molecule, as stearylamine increased pHi in Na+-containing and Na+-free buffer. The results show that SphPCho may activate Na+-H+ exchange, and that this effect is mediated via an amiloride-insensitive exchange mechanism.

Assessment of a secure/intensive care/forensic ward.

Recent trends in psychiatric hospitals have re-established the need for semi-secure, well-staffed acute wards. Such a ward is described and 400 consecutive admissions over a 34-month period reviewed. Using 13 beds all the acute patients requiring security and intensive treatment are managed from a total catchment area of 600,000. A wide range of referral agencies and diagnostic categories are encountered. Staff in the general psychiatric hospital where the ward is situated are satisfied with the service. A small group of chronically-disturbed patients require separate facilities.

Evaluation of the teratogenicity of ethylenediamine dihydrochloride in Fischer 344 rats by conventional and pair-feeding studies.

Ethylenediamine dihydrochloride (EDA.2HCl) was fed to groups of 20 (40 controls) timed-pregnant rats on Gestation Days 6 through 15 at 1.0, 0.25, 0.05, or 0 g/kg/day. The day of discovery of a vaginal plug was considered gestation day 0. On Gestation Day 21, the fetuses were delivered by cesarean section, and the standard endpoints for teratogenicity were evaluated. At 1.0 and 0.25 g/kg/day, reductions in maternal diet consumption and weight gain were observed during the exposure period. At 1.0 g/kg/day, fetal weight and crown-rump length were significantly reduced and the percentage of litters with resorptions, with skeletal variants, and with missing or shortened innominate arteries was increased. To circumvent the possible problem of palatability of the diet and/or the effects of reduced food intake, a probe study was performed in which 1.0 or 0 g/kg/day was given to pregnant rats by gavage on Gestation Days 6 through 15. However, food intake was also substantially reduced with gavage dosing of the test substance. To determine whether the arterial defects were the result of reduced food intake, a pair-feeding study was performed in which EDA.2HCl was fed on Gestation Days 6 through 15 at 1.0 g/kg/day. A pair-fed control group received the same amount of diet consumed by the EDA.2HCl-treated rats. An untreated control group was fed ad libitum. All groups contained 20 pregnant females. Maternal weight gain, fetal weight and length, and the length of the innominate artery were all reduced in the EDA.2HCl-treated group compared to both control groups. Two fetuses each in the EDA.2HCl-treated and pair-fed control groups had missing innominate arteries versus none in the untreated controls. The four affected fetuses were from four different litters. Ingestion of EDA.2HCl resulted in reduced maternal weight gain, fetal size, and length of the innominate artery, but the missing innominate artery was not a result of EDA.2HCl treatment. Therefore, there was no evidence of teratogenicity in Fischer 344 rats from EDA.2HCl ingestion during organogenesis.

ATP dependence is not an intrinsic property of Na+/H+ exchanger NHE1: requirement for an ancillary factor.

Na+/H+ exchange is a passive process not requiring expenditure of metabolic energy. Nevertheless, depletion of cellular ATP produces a marked inhibition of the antiport. No evidence has been found for direct binding of nucleotide to exchangers or alteration in their state of phosphorylation, suggesting ancillary factors may be involved. This possibility was tested by comparing the activity of dog red blood cells (RBC) and their resealed ghosts. Immunoblotting experiments using isoform-specific polyclonal and monoclonal antibodies indicated RBC membranes express Na+/H+ exchanger isoform 1 (NHE1). In intact RBC, uptake of Na+ was greatly stimulated when the cytosol was acidified. The stimulated uptake was largely eliminated by amiloride and by submicromolar concentrations of the benzoyl guanidinium compound HOE-694, consistent with mediation by NHE1. Although exchange activity could also be elicited by acidification in resealed ghosts containing ATP, the absolute rate of transport was markedly diminished at comparable pH. Dissipation of the pH gradient was ruled out as the cause of diminished transport rate in ghosts. This was accomplished by a "pH clamping" procedure based on continued export of base equivalents by the endogenous anion exchanger. These observations suggest a critical factor required to maintain optimal Na+/H+ exchange activity is lost or inactivated during preparation of ghosts. Depletion of ATP, achieved by incubation with 2-deoxy-D-glucose, inhibited Na+/H+ exchange in intact RBC, as reported for nucleated cells. In contrast, the rate of exchange was similar in control and ATP-depleted resealed ghosts. Interestingly, the residual rate of Na+/H+ exchange in ATP-depleted but otherwise intact cells was similar to the transport rate of ghosts. Therefore, we tentatively conclude that full activation of NHE1 requires both ATP and an additional regulatory factor, which may mediate the action of the nucleotide. Ancillary phosphoproteins or phospholipids or the kinases that mediate their phosphorylation are likely candidates for the regulatory factor(s) that is inactivated or missing in ghosts.

Three-generation reproduction and dominant lethal mutagenesis studies of ethylene glycol in the rat.

To assess the possible effects of ethylene glycol (EG) on reproductive performance and mutagenesis, three-generation reproduction and dominant lethal mutagenesis studies were performed in the Fischer 344 rat. EG was included in the diet at approximate dosages of 1.0, 0.2, and 0.04 g/kg/day during three generations of reproduction. Each generation was bred once. In a dominant lethal mutagenesis study, the F2 males from the reproduction study were bred to three consecutive lots of untreated females at weekly intervals. Concomitantly, another group of untreated F2 males that received a single ip injection of 0.50 mg/kg triethylenemelamine (TEM) were bred similarly to serve as a positive control group. No evidence of reduced fertility or increased fetal death was observed in any of the groups receiving EG. Dominant lethal effects in the TEM group confirmed the susceptibility of the rats to a known mutagen. In conclusion, there were no reproductive or dominant lethal effects associated with the inclusion of as much as 1.0 g/kg/day of EG in the diet.

Activation of the Na+/H+ antiporter during cell volume regulation. Evidence for a phosphorylation-independent mechanism.

A variety of cell types regulate their volume in anisotonic media by stimulating Na+/H+ exchange. Like growth factors, osmotic challenge activates the Na+/H+ antiport by increasing its sensitivity to intracellular [H+]. To investigate the molecular mechanism underlying this shift in pH sensitivity, the antiporter of 32P-labeled human bladder carcinoma cells and of Chinese hamster ovary cells was immunoprecipitated using antibodies raised against the cytosolic domain of the NHE-1 isoform of the Na+/H+ exchanger. Unlike the effects of growth promoters, activation of the antiport during volume regulation was not associated with increased phosphorylation. The possible coexistence of multiple antiporter isoforms was considered. The cytosolic alkalosis normally elicited by hypertonic media was found to be absent in Na+/H+ exchange-deficient fibroblasts. Responsiveness to osmotic challenge was restored by stable transfection of these cells with the cDNA encoding NHE-1. In these transfectants, phosphorylation of the antiporter was also unaffected during osmotic activation. The unchanged phosphate content of the antiporter might be explained by dephosphorylation of one site with concomitant phosphorylation at a different site. However, this possibility appears unlikely since phosphoamino acid analysis revealed that serine was the only residue phosphorylated in immunoprecipitated antiports of both control and osmotically stimulated cells. Moreover, phosphopeptide maps of control and hypertonically activated antiports were identical. These findings reveal a novel mode of activation of Na+/H+ exchange not requiring direct phosphorylation of the antiporter. We propose the existence of dual control of Na+/H+ exchange by phosphorylation-dependent and -independent mechanisms.