Hydrolysis of ATP at only one GyrB subunit is sufficient to promote supercoiling by DNA gyrase

Mutation of Glu42 to Ala in the B subunit of DNA gyrase abolishes ATP hydrolysis but not nucleotide binding. Gyrase complexes that contain one wild-type and one Ala42 mutant B protein were formed, and the ability of such complexes to hydrolyze ATP was investigated. We found that ATP hydrolysis was able to proceed independently only in the wild-type subunit, albeit at a lower rate. With only one ATP molecule hydrolyzed at a time, gyrase could still perform supercoiling, but the limit of this reaction was lower than that observed when both subunits can hydrolyze the nucleotide.     

The arterial to end-tidal carbon dioxide gradient increases with uncorrected but not with temperature-corrected PaCO2 determination during mild to moderate hypothermia

End-tidal carbon dioxide (PETCO2) monitoring is recommended as a basic standard of care and is helpful in adjusting mechanical ventilation. Gas solubility changes with temperature, which might affect the PaCO2 and thereby the gradient between PaCO2 and PETCO2 (PA-ETCO2) under hypothermic conditions. We investigated whether the PA-ETCO2 changes during mild to moderate hypothermia (36 degrees C-32 degrees C) using PaCO2 measured at 37 degrees C (uncorrected PaCO2) and PaCO2 corrected to actual body temperature. We preoperatively investigated 19 patients. After anesthesia had been induced, controlled ventilation was established to maintain normocarbia using constant uncorrected PaCO2 to adjust ventilation (alpha-stat acid-base regimen). Body core temperature was reduced without surgical intervention to 32 degrees C by surface cooling. Continuous PETCO2 was monitored with a mainstream PETCO2 module. The PA-ETCO2 was calculated using the uncorrected and corrected PaCO2 values. During body temperature reduction from 36 degrees C to 32 degrees C, the gradient between PETCO2 and uncorrected PaCO2 increased 2.5-fold, from 4.1 +/- 3.7 to 10.4 +/- 3.8 mm Hg (P < 0.002). The PA-ETCO2 remained unchanged when the corrected PaCO2 was used for the calculation. We conclude that when the alpha-stat acid-base regimen is used to adjust ventilation, the PA-ETCO2 calculated with the uncorrected PaCO2 increases and should be added to the differential diagnosis of widened PA-ETCO2. In contrast, when the corrected PaCO2 is used for the calculation of the PA-ETCO2, the PA-ETCO2 remains unaltered during hypothermia. Implications: We investigated the impact of induced hypothermia (36 degrees C-32 degrees C) on the gradient between PaCO2 and PETCO2 (PA-ETCO2). The PA-ETCO2 increased 2.5-fold when CO2 determinations were not temperature-corrected. Hypothermia should be added to the differential diagnosis of an increased PA-ETCO2 when the alpha-stat acid-base regimen is used.     

Command, control and communication in bacterial pathogenesis

Family violence is typically progressive. As a man realizes the effectiveness of physical and emotional coercion, he tends to use it more frequently. In the face of imbalance of power in the relationship, a victim becomes more fearful, more submissive, and more likely to face serious injury. This is the first of a two-part series on physicians helping patients cope with domestic violence.     

(Glyco)sphingolipids are sorted in sub-apical compartments in HepG2 cells: a role for non-Golgi-related intracellular sites in the polarized distribution of (glyco)sphingolipids

In polarized HepG2 cells, the fluorescent sphingolipid analogues of glucosylceramide (C6-NBD-GlcCer) and sphingomyelin (C6-NBD-SM) display a preferential localization at the apical and basolateral domain, respectively, which is expressed during apical to basolateral transcytosis of the lipids (van IJzendoorn, S.C.D., M.M. P. Zegers, J.W. Kok, and D. Hoekstra. 1997. J. Cell Biol. 137:347-457). In the present study we have identified a non-Golgi-related, sub-apical compartment (SAC), in which sorting of the lipids occurs. Thus, in the apical to basolateral transcytotic pathway both C6-NBD-GlcCer and C6-NBD-SM accumulate in SAC at 18 degreesC. At this temperature, transcytosing IgA also accumulates, and colocalizes with the lipids. Upon rewarming the cells to 37 degreesC, the lipids are transported from the SAC to their preferred membrane domain. Kinetic evidence is presented that shows in a direct manner that after leaving SAC, sphingomyelin disappears from the apical region of the cell, whereas GlcCer is transferred to the apical, bile canalicular membrane. The sorting event is very specific, as the GlcCer epimer C6-NBD-galactosylceramide, like C6-NBD-SM, is sorted in the SAC and directed to the basolateral surface. It is demonstrated that transport of the lipids to and from SAC is accomplished by a vesicular mechanism, and is in part microtubule dependent. Furthermore, the SAC in HepG2 bear analogy to the apical recycling compartments, previously described in MDCK cells. However, in contrast to the latter, the structural integrity of SAC does not depend on an intact microtubule system. Taken together, we have identified a non-Golgi-related compartment, acting as a "traffic center" in apical to basolateral trafficking and vice versa, and directing the polarized distribution of sphingolipids in hepatic cells.     

Neural responses to facial and vocal expressions of fear and disgust

Neuropsychological studies report more impaired responses to facial expressions of fear than disgust in people with amygdala lesions, and vice versa in people with Huntington's disease. Experiments using functional magnetic resonance imaging (fMRI) have confirmed the role of the amygdala in the response to fearful faces and have implicated the anterior insula in the response to facial expressions of disgust. We used fMRI to extend these studies to the perception of fear and disgust from both facial and vocal expressions. Consistent with neuropsychological findings, both types of fearful stimuli activated the amygdala. Facial expressions of disgust activated the anterior insula and the caudate-putamen; vocal expressions of disgust did not significantly activate either of these regions. All four types of stimuli activated the superior temporal gyrus. Our findings therefore (i) support the differential localization of the neural substrates of fear and disgust; (ii) confirm the involvement of the amygdala in the emotion of fear, whether evoked by facial or vocal expressions; (iii) confirm the involvement of the anterior insula and the striatum in reactions to facial expressions of disgust; and (iv) suggest a possible general role for the perception of emotional expressions for the superior temporal gyrus.     

Cre/loxP-mediated excision and amplification of large segments of the Escherichia coli genome

The isolation and amplification of large, predetermined segments of a genome from its host have been explored. The prototype of our approach was the excisional replication of some viruses such as the lambda-lysogen. Similar machinery was used to excise and amplify large genomic segments of Escherichia coli in its host. Two loxP sequences for a site-specific recombinase Cre, together with a conditional replication origin (pi-dependent gamma-ori), were inserted into the genome by homologous recombination at predetermined sites, 50-100 kb apart. Cre and pir200 which encodes the site-specific recombinase Cre and an ori-specific replication protein pi, respectively, were also introduced into the genome. The predetermined genomic segments flanked by the loxP sequences were excised and amplified upon induction of the cre and pir200 genes which were under the control of the tet promoter. This excised and amplified DNA could be easily purified as a large plasmid. This procedure can provide an alternative to conventional cloning methods by obtaining predetermined large genomic segments directly from the original organisms. In this study, using the Cre/loxP site-specific recombination and pi/gamma-ori replication system of plasmid R6K, a procedure was devised that could isolate a large segment of the E. coli genome and demonstrated the feasibility of the procedure by excising and amplifying the 50-kb trg-narZ and 100-kb trg-hipA regions of the E. coli W3110 genome.     

Endovascular occlusion with a new mechanical detachable coil

OBJECTIVE: Metallic coils have been used for vascular embolization for many years but controlled-release coils have only recently become commercially available. Most of these devices are microcoils that were manufactured primarily for the packing of intracerebral aneurysms; therefore, they lack radial force and are not ideal agents for the occlusion of high-flow lesions such as pulmonary arteriovenous malformations (PAVMs). The objective of this study was to review our experience with a new detachable coil based on the conventional Gianturco-Wallace coil. SUBJECTS AND METHODS: The new detachable coil was initially used for the treatment of varicocele in 20 patients. Subsequently, the coil was used in 48 patients during 90 procedures for the treatment of PAVMs. RESULTS: A total of 548 coils were used. Complete occlusion of the testicular vein was achieved in all patients with varicocele. Successful occlusion of the PAVM being treated was achieved in all patients, and no instances of recanalization were documented in any of the patients who returned for follow-up angiography. Forty-one coils had to be removed completely from the catheter before detachment because of inappropriate size or position. Eight coils failed to detach easily, and six of these had to be removed. Most of these device failures were associated with kinking of the screw thread mechanism between the coil and the delivery wire. CONCLUSION: The Jackson detachable coil allows safer, more accurate, and more distal embolization of PAVMs than is possible with nondetachable coils. Complications associated with its use have been few.     

Outcome of Graves' disease after antithyroid drug treatment in Taiwan

The outcome of Graves' disease after treatment with antithyroid drugs (ATDs) varies widely among countries, and large-scale studies in Asia are rare. We investigated the associations of various clinical and laboratory features with the outcome of ATD therapy for Graves' disease in Taiwan. A total of 210 patients (177 women, 33 men; mean +/- SD age, 41.7 +/- 15.1 yr) treated with ATD in Taiwan were included. ATD therapy started with methimazole 30 mg daily or propylthiouracil 300 mg daily and was continued until a euthyroid state was achieved. Afterwards, 154 patients received a maintenance dose of ATD alone, while 56 patients received a combination of an ATD and thyroxine (L-T4). Patients were considered to be in remission if they remained in a euthyroid state for more than 2 years after drug withdrawal. The mean follow-up periods were 45.0 +/- 20.9 months for patients with remission and 30.4 +/- 19.8 months for those with relapse. Relapse occurred in 126 (60%) patients during the follow-up period, within 3 months after drug withdrawal in 47 (37%), and within 6 months in 60 (46%). The relapse rate was 100% among patients with two or more previous relapses. Patients with a second occurrence had a higher relapse rate than those with a first occurrence (84% vs 43%). Past history of recurrence, goiter size, thyroid-stimulating hormone level and thyrotropin-binding inhibition immunoglobulin activity at the end of ATD treatment were independently associated with relapse. Prolonged duration of treatment did not yield better results in patients with larger goiters or a history of recurrence, or both. Combination therapy with L-T4 yielded similar results to those achieved with ATD treatment alone. In conclusion, the relapse rate of Graves' disease after ATD treatment in Taiwanese patients was high, especially in those with a history of recurrence. The treatment duration and drug regimen did not affect the outcome.