Relationship between mouth occlusion pressure and electrical activity of the diaphragm: effects of flow-resistive loading

We determined the relationship between mouth occlusion pressure and diaphragmatic electromyography during CO2 rebreathing with and without inspiratory flow resistance. Diaphragmatic electromyography was measured as a moving time average; occlusion pressures were measured 150 msec after onset of an inspiratory effort against a closed airway (P.15). P.15 versus diaphragmatic electromyographic plots during CO2 rebreathing with and without inspiratory flow resistance were linear. In 3 subjects the slope of P.15 versus diaphragmatic electromyography was unchanged with inspiratory flow resistance whereas in 3 others the slope increased, indicating greater inspiratory force for a given degree of diaphragmatic activity. We concluded that under unloaded conditions P.15 is a reliable index of respiratory neural output but may no longer reflect only inspiratory motoneuron drive during mechanical loading.     

In vitro transcription by isolated nuclei of Rhynchosciara americana salivary glands. Characteristics of incorporation and inhibition by alpha-amanitin

A method for the isolation of polytene nuclei from salivary glands cells of the Diptera Rhynchosciara americana is described. The stage-specific morphological pattern of the chromosome is maintained during the isolation. The isolated nuclei show two distinct RNA polymerase activities, namely I and II, characterized on the basis of ionic requirements and alpha-amanitin sensitivity. Studies of the product under the incubation conditions show that the system allows the synthesis of high-molecular weight RNA, beside a low molecular weight peak which may comprise pre-4S and 5S RNAs. Autoradiographic studies carried out in the presence or absence of the toxin alpha-amanitin showed that micronucleoli contain products of RNA polymerase type I activity (ribosomal RNA) and that the DNA puffs are engaged in alpha-amanitin sensitive RNA synthesis and thus are sites of polymerase type II activity.     

The complications of trauma and their associated costs in a level I trauma center

OBJECTIVES: To estimate the expected costs for acute trauma care, to quantify the costs associated with the development of complications in injury victims, and to determine the deficit incurred by patients in whom complications develop. DESIGN: A retrospective, cohort design. SETTING: A referral trauma center. PATIENTS: A total of 12,088 patients admitted to a single regional trauma center during a period of 5 years. INTERVENTIONS: This is an observational study, and no interventions specific to this study are included in the design. MAIN OUTCOME MEASURES: (1) The expected costs for injury victims based on readily available clinical data. (2) The costs associated with the most important complications of trauma. (3) The effect of complications on inadequate reimbursement for trauma care. RESULTS: The expected costs were estimated using a linear model incorporating demographic variables and measures of injury severity. The expected costs averaged $14,567, and the observed costs averaged $15,032. Six complications were important predictors of cost. These included adult respiratory distress syndrome, acute kidney failure, sepsis, pneumonia, decubitus ulceration, and wound infections. For 1201 individuals with these complications, the predicted costs averaged $23,266 and the observed costs averaged $47,457. The mean excess costs for a single complication ranged from $6669 to $18,052. Multiple complications led to greater increases in excess cost, averaging $110,007 for the 62 patients with 3 or more complications. Costs exceeded reimbursement to a much greater degree in those in whom any of the 6 complications developed. CONCLUSION: Expected hospital costs can be estimated using admission clinical data. Each of 6 complications was associated with enormous increases in costs, indicating their importance as a cause of avoidable expenditures in injury victims and identifying situations in which reimbursement may not be adequate.     

Intra-renal and subcellular distribution of the human chloride channel, CLC-5, reveals a pathophysiological basis for Dent's disease

Dent's disease, which is a renal tubular disorder characterized by low molecular weight proteinuria, hypercalciuria and nephrolithiasis, is associated with inactivating mutations of the X-linked chloride channel, CLC-5. However, the manner in which a functional loss of CLC-5 leads to such diverse renal abnormalities remains to be defined. In order to elucidate this, we performed studies to determine the segmental expression of CLC-5 in the human kidney and to define its intracellular distribution. We raised and characterized antisera against human CLC-5, and identified by immunoblotting an 83 kDa band corresponding to CLC-5 in human kidney cortex and medulla. Immunohistochemistry revealed CLC-5 expression in the epithelial cells lining the proximal tubules and the thick ascending limbs of Henle's loop, and in intercalated cells of the collecting ducts. Studies of subcellular human kidney fractions established that CLC-5 distribution was associated best with that of Rab4, which is a marker of recycling early endosomes. In addition, confocal microscopy studies using the proximal tubular cell model of opossum kidney cells, which endogenously expressed CLC-5, revealed that CLC-5 co-localized with the albumin-containing endocytic vesicles that form part of the receptor-mediated endocytic pathway. Thus, CLC-5 is expressed at multiple sites in the human nephron and is likely to have a role in the receptor-mediated endocytic pathway. Furthermore, the functional loss of CLC-5 in the proximal tubules and the thick ascending limbs provides an explanation for the occurrences of low molecular weight proteinuria and hypercalciuria, respectively. These results help to elucidate further the patho-physiological basis of the renal tubular defects of Dent's disease.     

Elevated levels of the IGF-binding protein protease MMP-1 in asthmatic airway smooth muscle

We characterized the changes in nitric oxide (NO) levels in the brain during global forebrain ischemia and reperfusion and tested the ability of the natural flavonoid, quercetin, and a synthetic flavonoid, FB277, to increase the amount of available NO by elimination of the superoxide radicals produced during reperfusion. In Sprague-Dawley rats, we used a four-vessel occlusion model of forebrain ischemia (15 min) and reperfusion (30 min). Brain NO was measured on samples of cerebral cortex and cerebellum ex vivo by electron paramagnetic resonance (EPR) spectroscopy. The spin trap used was diethyldithiocarbamate sodium salt (DETC) associated with ferrous citrate. The complex Fe(DETC)2NO was detected at 77 K as a triplet signal at g = 2.035. Groups of animals were treated with quercetin or FB277 (3-morpholinomethyl-3',4',5,7tetramethoxyflavone) or polyethylene glycol-conjugated superoxide dismutase (PEG-SOD). In control (intact anesthetized animals), the signal was about 3 times greater in the cortex than in the cerebellum. During ischemia, the signal rose to 110% in cortex (NS) and 283% in cerebellum (P < 0.05). In reperfusion, it fell again to 91% of control in cerebellum (NS) and 35% in cortex (P < 0.05). Treatment by quercetin (5 mg/kg i.v.) of intact and ischemia-reperfusion groups did not significantly change the signal amplitude in the cerebellum, but did double it in the cortex (to 76% of control) for the ischemia-reperfusion group (P < 0.05). In contrast, FB277 (3.75 mg/kg i.v.) did not increase the signal in the cortex during ischemia-reperfusion, but did do so in the cerebellum (to 152% of control, P < 0.05). The results obtained for PEG-SOD (10,000 U/kg i.v.) were similar to those for FB277. In separate in vitro measurements, we found that quercetin but not FB277 efficiently scavenged superoxide. We hypothesize that quercetin but not FB277 scavenged superoxide anions released in the cortex during reperfusion, thus diminishing the amount of NO removed by the formation of peroxynitrite. The lack of effect of PEG-SOD may be related to the need for chronic treatment to obtain protection.     

Permanent open shunt as a reason for impotence or reduced potency after surgical treatment of priapism in 26 patients

A permanent open shunt as a cause of impotence or impaired potency after a shunt operation for priapism is an unusual situation. In this series we studied the persistence of an open shunt in 26 patients who had developed impotence or impaired potency after operative treatment for priapism. All patients had been examined by cavernosography on the suspicion of an open shunt, giving a positive finding in five of 26 cases, in all of which impotence was cured by closure of the shunt. In five patients without a permanent open shunt potency returned to normal only after 6-12 months.     

Characterization of recombinant soybean leghemoglobin a and apolar distal histidine mutants

The cDNA for soybean leghemoglobin a (Lba) was cloned from a root nodule cDNA library and expressed in Escherichia coli. The crystal structure of the ferric acetate complex of recombinant wild-type Lba was determined at a resolution of 2.2 A. Rate constants for O2, CO and NO binding to recombinant Lba are identical with those of native soybean Lba. Rate constants for hemin dissociation and auto-oxidation of wild-type Lba were compared with those of sperm whale myoglobin. At 37 degrees C and pH 7, soybean Lba is much less stable than sperm whale myoglobin due both to a fourfold higher rate of auto-oxidation and to a approximately 600-fold lower affinity for hemin. The role of His61(E7) in regulating oxygen binding was examined by site-directed mutagenesis. Replacement of His(E7) with Ala, Val or Leu causes little change in the equilibrium constant for O2 binding to soybean Lba, whereas the same mutations in sperm whale myoglobin cause 50 to 100-fold decreases in K(O2). These results show that, at neutral pH, hydrogen bonding with His(E7) is much less important in regulating O2 binding to the soybean protein. The His(E7) to Phe mutation does cause a significant decrease in K(O2) for Lba, apparently due to steric hindrance of the bound ligand. The rate constants for O2 dissociation from wild-type and native Lba decrease significantly with decreasing pH. In contrast, the O2 dissociation rate constants for mutants with apolar E7 residues are independent of pH, suggesting that hydrogen bonding to the distal histidine residue in the native protein is enhanced under acid conditions. All of these results support the hypothesis that the high affinity of Lba for oxygen and other ligands is determined primarily by enhanced accessibility and reactivity of the heme group.