Acceleration of plasma bicarbonate conversion to carbon dioxide by pulmonary carbonic anhydrase.

In isolated rabbit lungs perfused with solutions containing little or no carbonic anhydrase activity, nearly complete equilibration between H14CO3- and 14CO2 occurs during a single circulation. This equilibration can be inhibited by blocking pulmonary carbonic anhydrase with acetazolamide.

Malaria resistance: artificial induction with a partially purified plasmodial fraction.

Plasmodium berghei were released from mouse erythrocytes by passage through a French pressure cell. The released organisms were washed and disintegrated; the soluble portion was chromatographed on a Sephadex (G-200) column. The void-volume eluate contained an erythrocyte-free plasmodial fraction which behaved as a vaccine, preventing parasitemia, anemia, and death in mice subsequently challenged with living Plasmodium berghei.

Effect of tumor necrosis factor on the human fibrinolytic system.

We report the effects on the fibrinolytic system of intravenous (IV) recombinant tumor necrosis factor-alpha (rTNF-alpha) infusions in patients with advanced cancers. During a phase I clinical trial of rTNF-alpha, the plasma fibrinolytic system was closely monitored, measuring tissue-type plasminogen activator (tPA) antigen, plasminogen activator (PA) inhibitor activity, and plasma fibrinolytic activity. Thirteen patients with refractory malignancies received 40, 80, or 160 micrograms/m2 rTNF-alpha as 2-hour IV infusions. After a 1-week rest, the same dose was repeated daily for 5 days every 3 weeks for a maximum of four courses. The serum rTNF-alpha levels peaked at the completion of the IV infusion and rapidly declined thereafter, becoming unmeasurable within 1 hour in all patients. rTNF-alpha infusion markedly alters the plasma fibrinolytic system. During the 2-hour infusion, significant increases in the tPA antigen and plasma fibrinolytic activity were seen. After the infusion, PA inhibitor activity increased, neutralizing the plasma fibrinolytic activity. The increase in PA inhibitor activity was maximal 6 hours after the onset of the rTNF-alpha infusion. Fibrinolytic properties returned to pretreatment values within 24 hours. Daily rTNF-alpha infusions caused changes in plasma tPA antigen and PA inhibitor similar to those of single infusions. We conclude from these observations that the administration of rTNF-alpha in vivo to cancer patients causes profound alterations of endothelial cell-derived components of the fibrinolytic system.

Phase I trial of high-dose mitoxantrone plus cyclophosphamide and filgrastim in patients with advanced breast carcinoma.

PURPOSE: To determine the maximum-tolerated dose (MTD) of mitoxantrone that could be safely used in combination with cyclophosphamide and filgrastim in patients with advanced breast carcinoma. PATIENTS AND METHODS: Twenty-seven patients with metastatic (n = 24) or locally advanced (n = 3) breast carcinoma received escalating doses of mitoxantrone (16, 20, 24, 28, or 32 mg/m2) plus cyclophosphamide at one of three dose levels: group 1, 1,200 mg/m2; group 2, 2,400 mg/m2; and group 3,600 mg/m2. All patients also received filgrastim 5 micrograms/kg administered subcutaneously beginning on day 2 and continuing until the post-nadir absolute neutrophil count (ANC) was > or = 10,000/microL. Treatment was repeated every 3 weeks if the ANC was > or = 2,000/microL and platelet count > or = 90,000/microL for a maximum of six cycles. Dose escalation occurred within each group if zero of three or one of four patients had dose-limiting toxicity during the first cycle. RESULTS: The MTD of mitoxantrone was 24 mg/m2 in group 1, less than 16 mg/m2 in group 2, and 28 mg/m2 in group 3. Neutropenia was dose-limiting, and cumulative neutropenia and thrombocytopenia occurred with continued therapy. Nonhematologic toxicity consisted predominantly of nausea, vomiting, alopecia, and fatigue. Three patients (11%) had a > or = 10% decrease in the left ventricular ejection fraction (LVEF), one patient (4%) had a decrease in the LVEF below normal, and none developed clinical congestive heart failure. Of patients with stage IV breast carcinoma who had not received prior chemotherapy for advanced disease, objective responses occurred in nine of 20 (45%), and the median response duration was 5 months. CONCLUSION: In combination with 600 mg/m2 of cyclophosphamide and filgrastim, the MTD of mitoxantrone is 28 mg/m2, a dose that is approximately twofold to 2.8-fold higher than the conventional dose used without a hematopoietic growth factor.

Safety and efficacy of a new transpulmonary ultrasound contrast agent: initial multicenter clinical results.

Myocardial contrast echocardiography has been found to be a safe and useful technique for evaluating relative changes in myocardial perfusion and delineating areas at risk. Although earlier contrast agents required direct delivery into the coronary arteries or aortic root, a new echocardiographic contrast agent, sonicated albumin microspheres (Albunex), has been found to cross the pulmonary circulation in experimental models. To determine the safety and preliminary efficacy of intravenous injections of Albunex in humans, 71 patients at three independent medical institutions underwent two-dimensional echocardiographic examination before, during and after the administration of three intravenous doses of Albunex, ranging from 0.01 to 0.12 ml/kg body weight. All patients provided a complete history and underwent physical and neurologic examination and laboratory and electrocardiographic evaluation before the injections; all evaluations (except for the history) were repeated at 2 h and 3 days after the injections of Albunex. The efficacy of the injections was qualitatively assessed by two independent blinded observers using a grading system of 0 to +3, with 0 indicating an absence of contrast effect and +3 indicating full opacification of the cavities examined. All injections were well tolerated and no serious side effects were noted in any of the patients. Irrespective of dose group, a cavity opacification greater than or equal to +2 was seen in the right ventricle in 212 (88%) of 240 injections and in the left ventricle in 151 (63%) of 240 injections as judged by the independent observers. The degree of ventricular cavity opacification appeared to be dose and concentration related.(ABSTRACT TRUNCATED AT 250 WORDS)

The cpx proteins of Escherichia coli K12. Structure of the cpxA polypeptide as an inner membrane component.

Gene cpxA of Escherichia coli K12 encodes the 52,000 Mr CpxA polypeptide. The complete cpxA nucleotide sequence, reported here, predicted that CpxA contains two extended, hydrophobic segments in its amino-terminal half and could therefore be a membrane protein. Using a lac-cpxA operon fusion plasmid to overproduce CpxA and an immunochemical assay to detect the polypeptide, we show that CpxA fractionated with the bacterial inner membrane during differential and isopycnic sedimentation. Moreover, the protein could be solubilized by extraction of crude membranes with non-ionic detergents but not with KCl or NaOH, indicating that Cpx is an intrinsic membrane component. Analysis of TnphoA insertions in cpxA indicated that the region between the hydrophobic segments of CpxA is periplasmic, whereas the region carboxy-terminal to the second such segment is cytoplasmic. Based on these structural data, we propose that CpxA functions as a trans-membrane sensory protein. The DNA sequence data also indicate that cpxA is the 3' gene of an operon.

Regulation of the F plasmid traY promoter in Escherichia coli by host and plasmid factors.

F plasmid DNA transfer (tra) gene expression in Escherichia coli is regulated by chromosome- and F-encoded gene products. To study the relationship among these regulatory factors, we constructed low-copy plasmids containing a phi(traY'-'lacZ)hyb gene that couples beta-galactosidase and Lac permease synthesis to the F plasmid traY promoter. Wild-type transformants maintained high levels of beta-galactosidase over a broad range of culture densities. Primer extension analysis of tra mRNA from F'lac and phi(traY'-'lacZ)hyb strains indicated very similar, though not identical, transcription initiation sites. Moreover, phi(traY'-'lacZ)hyb gene expression required both TraJ and SfrA, as does tra gene expression in F+ strains. beta-Galactosidase activity was reduced approximately 30-fold in the absence of TraJ, which could be supplied in cis or in trans. In a two-plasmid system in which TraJ was supplied in trans by a lac-traJ operon fusion, phi(traY'-'lacZ)hyb expression was a linear, saturable function of traJ expression. Enzyme activity was reduced approximately tenfold in sfrA mutants. That reduction could not be attributed to an effect on the TraJ level. Several other cellular or environmental variables had only a modest effect on phi(traY'-'lacZ)hyb expression. Hyperexpression was observed at high cell density (twofold) and in anaerobic cultures (1.2- to 1.5-fold). In contrast, expression was reduced twofold in integration host factor mutants.

Regulation of the F plasmid traY promoter in Escherichia coli K12 as a function of sequence context.

TraJ and SfrA are, respectively, plasmid and host (Escherichia coli)-encoded proteins normally required for F plasmid traY promoter function. Beginning with plasmids in which a traY-lacZ fusion gene, designated phi (traY'-'lacZ)hyb, and lacY are expressed from the F plasmid traY promoter, we isolated mutants in which lac gene expression was SfrA or TraJ-independent. A total of 45 of 50 SfrA-independent isolates obtained after 2-aminopurine mutagenesis proved to have chromosomal mutations, whereas four out of four isolates obtained without mutagenesis had plasmid mutations. All of 17 isolates selected for TraJ-independent expression after mutagenesis had plasmid mutations. By restriction endonuclease digestions, 25 of 26 SfrA-independent and TraJ-independent plasmid mutations were insertions. Four of the former and three of the latter were examined further. By sequence analysis, all seven proved to be IS1 or IS2 insertions defining five insertion sites between base-pairs -49 and -82 with respect to the major traY transcription initiation site. In two cases, the same insertion allele was obtained from the two selection schemes. All three of the mutants selected for TraJ-independent gene expression manifested SfrA-independent expression as well, and levels of beta-galactosidase in different plasmid mutant strains lacking TraJ and SfrA were indistinguishable. By primer extension analysis, transcription initiation sites for traY mRNA synthesis were unaltered by the mutations. Replacing the tra sequence upstream from base-pair -78, without genetic selection, increased beta-galactosidase activity in the absence of TraJ and SfrA greater than tenfold. Activity increased two- to threefold more in a traJ+ sfrA mutant strain, and fivefold more in a traJ+ sfrA+ strain. Activity was unaltered in an sfrA+ strain without TraJ. By primer extension analysis, the traY promoter was utilized under all conditions. The data indicate that regulation of traY promoter activity is strongly dependent on sequence context.

Race-Specific Elicitors of Cladosporium fulvum Induce Changes in Cell Morphology and the Synthesis of Ethylene and Salicylic Acid in Tomato Plants Carrying the Corresponding Cf Disease Resistance Gene.

Defense responses mediated by the genetically unlinked Cf-9 and Cf-2 genes were compared with those involving no Cf gene (Cf0). Compatible tomato (Lycopersicon esculentum)-Cladosporium fulvum intercellular washing fluids were injected into tomato cotyledons, and the kinetics of responses was monitored under conditions of 70 and 98% relative humidity. The latter conditions suppressed the normal macroscopic responses. For the Cf-9-Avr9 interaction, stomatal opening was induced within 3 to 4 h and after 9 h mesophyll cell death commenced. A burst of ethylene production occurred between 9 and 12.5 h and remained elevated. Free salicylic acid levels increased after 12 h, peaked at 24 h, and thereafter declined. For the Cf-2-Avr2 interaction, stomata became plugged after 8 h, and salicylic acid and ethylene levels increased by 12 and 18 h, respectively, and thereafter declined. Host cell death commenced around vascular tissue by 24 h. Cell death in both incompatible interactions was frequently preceded by cell enlargement. For Cf0-injected plants, no significant responses were detected. High humidity delayed and reduced the Cf-Avr-gene-dependent cell death and ethylene synthesis, whereas induced salicylic acid levels were unaffected for Cf-2-Avr2 and reduced in magnitude only for Cf-9-Avr9.

Jasmonate-Inducible Genes Are Activated in Rice by Pathogen Attack without a Concomitant Increase in Endogenous Jasmonic Acid Levels.

The possible role of the octadecanoid signaling pathway with jasmonic acid (JA) as the central component in defense-gene regulation of pathogen-attacked rice was studied. Rice (Oryza sativa L.) seedlings were treated with JA or inoculated with the rice blast fungus Magnaporthe grisea (Hebert) Barr., and gene-expression patterns were compared between the two treatments. JA application induced the accumulation of a number of pathogenesis-related (PR) gene products at the mRNA and protein levels, but pathogen attack did not enhance the levels of (-)-JA during the time required for PR gene expression. Pathogen-induced accumulation of PR1-like proteins was reduced in plants treated with tetcyclacis, a novel inhibitor of jasmonate biosynthesis. There was an additive and negative interaction between JA and an elicitor from M. grisea with respect to induction of PR1-like proteins and of an abundant JA-and wound-induced protein of 26 kD, respectively. Finally, activation of the octadecanoid signaling pathway and induction of a number of PR genes by exogenous application of JA did not confer local acquired resistance to rice. The data suggest that accumulation of nonconjugated (-)-JA is not necessary for induction of PR genes and that JA does not orchestrate localized defense responses in pathogen-attacked rice. Instead, JA appears to be embedded in a signaling network with another pathogen-induced pathway(s) and may be required at a certain minimal level for induction of some PR genes.

Salicylic Acid in Rice (Biosynthesis, Conjugation, and Possible Role).

Salicylic acid (SA) is a natural inducer of disease resistance in some dicotyledonous plants. Rice seedlings (Oryza sativa L.) had the highest levels of SA among all plants tested for SA content (between 0.01 and 37.19 [mu]g/g fresh weight). The second leaf of rice seedlings had slightly lower SA levels than any younger leaves. To investigate the role of SA in rice disease resistance, we examined the levels of SA in rice (cv M-201) after inoculation with bacterial and fungal pathogens. SA levels did not increase after inoculation with either the avirulent pathogen Pseudomonas syringae D20 or with the rice pathogens Magnaporthe grisea, the causal agent of rice blast, and Rhizoctonia solani, the causal agent of sheath blight. However, leaf SA levels in 28 rice varieties showed a correlation with generalized blast resistance, indicating that SA may play a role as a constitutive defense compound. Biosynthesis and metabolism of SA in rice was studied and compared to that of tobacco. Rice shoots converted [14C]cinnamic acid to SA and the lignin precursors p-coumaric and ferulic acids, whereas [14C]benzoic acid was readily converted to SA. The data suggest that in rice, as in tobacco, SA is synthesized from cinnamic acid via benzoic acid. In rice shoots, SA is largely present as a free acid; however, exogenously supplied SA was converted to [beta]-O-D-glucosylSA by an SA-inducible glucosyltransferase (SA-GTase). A 7-fold induction of SA-GTase activity was observed after 6 h of feeding 1 mM SA. Both rice roots and shoots showed similar patterns of SA-GTase induction by SA, with maximal induction after feeding with 1 mM SA.

Inhibition of erythropoiesis by a soluble factor in murine malaria.

To study the cellular mechanisms involved in the ineffective erythropoiesis associated with malaria, an in vitro proliferative assay was used to measure the response to erythropoietin (Epo) of erythroid progenitor cells from malaria-infected mice. In this assay, spleen (SP) cells from phenylhydrazine (PHZ)-treated mice (PHZ-SP), enriched for erythroid progenitor cells, respond to Epo in a dose-dependent manner. Despite a similar degree of anemia, SP and bone marrow (BM) cells from Plasmodium berghei- or P. vinckei-infected mice did not show a significant response to Epo in this assay. When SP or BM cells from malaria-infected mice were added to cultures of SP or BM cells from PHZ-treated mice the response to Epo of these cells was significantly inhibited. Removal of parasitized red blood cells (pRBC) from SP cells of P. berghei-infected mice had no effect on the ability of the cells to inhibit the response to Epo. Adherent SP cells and SP cells positive for the Mac-1 antigen, from malaria-infected mice, were shown to be enriched for cells that could inhibit the response to Epo. Cell-free conditioned media (CM) prepared from SP cells of P. berghei- or P. vinckei-infected mice or from normal SP cells incubated with pRBC were also able to inhibit the response to Epo of SP cells from PHZ-treated mice. These investigations have shown that during the course of malaria infection, cells appear in the SP and BM capable of inhibiting, via soluble mediators, the response to Epo of erythroid progenitor cells. The cells responsible are probably macrophages. The nature of the factor(s) and its mechanism of action are not known. Through the ability to inhibit erythropoiesis, soluble factors may, in part, mediate the anemia associated with malaria.

Can myocardial contrast echo determine coronary flow reserve?

OBJECTIVE: The aim was to evaluate the applicability of myocardial contrast echocardiography in the measurement of coronary flow reserve. METHODS: Eleven anaesthetised open chest pigs were studied, in which coronary atherosclerosis had been induced by abrasion of the left anterior descending coronary artery at one month, followed by an atherogenic diet for eight months. Coronary flow reserve was determined by electromagnetic flow measurement and contrast echocardiography before and after partial occlusion of the left anterior descending coronary artery, using papaverine as a coronary vasodilator. Coronary blood flow was reduced by tightening a clamp placed around the coronary artery. Systemic haemodynamics and myocardial wall thickness (epicardial ultrasound 5 MHz transducer) were recorded simultaneously. Echocardiograms were recorded on VHS tape and analysed by digitised videodensitometry off line for construction of the time v videointensity curve (time-intensity curves). From these curves washout time (T50), area under the curve, peak contrast intensity, and time to peak intensity were calculated. RESULTS: Following papaverine, coronary blood flow increased significantly from 47 (SD 23) ml.min-1 at baseline to 88(39) ml.min-1 (p less than 0.05). During the stenosis, flow decreased to 19(16) ml.min-1 (p less than 0.01), and increased to 38(29) ml.min-1 (p less than 0.05 v stenosis) after administration of papaverine. Correlations between coronary blood flow and indices calculated from the quantitative videodensitometric analysis were poor, varying between r = 0.03 for area at control flow to r = 0.62 for T50 during stenosis. The same was true for coronary flow reserve: r = 0.09 for peak to r = 0.75 (p less than 0.05) for time to peak without the stenosis. CONCLUSIONS: Current limitations in injection, imaging, and analysis techniques cause variability in data from time-intensity curves, which precludes accurate quantification of coronary flow (reserve) by myocardial contrast echocardiography.

Metabolic emergencies in clinical oncology.

As antineoplastic therapy improves, patients with cancer will have the potential for prolonged survival. Tumor products may produce metabolic complications, such as hypercalcemia, hyponatremia, and hypoglycemia, and these conditions must be promptly recognized so that treatment can be initiated before severe or life-threatening symptoms occur. Tumor-lysis syndrome is a metabolic complication that occurs most frequently when effective treatment for rapidly proliferating tumors is initiated. It is a syndrome that can be prevented or ameliorated with appropriate anticipatory treatment. Recognition of metabolic emergencies common to cancer patients will improve quality of life and improve survival, allowing patients to benefit from definitive cancer treatment.

Improved detection of small cavernous hemangiomas of the liver with high-resolution three-headed SPECT.

The purpose of this study was to review our experience with 99mTc-red blood cell scintigraphy for diagnosis of cavernous hemangiomas of the liver using a new three-headed, high-resolution dedicated SPECT system. Of 19 patients referred with a total of 38 lesions seen on CT, US, or MRI, 14 patients had 24 lesions that were hemangioma-positive with SPECT (all true-positives). Six of these 14 patients also had 9 hemangioma-negative lesions; all were less than or equal to 1.3 cm in size and false-negative. The remaining five patients had hemangioma-negative lesions only (1 false-negative, 4 true-negatives). Two hemangiomas were seen by SPECT that were not detected by CT, US, or MR. The sensitivity for hemangiomas greater than or equal to 1.4 cm. was 100% (20/20). The sensitivity was 33% for lesions 0.9-1.3 cm, and 20% for lesions less than or equal to 0.8 cm. The smallest hemangioma detected was 0.5 cm. These results show a definite improvement in sensitivity with high-resolution triple-headed SPECT over previously reported results using single-headed SPECT. High-resolution SPECT has improved our ability to detect small cavernous hemangiomas of the liver.

Computed tomography of the normal pericardium.

Computed tomographic (CT) scans of 100 patients were reviewed to establish the normal thickness of the pericardium. The pericardium was identified in 95 of 100 patients. The mean width of the thinnest portion of the pericardium was 2.0 +/- 0.6 mm (1 SD) in males (N = 46) and 2.3 +/- 0.5 mm (1 SD) in females (N = 49). The mean width of the combined series was 2.2 +/- 0.6 mm (N = 95). In none of the 95 hearts reviewed was the width of the thinnest portion of the pericardium greater than 4 mm. Focal changes in width of the pericardium were a normal finding in patients without documented pericardial disease.

High-resolution multiplanar CT images of the larynx.

The standard technique for computed tomographic evaluation of the larynx utilizes 5-mm contiguous transaxial sections. Multiplanar images reformatted with these sections have not been of clinical use. We have evaluated the practicality of utilizing coronal and sagittal reformatted images produced from contiguous 1.5-mm transaxial sections. The technique of rapid sequential scanning with automatic table incrementation allows 36 contiguous thin section scans to be acquired in less than 9 minutes. Phantom studies showed a marked improvement in spatial resolution with thin section reconstructions. Preliminary clinical evaluation shows visualization of smaller structures with improved edge definition of both low- and high-contrast structures.

Computed tomography in the detection of abdominal metastases from malignant melanoma.

Malignant melanoma is an unpredictable and often virulent cutaneous malignancy. Although computed tomography is the most sensitive method for detection of intrathoracic metastases, its value in the assessment of abdominal spread has not been fully defined. We evaluated the sites of CT-identified metastases in 70 patients with pathologically confirmed malignant melanoma. Results were correlated with Clark's level, Breslow thickness, site of the primary, and clinicopathologic stage. CT detected enlarged abdominal or pelvic lymph nodes in 75% of patients with Clark's level 5 lesions but only in 24% and 33% of those with level 3 and 4, respectively. Patients with deep primary lesions of the lower extremities had a high frequency of pelvic node metastases. Liver and splenic metastases were detected in up to 25% of patients with level 4 or 5 melanoma. Adrenal and subcutaneous metastases were frequently discovered as were unsuspected nodules at the lung bases.

A CT technique to evaluate the development of carcinogen-induced neoplasia in the rat liver.

Hepatocellular neoplasms, including neoplastic nodules (NN), are the most commonly induced tumors resulting from chemical carcinogen evaluation. Our objective was to image neoplastic nodules using computed tomography. In a preliminary study using rats with diethylnitrosamine (DEN) induced tumors, lesions smaller than 1.5 cm were difficult to identify by CT. Since NN do not take up excess iron whereas normal liver does accumulate iron, we studied iron as a CT contrast material. Hemochromatosis was induced in 15 control rats by subcutaneous injections of iron dextran. A linear increase in iron-loading dose produces a linear CT liver enhancement (r = 0.97): 68, 80, 84, 94, and 104 HU at 0, one, two, four, and six weeks, respectively. No loss of enhancement was noted ten weeks later. Rat hepatic tissue was chemically analyzed after a similar iron-loading regimen. The iron concentration (microgram/g hepatic tissue) progressively increased during the first four weeks of loading and remained stable for three weeks following iron-loading. Four animals that had been given DEN and iron were examined by CT scanning to detect small NN. Iron-enhanced CT allowed the visualization of small lesions (less than 5 mm). Histopathologic sections confirmed a homogeneous pattern of iron uptake in normal liver with a deficiency of iron in NN. We conclude that CT scanning following iron-loading is a noninvasive method to detect small nodules and may provide a method to study the progression or regression of small liver nodules in rodents.

Computed tomographic evaluation of an experimental model for pyogenic liver abscesses.

Computed tomography (CT) was used to evaluate 15 rabbits with experimentally induced liver abscesses. The animals were examined both before and after intravenous contrast injection. After sacrificing the animals, postfreeze CT scans were made to mark the abdomen for 1-cm thick whole body sections for correlating the gross pathology with the results of the CT scans. CT detected 15 abscesses in 13 of the 14 rabbits with true positive lesions. Ten abscesses less than 1.4 cm in diameter were not detected by CT. Contrast agent enhancement was helpful in 70% of the studies. These abscesses have characteristics similar to human liver abscesses, but there was more gas and calcium in the experimentally induced abscesses than is encountered in humans with hepatic abscesses. The model and its CT characteristics appear well suited for future studies in the diagnosis and treatment of liver abscesses.