Automatable lipid bilayer formation and ion channel measurement using sessile droplets.

Artificial lipid bilayer membranes have been used to reconstitute ion channels for scientific and technological applications. Membrane formation has traditionally involved slow, labor intensive processes best suited to small scale laboratory experimentation. We have recently demonstrated a high throughput method of membrane formation using automated liquid-handling robotics. We describe here the integration of membrane formation and measurement with two methods compatible with automation and high throughput liquid-handling robotics. Both of these methods create artificial lipid bilayers by joining lipid monolayers self-assembled at the interface of aqueous and organic phases using sessile aqueous droplets in contact with a measurement electrode; one using a pin tool, commonly employed in high throughput fluid handling assays, and the other using a positive displacement pipette. Membranes formed with both methods were high quality and supported measurement of ion channels at the single molecule level. Full automation of bilayer production and measurement with the positive displacement pipette was demonstrated by integrating it with a motion control platform.

Hepatic alpha-smooth muscle actin expression in hepatitis C patients before and after interferon therapy.

BACKGROUND/AIMS: The mechanism of hepatic fibrogenesis with chronic viral hepatitis is not well understood. Persistent activation of hepatic stellate cells is felt to play a role in the development of fibrogenesis and progression to cirrhosis. METHODOLOGY: We determined the expression of hepatic alpha-smooth muscle actin, a marker of hepatic stellate cell activation, in 29 patients with chronic hepatitis C and varying degrees of liver injury and fibrosis. In addition to a baseline evaluation, we assessed the effect of interferon therapy on alpha-smooth muscle actin expression in 11 patients, including 6 with a sustained response to therapy. Specimens were evaluated by light microscopy for grade of inflammation and stage of fibrosis. Expression of alpha-smooth muscle actin was assessed semiquantitatively by immunohistochemical staining. RESULTS: At baseline, all patients had alpha-smooth muscle actin expressed within the liver without an obvious correlation with the severity of liver injury. However, among sustained responders, a reduction in hepatic necroinflammatory activity was associated with a trend towards a decrease in alpha-smooth muscle actin expression. This however did not reach statistical significance. CONCLUSIONS: Hepatic alpha-smooth muscle actin expression, as a marker of hepatic stellate cell activation appears reversible and tends to correlate with necroinflammatory activity.

Repetitive self-limited acute pancreatitis induces pancreatic fibrogenesis in the mouse.

The role of repetitive acute injury in the pathogenesis of chronic pancreatitis remains unknown. To determine if repetitive injury induced by pancreatic hyperstimulation would reproduce the characteristic features of human chronic pancreatitis, acute reversible pancreatic injury was induced in mice by twice weekly cerulein treatment, 50 microg/kg/hr x 6 hr, for 10 weeks. Procollagen alpha1(I) mRNA was markedly increased by week 2. Sirius red staining of interstitial collagen demonstrated progressive accumulation of extracellular matrix surrounding acinar units and in interlobular spaces. Atrophy, transdifferentiation of acinar units to ductlike tubular complexes, and dilatation of intraacinar lumina also developed. Electron microscopy demonstrated the presence of stromal cells in areas of fibrosis with morphologic characteristics of pancreatic stellate cells. These findings demonstrate that, in a murine model, repetitive acute injury to the pancreas by hyperstimulation can reproduce the major morphological characteristics of human chronic pancreatitis.

Comparison of genetic heterogeneity of hepatitis C viral RNA in liver tissue and serum.

OBJECTIVE: Hepatitis C virus (HCV) is known to be heterogeneous and to circulate as a group of closely related quasispecies in individual patients, although hepatic viral genetic characteristics have not been well documented. METHODS: Matched serum and liver samples were tested by reverse transcription polymerase chain reaction amplification and single stranded conformation polymorphism analysis of the hypervariable portion of the E2/NS1 region of the HCV genome. The number of quasispecies was compared with the amount of HCV RNA, HCV genotyping, and infection with the hepatitis G virus. RESULTS: Sixteen of 40 patients had HCV RNA detectable in serum and liver. The HCV genotype was identical in serum and liver of all but one case. HCV RNA levels were approximately 10-fold higher in liver than serum. The number of HCV quasispecies in serum ranged between two and six (median 3.0) and in the liver between 2 and 19 (median 3.5, mean liver/serum ratio 1 to 6.3, median 1.8). The number of quasispecies in liver was equal to or greater than that in serum in all cases. HGV infection was found in 14 cases and did not influence serum or hepatic levels of HCV RNA. CONCLUSIONS: The number of hepatic HCV quasispecies usually exceeds that in serum, independent of the amount of HCV RNA and HCV genotype. This finding is compatible with clearance of some quasispecies from serum, but not liver, by putative neutralizing antibodies.

Nefazodone-induced liver failure: report of three cases.

BACKGROUND: Liver failure is a rare but devastating result of drug toxicity. OBJECTIVE: To describe three cases of subfulminant liver failure that were probably caused by nefazodone, a new antidepressant that is a synthetically derived phenylpiperazine. DESIGN: Case series. SETTING: Two university medical centers and a children's hospital. PATIENTS: Three women 16 to 57 years of age. INTERVENTION: Two patients underwent liver transplantation; the third was listed for transplantation but subsequently improved. MEASUREMENT: Liver biopsy. RESULTS: Nefazodone was administered for 14 to 28 weeks before the onset of symptoms. The duration of jaundice before onset of encephalopathy ranged from 4 to 6 weeks. All cases of liver failure had similar histologic appearance, with prominent necrosis in the centrolobular areas (zone 3). One patient had successful liver transplantation, one underwent transplantation but died, and one improved without transplantation. The temporal onset of disease after the start of nefazodone therapy suggested severe hepatocellular injury caused by the drug. CONCLUSIONS: Because nefazodone seems to cause severe hepatocellular injury in an idiosyncratic manner, routine liver chemistries should be performed before starting nefazodone therapy and patients should be monitored regularly. Therapy should be discontinued if liver enzyme concentrations become abnormal.

Fibronectin and cytokines increase JNK, ERK, AP-1 activity, and transin gene expression in rat hepatic stellate cells.

Cytokines, growth factors, and alterations in the extracellular matrix composition may play a role in maintaining hepatic stellate cells (HSC) in the activated state that is responsible for hepatic fibrogenesis. However, the signal transduction pathways that are stimulated by these factors in HSC remain to be fully elucidated. Recent evidence indicates that the mitogen-activated protein kinase (MAPK) family, including c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK), plays an important role in the cellular response to stress. The aims of this study were to investigate whether fibronectin (FN) or the inflammatory cytokines interleukin-1alpha (IL-1alpha) and tumor necrosis factor-alpha (TNF-alpha) activate JNK, ERK, and AP-1 activity in HSC and induce the gene expression of the matrix metalloproteinase transin. Treatment of HSC with FN resulted in an up to 4.5-fold increase in ERK activity and a 2.1-fold increase in JNK activity. IL-1alpha and TNF-alpha produced up to a fourfold increase in JNK activity and a twofold increase in ERK activity. We then compared the effects of FN, IL-1alpha, and TNF-alpha on AP-1 activity and metalloproteinase mRNA induction. All three compounds increased AP-1 binding and promoter activity, and transin mRNA levels were increased 1.8-fold by FN, 2.2-fold by IL-1alpha, and 2.8-fold by TNF-alpha. Therefore, FN and inflammatory cytokines increase MAPK activity, stimulate AP-1 activity, and increase transin gene expression in HSC. Signal transduction pathways involving the MAPK family may play an important role in the regulation of matrix metalloproteinase expression by cytokines and FN in HSC.

Disinfection of impressions. Methods and effects on accuracy.

An understanding of the methods and mechanics of sterilization and disinfection is necessary to prevent bacterial cross-contamination. Since sterilization of impressions is not possible, because of the high temperature and time needed, disinfection is the method of choice. The most effective means of disinfecting impressions is to immerse them in disinfectant solution for 30 minutes. Most hydrophobic impression materials exhibit minimal distortion when disinfected in this manner. Irreversible hydrocolloid (alginate) and hydrophilic silicones distort when immersed and can be disinfected by spraying and placing in a sealed plastic bag for manufacturer's recommended time. Internal disinfection (replacing water with disinfectant before impression taking) is the method of choice for alginate, since it allows immediate pouring of the impression after removal from the oral cavity.

Presentation and management of lymphatic cyst of the colon: report of a case.

PURPOSE: We report a case of lymphatic cyst of the colon in a 43-year-old white female previously treated with chemotherapy for a B cell lymphoma. METHODS: Radiographic, endoscopic, and pathologic evaluation of the cyst is presented. Surgical resection of this lesion was undertaken because of the size of the lesion, risk of obstruction, and history of an underlying malignancy. A recent review of the literature in relation to clinical symptoms, signs, diagnosis, and treatment of lymphatic cysts is presented. CONCLUSION: Lymphatic cysts of the colon are benign submucosal lesions that are being recognized with increasing frequency. Treatment options include endoscopic polypectomy for lesions smaller than 2 cm and surgical resection for larger lesions if there is a threat of obstruction or question of an underlying colorectal malignancy.

Curing lights: changes in intensity output with use over time.

Intensity output of curing lights affects cure depth and time needed for complete polymerization of resin composites. In this study, changes in intensity output of Max Curing Lights (L.D. Caulk Co., Milford, DE) were compared over time. With a Demetron Model 100 Curing Radiometer (Demetron Research Corp., Danbury, CT), 201 curing lights (from 1 to 3 years old) were tested. The 1- and 2-year-old lights had mean outputs of 423 mW/cm2; the 3-year-old lights had a mean output of 376 mW/cm2. An analysis of variance (ANOVA) and post-hoc Scheffe test demonstrated a significant effect of use over time on intensity output of these lights.

Effect of vitamin E supplementation on hepatic fibrogenesis in chronic dietary iron overload.

It has been suggested that lipid peroxidation plays an important role in hepatic fibrogenesis resulting from chronic iron overload. Vitamin E is an important lipid-soluble antioxidant that has been shown to be decreased in patients with hereditary hemochromatosis and in experimental iron overload. The aim of this study was to determine the effects of vitamin E supplementation on hepatic lipid peroxidation and fibrogenesis in an animal model of chronic iron overload. Rats were fed the following diets for 4, 8, or 14 mo: standard laboratory diet (control), diet with supplemental vitamin E (200 IU/kg, control + E), diet with carbonyl iron (Fe), and diet with carbonyl iron supplemented with vitamin E (200 IU/kg. Fe + E). Iron loading resulted in significant decreases in hepatic and plasma vitamin E levels at all time points, which were overcome by vitamin E supplementation. Thiobarbituric acid-reactive substances (an index of lipid peroxidation) were increased three- to fivefold in the iron-loaded livers; supplementation with vitamin E reduced these levels by at least 50% at all time points. Hepatic hydroxyproline levels were increased twofold by iron loading. Vitamin E did not affect hydroxyproline content at 4 or 8 mo but caused an 18% reduction at 14 mo in iron-loaded livers. At 8 and 14 mo, vitamin E decreased the number of alpha-smooth muscle actin-positive stellate cells in iron-loaded livers. These results demonstrate a dissociation between lipid peroxidation and collagen production and suggest that the profibrogenic action of iron in this model is mediated through effects which cannot be completely suppressed by vitamin E.

Congestive heart failure: increased cardiac and extracardiac atrial natriuretic peptide gene expression.

OBJECTIVES: The present investigation was designed to determine if atrial natriuretic peptide (ANP) gene expression increases in extracardiac as well as within the heart in congestive heart failure. METHODS: Congestive heart failure (CHF) was induced by producing cardiac hypertrophy secondary to an aortocaval fistula in Sprague-Dawley rats. To characterize this model, control and CHF rats had cardiac catheterizations and transthoracic echocardiography. ANP messenger RNA was measured by RNAase protection analysis in atria, ventricles, liver, colon, and stomach of CHF and sham rats and quantitated by 2-D scanning. The product of ANP gene expression was determined in each of these tissues with high performance-gel permeation chromatography. To help determine if increased degradation of atrial natriuretic peptides occur in congestive heart failure, the circulating concentrations and the excretion of the atrial natriuretic peptides into urine were measured by specific radioimmunoassays. RESULTS: ANP steady-state mRNA increased 4.2 +/- 0.05 and 4.3 +/- 0.06-fold, respectively, in the antrum of the stomach and within the heart ventricle of CHF rats compared with age-matched sham rats. ANP gene expression was present but not increased in atria, liver, and gastrointestinal tract of the CHF rats. High-performance gel permeation chromatography revealed that the product of this ANP gene expression within the stomach and heart ventricle in CHF animals was the ANP prohormone. There was not any decrease in the metabolism of these peptides by the kidney in CHF. CONCLUSIONS: ANP steady-state mRNA increases in extracardiac (i.e., stomach antrum) tissue as well as in the ventricle of the heart in CHF. The product of the ANP gene expression, i.e., the ANP prohormone is the same in the extracardiac tissues as within the heart. Whether the increased extracardiac ANP steady-state mRNA and its resultant increased atrial natriuretic peptides helps prevent bowel wall edema in CHF needs to be elucidated.

Liver transplantation for hereditary hemochromatosis.

Although the experience of orthotopic liver transplantation (OLT) for hereditary hemochromatosis (HHC) is limited, the existing data indicate that it carries a higher mortality when compared to transplantation for other causes of end-stage liver disease. Posttransplantation deaths are usually related to infectious or cardiac complications. HHC is often not diagnosed prior to OLT and one series has shown a high incidence of primary liver cancer diagnosed incidentally only at the time of transplantation. Factors that may account for the increase in postoperative mortality for HHC are the extent of iron deposition in extrahepatic sites in patients undiagnosed and thus untreated prior to transplantation. A high index of suspicion in subjects with end-stage liver disease should lead to improved diagnosis and allow for the prompt institution of either phlebotomy therapy or iron chelation therapy prior to transplantation. It is expected that these changes would reduce postoperative complications as well as improve long-term survival.

Immediate provisional and long-term anterior prosthodontics: a comprehensive approach.

The success of any anterior prosthodontic restoration depends on many factors. Considering those multiple influences during treatment planning can profoundly affect the long-term success of such cases. Comprehensive dental care involves evaluating all available restoration options for treating any given patient's condition. The dentist is obligated to present all of these options to the patient without bias. The following case reports provide a perspective from which decision-making occurs before treatment planning is accomplished.

The incidence of neurovascular complications following axillary brachial plexus block using a transarterial approach. A prospective study of 1,000 consecutive patients.

BACKGROUND AND OBJECTIVES: The present study was performed at the Crystal Clinic Surgery Center, an outpatient free-standing surgicenter specializing in orthopedic surgery, to determine the incidence of both neurologic and vascular sequelae associated with exclusive use of a transarterial approach to axillary brachial plexus block in order to assess the technique's safety and efficacy. METHODS: The prospective consecutive study involved 1,000 adult patients scheduled for surgery using axillary brachial plexus block. The transarterial approach was performed on all patients using a medium-bevel 24-gauge Jelco 1-5-inch needle. Data tabulated included the incidence of neurovascular complications and the outcome of successful axillary brachial plexus anesthesia. RESULTS: Two patients presented with a sensory paresthesia (0.2%) in the distribution of the ulnar nerve and the musculocutaneous nerve that most likely occurred during supplementation of an incomplete block. Three patients presented with upper-arm myalgias (0.3%) related to tourniquet injury. After the operation, two patients developed reflex sympathetic dystrophy, which responded to stellate ganglion blocks. Vascular complications, including transient arterial spasm in 10 of 996 (1%), unintentional intravascular injection in 2 of 996 (0.2%), and small (0-2 cm) hematoma formation in 2 of 996 (0.2%), were recognized but did not require any intervention other than close observation. The study revealed a complete block in 88.8% of cases, an incomplete block requiring supplemental local anesthesia in 10% of cases, and a complete block failure in 1.2%. CONCLUSIONS: This study demonstrated the safety and efficacy of the transarterial technique in achieving brachial plexus block.

Atrial natriuretic peptide gene expression within invertebrate hearts.

The present investigation was designed to (1) determine if atrial natriuretic factor gene expression occurs within invertebrates as well as within vertebrates; (2) determine whether the product of this gene expression is the 126-amino-acid atrial natriuretic factor prohormone or some other molecular species; and (3) evaluate within the same invertebrates if the products of atrial natriuretic factor gene expression are released into their circulation. Utilizing a very sensitive RNase protection assay it was found that atrial natriuretic peptide gene expression occurs within the heart of the oyster, Crassostrea virginica, and within the heart of the blue crab, Callinectes sapidus, but was expressed sevenfold less than in a vertebrate heart (i.e., rat, Rattus norvegiucs). High-performance gel-permeation chromatography followed by N-terminal and C-terminal atrial natriuretic factor prohormone radioimmunoassays indicated that the molecular species synthesized within the oyster and blue crab hearts was the atrial natriuretic factor prohormone. The product(s) of this atrial natriuretic factor gene expression (i.e., atrial natriuretic peptides) was found to be released into the circulation, i.e., hemolymph, of both the oyster and the blue crab.

Case report: hypoglycemia secondary to a meningioma.

Hypoglycemia secondary to a meningioma that has not metastasized to the liver has not been reported previously. A 41-year-old woman with a spinal cord meningioma first diagnosed 5 years previously with 3 recurrences in the spinal cord resulting in 4 neurosurgical procedures was admitted with a serum glucose of 23 mg/dL. Six months before the current admission, the patient was noted to have an abdominal mass of 10 cm not present on previous computed tomography. Three months later, the mass was 15.2 cm, and on the current admission, had increased to 23 cm and encased both the aorta and inferior vena cava. A needle biopsy of this mass before referral to the authors' hospital with hypoglycemia revealed that it was a meningioma. Evaluation of the etiology of the hypoglycemia, which required continuous intravenous glucose therapy, revealed that circulating insulin, C-peptide (i.e., connecting peptide), insulin-like growth factor-I (i.e., somatomedin-C) and insulin-like growth factor-II were all normal or low. Serum cortisol also was not low. Based on her endocrine evaluation, the hypoglycemia was secondary to the large mass of tumor cells, requiring a large glucose uptake to sustain its growth. After radiation therapy of 3,770 CGy to the meningioma, the patient became euglycemic without glucose supplementation.

Cirrhosis with ascites: increased atrial natriuretic peptide messenger RNA expression in rat ventricle.

BACKGROUND/AIMS: Atrial natriuretic peptides (ANPs) are increased in the circulation of cirrhotics with ascites; however, it is unknown whether this increase is caused by increased synthesis or a decrease in the metabolic processing of these peptides. ANP gene expression in the liver, atria, ventricles, and gastrointestinal tract of cirrhotic vs. control rats was studied as was their metabolism. METHODS: Sprague-Dawley rats developed cirrhosis with ascites approximately 20 weeks after weekly intragastric instillation of carbon tetrachloride. Their circulating, ascitic, and urinary levels of ANPs were measured by radioimmunoassays. ANP gene expression was measured by a ribonuclease protection assay. RESULTS: ANP gene expression was increased 2.8- to 4.1-fold in the ventricles of cirrhotic rats compared with age-matched healthy rats. ANP gene expression was present but not increased in the liver, atria, and gastrointestinal tract of cirrhotic rats. No increase of metabolic processing of these peptides was found in the circulation. Cardiac ultrasonography and catheterization revealed no ventricular dilation or increased ventricular pressure. CONCLUSIONS: Elevation of circulating ANPs with cirrhosis was associated with increased ventricular steady-state ANP messenger RNA concentrations. The increased ANP gene expression in cirrhosis seems to involve a novel mechanism not related to stretch because neither increased ventricular pressure nor dilation was present.

Circulating insulin-like growth factor I concentrations in clinically severe obese patients with and without NIDDM in response to weight loss.

Earlier studies have demonstrated decreased levels of circulating Insulin-Like Growth Factor-I (IGF-I) in patients with NIDDM and IDDM (Yde 1969; Rieu and Binoux 1985), with a return to normal in those diabetics who achieve improved metabolic control (Rieu and Binoux 1985; Ameil, Sherwin, Hintz, Gertner, Press and Tamborlane 1984) following insulin therapy. One method of improving metabolic control in clinically severe obese NIDDM patients is the gastric bypass procedure (GBP). This study revealed a significant decrease in serum IGF-I concentrations in clinically severe obese patients with NIDDM (obese NIDDM) (105 ng/dl +/- 11; n = 29) as compared with clinically severe obese patients with normal glucose tolerances (obese control) (143 +/- 11; n = 21) and lean controls (177 +/- 14; n = 19) (p < 0.001). Following a GBP, IGF-I levels increased in the NIDDM group (142 ng/dl +/- 13.0; n = 20) to the extent that no significant difference was seen between postoperative NIDDM, obese controls, and lean controls. Postoperative IGF-I levels in the obese controls (151 +/- 14; n = 9) revealed no difference from preoperative levels. Postoperative obese NIDDM and obese control had a 28% and 29% decrease, respectively, in weight, with no difference between the groups in respect to Body Mass Indices. The NIDDM postoperative group revealed reductions in levels of HbA1C, insulin, and glucose concurrent with elevations in IGF-I when compared with controls. We conclude that improvement in glucose control led to the increase in IGF-I levels.