PVX-tolerant potato development using a nucleic acid-hydrolyzing recombinant antibody.

3D8 scFv, a catalytic recombinant antibody developed in the MRL mouse, exhibits nucleic acid-hydrolyzing activity. Previous studies have demonstrated that tobacco plants harboring 3D8 scFv antibodies showed broad-spectrum resistance to infection by both DNA and RNA viruses. In this study, potatoes were transformed with the 3D8 scFv gene and screened by potato virus X (PVX) challenge. Starting with the T0 and T1 potato lines, PVX-tolerant T1 potatoes were identified in the field and characterized by ELISA and RT-PCR analysis. T2 potatoes were propagated for T3 generation and additional virus challenges in the field, and 44% of the 3D8 scFv T3 transgenic potatoes grown in GMO fields were found to be tolerant to PVX infection. Tubers from PVX-tolerant T3 lines were 60% bigger and 24% heavier, compared with tubers from PVX-susceptible transgenic lines and wild-type potatoes. Three-step virus challenge experiments and molecular characterization techniques were used for plants grown in growth chambers or fields to identify 3D8 scFv-transgenic, PVX-tolerant potatoes. These studies also revealed that the viral tolerance enabled by 3D8 scFv persisted during asexual propagation.

Biological Behavior of Osteoblast Cell and Apatite Forming Ability of the Surface Modified Ti Alloys.

Titanium as one kind of biomaterials comes in direct contact with the body, making evaluation of biocompatibility an important aspect to biomaterials development. Surface chemistry of titanium plays an important role in osseointegration. Different surface modification alters the surface chemistry and result in different biological response. In this study, three kinds of mixed acid solutions were used to treat Ti specimens to induce Ca-P formation. Following a strong mixed acid activation process, Ca-P coating successfully formed on the Ti surfaces in simulated body fluid. Strong mixed acid increased the roughness of the metal surface, because the porous and rough surface allows better adhesion between Ca-P coatings and substrates. After modification of titanium surface by mixed acidic solution and subsequently H2O2/HCL treatment evaluation of biocompatibility was conducted from hydroxyapatite formation by biomimetic process and cell viability on modified titanium surface. Nano-scale modification of titanium surfaces can alter cellular and tissue responses, which may benefit osseointegration and dental implant therapy. Results from this study indicated that surface treatment methods affect the surface morphology, type of TiO2 layer formed and subsequent apatite deposition and biological responses. The thermo scientific alamarblue cell viability assay reagent is used to quantitatively measure the viability of mammalian cell lines, bacteria and fungi by incorporating a rapid, sensitive and reliable fluorometric/colorimetric growth indicator, without any toxic and side effect to cell line. In addition, mixed acid treatment uses a lower temperature and shorter time period than widely used alkali treatment.

Biomimetic Deposition of Hydroxyapatite by Mixed Acid Treatment of Titanium Surfaces.

A simple chemical method was established for inducing bioactivity of Ti metal. In the present study, two kinds of mixed acid solutions were used to treat Ti specimens to induce Ca-P formation. Following a strong mixed acid activation process, Ca-P coatings successfully formed on the Ti surfaces in the simulated body fluid. Strong mixed acid etching was used to increase the roughness of the metal surface, because the porous and rough surfaces allow better adhesion between Ca-P coatings and substrate. Nano-scale modification of titanium surfaces can alter cellular and tissue responses, which may benefit osseointegration and dental implant therapy. Some specimens were treated with a 5 M NaOH aqueous solution, and then heat treated at 600 °C in order to form an amorphous sodium titanate layer on their surface. This treated titanium metal is believed to form a dense and uniform bone-like apatite layer on its surface in a simulated body fluid (SBF). This study proved that mixed acid treatment is not only important for surface passivation but is also another bioactive treatment for titanium surfaces, an alternative to alkali treatment. In addition, mixed acid treatment uses a lower temperature and shorter time period than alkali treatment.

Peripheral venous pressure as an alternative to central venous pressure in patients undergoing laparoscopic colorectal surgery.

BACKGROUND: Peripheral venous pressure (PVP) is strongly correlated with central venous pressure (CVP) during various surgeries. Laparoscopic surgery in the Trendelenburg position with pneumoperitoneum typically increases CVP. To determine whether PVP convincingly reflects changes in CVP, we evaluated the correlation between PVP and CVP in patients undergoing laparoscopic colorectal surgery. METHODS: Both CVP and PVP were measured simultaneously at predetermined time intervals during elective laparoscopic colorectal surgery in 42 patients without cardiac disease. The pairs of venous pressure measurements were analysed for correlation, and the Bland-Altman plots of repeated measures were used to evaluate the agreement between CVP and PVP. RESULTS: A total of 420 data pairs were obtained. The overall mean CVP was 11.3 (sd 4.5) mm Hg, which was significantly lower than the measured PVP of mean 12.1 (4.5) mm Hg (P=0.005). There was a strong positive correlation between overall CVP and PVP (correlation coefficient=0.96, P<0.0001). The mean bias (PVP-CVP) corrected for repeated measurements using random-effects modelling was 0.9 mm Hg [95% confidence interval (CI) 0.54-1.19 mm Hg] with 95% limits of agreement of -1.2 mm Hg (95% CI -1.75 to -0.62 mm Hg) to 2.9 mm Hg (95% CI 2.35-3.48 mm Hg). CONCLUSIONS: PVP displays a strong correlation and agreement with CVP under the increased intrathoracic pressure of pneumoperitoneum in the Trendelenburg position and may be used as an alternative to CVP in patients without cardiac disease undergoing laparoscopic colorectal surgery.

Effect of glass phase on the dissolution of hydroxyapatite.

Nano size defect formation at grain boundary during the dissolution of hydroxyapatite in water was evaluated by adding several sintering additives for sinterability enhancement. In the case of sintered pure hydroxyapatite, significant dissolution occurred after immersion in distilled water or in simulated body fluid. The dissolution initiated at the grain boundaries creating nano-size defects like small pores that afterwards grew up to micro scale by increasing immersion time. This dissolution resulted in grain separation at the surfaces and finally in fracture. The dissolution concentrated on the grains adjacent to pores rather than those in the dense region. So hydroxyapatite ceramics containing glass powders were prepared to prevent the dissolution by strengthening grain boundary. Calcium silicate and phosphate glasses were added at 0 to 10 mass% and sintered at 1200 degrees C for 2 h in air with moisture protection. Glass phase was incorporated into hydroxyapatite to act as the sintering aid followed by crystallization in order to improve the mechanical properties without reducing biocompatibility. Dissolution tests, as well as X-ray diffraction and SEM showed little decomposition of hydroxyapatite to secondary phases and the fracture toughness increased compared to pure hydroxyapatite.

The presence of a natriuretic factor in urine of patients with chronic uremia. The absence of the factor in nephrotic uremic patients.

A gel filtration fraction of serum from chronically uremic patients has been shown previously to produce natriuresis in the rat. In the present studies, the same fraction from urine of uremic patients and normal subjects was studied for its natriuretic activity. Urine samples were obtained from 17 chronically uremic patients (mean glomerular filtration rate [GFR], 8.7 ml/min; mean fractional sodium excretion [FE(Na)], 5.7%), and 14 normal subjects. The fraction from the uremic patients produced a significant increase in absolute sodium excretion (U(Na)V) and FE(Na); the fraction from normal subjects had no statistically significant effect on either U(Na)V or FE(Na); and the difference between the response to the uremic vs. normal fractions was highly significant for both parameters of sodium excretion. When a more concentrated urine fraction from uremic patients was administered, a striking natriuresis was observed with values for FE(Na) rising to levels as high as 12%. Studies also were performed on eight patients with far advanced chronic renal insufficiency and the nephrotic syndrome. The serum fraction was studied in each of these patients and the urine fraction in three. For the group, U(Na)V in the assay rats decreased by 0.87 mueq/min and FE(Na) decreased by 1.35% after infusion of the serum fraction. These results differ significantly from those of patients with chronic uremia without the nephrotic syndrome. The data are consistent with the view that the increased activity of the natriuretic factor in the serum of chronically uremic patients is not due to failure of excretion; rather it relates either to an increased rate of production and/or a decreased rate of degradation. The data also show that the inhibitor is detectable when FE(Na) is increased, but not when uremia is associated with a sodium-retaining state.

A natriuretic factor in the serum of patients with chronic uremia.

Sera from chronically uremic and normal individuals were subjected to gel filtration with Sephadex G-25 and the same fraction of both was infused into rats with a decreased nephron population to determine the effects on sodium excretion. Sodium excretion rate and fractional sodium excretion increased slightly with the normal fractions; but the increase in both functional parameters produced by the uremic fractions was substantially and significantly greater. The natriuresis could not be explained by associated changes in glomerular filtration rate (GFR), para-aminohippurate (PAH) clearance, filtration fraction, hematocrit, or blood pressure. The possibility thus exists that the inhibitor affected some component part of the transepithelial sodium transport system. The elution characteristics of the fraction plus certain of its physicochemical properties suggest that the inhibitor of sodium reabsorption by the rat nephron may be identical with the inhibitor of PAH uptake by kidney slices and the inhibitor of transepithelial sodium transport by the frog skin and toad bladder previously found in the serum of chronically uremic patients.

On the influence of the natriuretic factor from patients with chronic uremia on the bioelectric properties and sodium transport of the isolated mammalian collecting tubule.

A gel filtration fraction of urine from patients with chronic renal disease (natriuretic factor) has been shown previously to cause natriuresis in rats and to inhibit sodium transport in the isolated toad bladder. The effect of this fraction on transtubular potential difference and sodium transport was examined on the isolated perfused cortical collecting tubule of the rabbit. A rapid inhibition of potential difference from -22.5 mV to -12 mV (P less than 0.001) was observed when the fraction was applied to the peritubular surface. This effect was accompanied by a decrease in net sodium flux from 6.29 to 3.21 pmol/cm per s (P less than 0.001). Unidirectional fluxes using isotopic sodium revealed that the inhibition of net sodium transport was due to a decrease in flux from the lumen to the peritubular surface, i.e., an inhibition of active sodium transport. There was no change in sodium flux in the reverse direction. These changes were all rapidly reversed by removal of the fraction from the peritubular surface. The addition of the fraction to the lumen had no effect on potential difference or net sodium flux. Control studies using the same fraction from the urine of normal subjects had no effect on any of the parameters studies. Where both a uremic and a normal fraction were sequentially applied to the peritubular surface of the same tubule, inhibition of potential difference was obtained only with the former. In the light of evidence implicating the collecting duct fraction from normal animals, the data are consistent with the view that the natriuretic factor may be biologically important in the regulation of sodium balance via it's regulatory role in active sodium transport in the collecting tubule.

An inhibitor of sodium transport in the urine of dogs with normal renal function.

The urine and serum of chronically uremic patients and dogs contain an inhibitor of sodium transport that reduces short-circuit current (SCC) in the toad bladder and produces natriuresis in the rat. The present studies represent an effort to determine whether the same inhibitor is detectable in urine of normal dogs maintained on a dosium intake varying from 3 to 258 meq/day. Observations were made with and without fludrocortisone. The same Sephadex G-25 gel filtration fraction previously shown to contain the "uremic" inhibitor was tested in both the isolated toad bladder and rat bioassay systems. The fraction from dogs maintained on 258 meq qodium plus 0.2 mg fludrocortisone/day consistently inhibited SCC in the toad bladder and induced a natriuresis in the rat (P less than 0.001). The fraction from dogs on the same sodium intake without fludrocortisone was also natriuretic (P less than 0.01) but did not inhibit SCC significantly. In contrast, the fraction from dogs fed 3 meq sodium with fludrocortisone or 91 meq sodium without fludrocortisone had no significant effect in either assay system. Thus, an inhibitor of sodium transport has been found in the urine of nonuremic dogs. Both the degree of natriuresis in the rat and the degree of inhibition of SCC in the toad bladder correlated with the state of sodium balance which ensued in the dog.

Sodium transport in turtle erythrocytes. Apparent stimulation of exchange diffusion by anaerobiosis.

Studies were performed on Na and K transport by red blood cells of the freshwater turtle under anaerobic and aerobic conditions. Although it had previously been assumed that cation transport in turtle red blood cells was dependent on respiration, the present data show greater Na efflux rates in N(2) than in O(2). However, ouabain inhibited Na transport by the same amount quantitatively in O(2) and N(2) gas phases. Thus there was no difference in ouabain-sensitive or "pump" Na transport rates. Na influx rates were higher in nitrogen than in air and potassium influx rates were not significantly different under aerobic and anaerobic conditions. Moreover in the absence of sodium in the bathing medium no difference between air and nitrogen could be discovered. Finally with ethacrynic acid plus ouabain there was an additional decrease in Na efflux but there was a persisting difference between air and nitrogen. These studies do not rule out the existence of a ouabain-insensitive ethacrynic acid-inhibitable flux; however, they suggest that at least part of the activation of Na efflux observed in N(2) was due to increased exchange diffusion.

Overexpression of the transforming growth factor-beta-inducible gene betaig-h3 in anterior polar cataracts.

PURPOSE: In anterior polar cataracts and the fibrosis that can occur after cataract surgery, lens epithelial cells synthesize abundant extracellular matrix molecules and transdifferentiate into myofibroblast-like cells. Transforming growth factor (TGF)-beta has been implicated as a key player in these cataractous changes. The purpose of this study was to determine whether the TGF-beta-inducible gene h3 (betaig-h3) is expressed in lens epithelial cells from patients with anterior polar cataracts and to test whether betaig-h3 is induced by TGF-beta in cultured lens epithelial cells. METHODS: Lens epithelial cells attached to the anterior capsules of human cataractous lenses and noncataractous lenses were examined for the expression of betaig-h3 mRNA and protein using reverse transcription-polymerase chain reaction and immunohistochemical analyses. The effect of TGF-beta on betaig-h3 gene expression was also tested in human lens epithelial B-3 cells using Northern and Western blot analyses. RESULTS: betaig-h3 mRNA was not detected in lens epithelial cells from patients with clear lenses or patients with nuclear cataracts. Significant expression of mRNA for betaig-h3 was observed in lens epithelial cells from patients with anterior polar cataracts. Immunohistochemical analysis using anti-betaig-h3 antiserum indicated that betaig-h3 protein was present within the subcapsular plaques of anterior polar cataracts. Treatment of human lens epithelial B-3 cells with TGF-beta1 led to an increase in betaig-h3 mRNA and the secretion of betaig-h3 protein into the culture medium. CONCLUSIONS: betaig-h3 may serve as a marker for anterior polar cataracts in addition to previously known proteins, fibronectin, type I collagen, and alpha-smooth muscle actin. The functions of this protein in lens pathology need to be further investigated.

Effect of endogenous nitric oxide on tumour necrosis factor-alpha-induced leukosequestration and IL-8 release in guinea-pigs airways in vivo.

1. Tumour necrosis factor-alpha (TNF-alpha) is implicated in the pathogenesis of many pulmonary and airway diseases. TNF-alpha stimulation may release interleukin-8 (IL-8) in airways mediated via an increase in intracellular oxidant stress. In the present study, we have assessed leukosequestration and IL-8 release in the airways in response to intratracheal administration of human recombinant TNF-alpha, and examined the modulatory role of endogenous NO by pretreatment with a NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME). 2. TNF-alpha (10(2)-10(-4) u) was administered intratracheally in male guinea-pigs which were anaesthetized with urethane and were ventilated artificially. TNF-alpha induced a time- and dose-related increase in neutrophil numbers and a concomitant increase in human IL-8 equivalent level retrieved from bronchoalveolar lavage (BAL) with the peak effect at 10(3) u at 6 h of TNF-alpha injection (late phase). Intratracheal administration of recombinant human (rh)IL-8 (0.025, 0.25, 2.5 ng) producing a similar range of human IL-8 equivalent levels in BAL as measured in our results induced neutrophil recovery in BAL fluid to a similar extent. Administration of anti-IL-8 antibody prevented the late phase of neutrophil recruitment induced by TNF-alpha or rhIL-8. 3. Pretreatment with L-NAME significantly enhanced the TNF-alpha (10(3) u)-induced neutrophil recruitment and human IL-8 equivalents production at 6 h, but not at 1 h of TNF-alpha administration (early phase). L-Arginine reversed the responses to L-NAME. Pretreatment with 0.2% DMSO (i.v.) significantly inhibited TNF-alpha-induced neutrophil recruitment and human IL-8 equivalents release both in the early and late phase of the responses. Pretreatment with DMSO also inhibited the enhancement effect of L-NAME on the late phase of TNF-alpha-induced responses. DMSO failed to modify exogenous rhIL-8-induced neutrophil recruitment. Neither L-NAME nor DMSO alone induced any significant change in neutrophil numbers or human IL-8 equivalent level in BAL fluid. 4. Neutrophil depletion by cyclophosphamide pretreatment failed to modify TNF-alpha-induced human IL-8 equivalent release. 5. The expression of beta 2-integrin, CD11b/CD18 on neutrophils was increased only in the late but not early phase of TNF-alpha stimulation. L-NAME failed to modify these responses. 6. In conclusion, we demonstrated that NO may be an important endogenous inhibitor of TNF-alpha-induced leukocyte chemotaxis via inhibition of IL-8 production. Thus, the production of NO in airway inflammatory diseases may play a negative feedback role in self-limiting the magnitude of inflammatory responses.

Lipopolysaccharide enhances neurogenic plasma exudation in guinea-pig airways.

1. Lipopolysaccharide (LPS) is implicated in many pulmonary and airway inflammatory diseases. Tachykinins released from nerve endings increase vascular permeability. In this study, we have assessed the enhancement by LPS of tachykinin-mediated plasma exudation in guinea-pig airways, and examined the role of oxidants as well as leukocyte adherence. 2. LPS (100 microg kg(-1), i.v.) was administered 0-3 h before bilateral electrical stimulation of the cervical vagus nerves in animals anaesthetized with urethane and ventilated. Vagal stimulation increased vascular permeability in the airways. LPS enhanced the vagally-mediated plasma exudation with the peak effect at 1 h after LPS administration. LPS alone induced no significant plasma exudation. LPS also enhanced exogenous substance P (10(-8) mol kg(-1), i.v.)-induced plasma exudation. 3. The NK-1 receptor antagonist L-732,138 abolished vagally-induced plasma exudation and significantly inhibited the enhancement by LPS. Pretreatment with superoxide dismutase (SOD, 5000 U kg(-1), i.p.) did not affect the vagally-induced plasma exudation, but inhibited the LPS-enhanced neurogenic plasma leakage. The LPS-enhanced vagally-induced plasma exudation was not completely inhibited by either L-732,138 or SOD pretreatment alone, but was blocked by the combination of both pretreatments. 4. Neutrophil depletion by cyclophosphamide alone did not influence vagally-induced plasma exudation, but significantly inhibited the LPS-enhanced response. 5. In conclusion, we have demonstrated LPS enhanced neurogenic plasma exudation by augmenting the response to tachykinins, partly through NK-1 receptors, to directly increase vascular permeability or to enhance leukocyte adhesion-mediated endothelial cell injury. Tachykinins released from nerve endings may contribute to endotoxin-related airway inflammatory responses.

Renal function of baboons (Papio hamadryas) with a remnant kidney, and impact of different protein diets.

To assess progression of renal disease and the effects of protein intake in a species phylogenically close to man, 10 young adult baboons (Papio hamadryas) were subjected to 20 to 30% infarction of the left kidney and, two months later, to right nephrectomy. They were then randomized to a synthetic diet containing either 8% or 25% protein. Hemodynamic and metabolic measurements were obtained in awake animals every four months. Baseline mean blood pressure, inulin clearance, protein and urea nitrogen excretion in intact animals on 15% protein averaged 75.5 +/- 3.5 (SE) mm Hg, 42.9 +/- 2.7 ml/min, 52 +/- 4.3 mg/24 hr, and 3.8 +/- 0.4 g/24 hr, respectively. At 12 months, values in the same baboons with a remnant kidney on 8% versus 25% protein averaged 100.6 versus 96.7 mm Hg, 29.2 versus 54.9 ml/min (P less than 0.01), 111 versus 108 mg/24 hr, and 3.4 versus 11.0 g/24 hr (P less than 0.001), respectively. Electron microscopic examination of renal biopsies obtained eight months after nephrectomy was normal but for slightly increased mesangial matrix in three animals. Thus, blood pressure increased (P less than 0.01), proteinuria doubled (P less than 0.01) and adaptations in GFR developed within four months of renal mass reduction, without significant changes occurring between four and 12 months. The adaptations in GFR were markedly attenuated by low protein intake. Further follow-up is necessary to assess progression of renal disease and the impact of different protein diets.

Characterization, stability and in-vivo distribution of asialofetuin glycopeptide incorporating DSPC/CHOL liposomes prepared by mild cholate incubation.

In this study, a small triantennary asialoglycopeptide of fetuin (A-F2) was used as a ligand to direct liposomes to hepatocytes. A-F2 was cleaved from asialofetuin, purified, conjugated with fatty acids and incorporated into pre-formed sonicated DSPC/Chol (2:1) liposomes. A mild cholate incubation method for incorporating the A-F2 ligand on pre-formed vesicles was used. In preliminary in vivo experiments 111In3+ encapsulated in A-F2/palmityl liposomes was seen to accumulate in the liver of mice significantly faster than when encapsulated in non-ligand bearing liposomes of the same lipid composition (studied before), justifying further investigation of this system. The presence of the A-F2/fatty acid conjugate in a functional form on the vesicle surface was confirmed by their reversible agglutination in the presence of Ricinus communis agglutinin (RCA120). Effects of ligand incorporation on the vesicle size distribution, z-potential, membrane integrity and stability were monitored. The results demonstrate that highest ligand incorporation was achieved when liposomes and ligand were co-incubated in the presence of 1 mM sodium cholate. Incorporation increased with the length of the fatty acid used for A-F2 conjugation. Ligand-bearing liposomes were demonstrated to be smaller in diameter (about 30%) with a more positive z-potential in comparison to control vesicles while ligand incorporation did not influence the liposome membrane integrity. The size of the ligand-incorporating vesicles was maintained after 24 hours of incubation in isotonic buffer, proving that the vesicles do not aggregate. Although the preliminary biodistribution results may suggest that ligand bearing liposomes are accumulating in the liver, further cell culture, in vivo distribution and especially liver fractionation studies are required in order to clarify the intrahepatic localization of these liposomes and the ability to target liver hepatocytes in vivo.

Complete congenital duplication with incomplete separation of a lower extremity.

A case of a boy with complete duplication of the lower extremity and separation below the lower leg was described. Although x-ray examination revealed no femur, operation revealed a cartilage expected to become the femur in the future. A classification of duplication and separation of the lower extremity is proposed. The method and course of treatment in the present case were described in detail, including the application of tissue expanders on the deformity of the knee joint accompanied by a shortening of the sciatic nerve.

Transplantation of long-term cryopreserved allocutaneous tissue by skin graft or microsurgical anastomosis: experimental studies in the rat.

A step-wise method of freezing cutaneous soft tissue containing vessels to -196 degrees C for permanent preservation was developed. The methods of embryo cryopreservation were modified for soft-tissue preservation. Cutaneous tissue obtained from the abdominal wall of a Lewis rat was preserved for more than 3 weeks by this method and was then transplanted to a Brown Norway rat with the largest difference in major histocompatibility complex. Rejection reaction against the epidermal layer was markedly delayed, followed by a decrease in antigenicity of the dermis, with survival of the majority of the tissue for more than 6 months. A concept of artificial skin was formed based on these experimental results. A skin flap from the abdominal wall with a vascular pedicle consisting of the femoral artery and vein was similarly preserved and transplanted by vascular anastomosis. This transplant was made viable over a long period through the additional use of an immunosuppressant. Replantation of a rat hindleg also was successfully accomplished by using a skin flap from the abdominal wall with a vascular pedicle consisting of the femoral artery and vein, which had been cryopreserved for more than 3 weeks. These results with 65 pieces in five different treatment groups indicate the reliability of this method of preservation in maintaining the tissue in a state adequate for transfer without loss of viability over a long period. The applicability of this method of allograft preservation to the field of free-tissue transfer combined with microsurgical technique may introduce a new concept to the field of reconstructive surgery in the future. According to the results of the present experiment, the great potential for clinical application is described for the deepithelialized cryopreserved allocutaneous flap transfer.

Inhibition of calmodulin-dependent calcium-ATPase and phosphodiesterase by various cyclopeptides and peptide alkaloids from the Zizyphus species.

The effects of various sedative cyclopeptides and peptide alkaloids from the Zizyphus species on calmodulin-dependent Ca2+-ATPase and phosphodiesterase were investigated. Calmodulin-induced activation of Ca2+-ATPase was strongly inhibited by sanjoinine-A dialdehyde (IC0O, 2.3 microM), -Ah1 (IC50, 4.0 microM), -A (IC50, 4.6 microM), and -G2 (IC50, 7.2 microM), while calmodulin-induced activation of phosphodiesterase was strongly inhibited by both deachuine-S10 (IC30, 4.9 microM) and sanjoinine-D (IC50, 9.0 microM). The inhibitory activity of the various cyclopeptides and peptide alkaloids on Ca2+-ATPase was found to correlate well with their sedative activity.

Determination of recombinant human epidermal growth factor (rhEGF) in a pharmaceutical formulation by high performance liquid chromatography with electrochemical detection.

A novel HPLC method with electrochemical detection has been developed for the determination of recombinant human epidermal growth factor (rhEGF) in pharmaceutical products. rhEGF was separated from other components in formulation on a reversed-phase C18 column with 24% acetonitrile in 0.1 M phosphate buffer (pH 4.75). The optimum electrochemical oxidation of EGF was obtained at 0.85 V vs. Ag/AgCl in a glassy carbon working electrode due to electroactive tyrosine, tryptophan, methionine, and arginine residues. The quantitation range was from 1.0 to 200 ng of rhEGF with the linear correlation coefficient greater than 0.999. The method was successfully applied for the quantitation of rhEGF in a pharmaceutical preparation.

Determination of recombinant human epidermal growth factor (rhEGF) in a pharmaceutical preparation by capillary electrophoresis.

A simple assay method of recombinant human epidermal growth factor (rhEGF) in a pharmaceutical preparation was studied and validated by capillary electrophoresis (CE) using micellar electrokinetic chromatography (MEKC) techniques. Factors affecting the migration behavior and separation performances of the peptide; type of buffer, pH, buffer concentration, and concentration of sodium dodecyl sulfates (SDS) were investigated to optimize the analytical performance. CE was performed using running buffer, 50.0 mM borate (pH 8.5) containing 12.5 mM SDS at 20 kV of the applied voltage. Calibration curves for the rhEGF showed good linearity (r>0.999) over the wide dynamic range from 1.25 to 100 microg/ml. Sample analysis was performed by using standard addition method to eliminate the matrix effects of dosage vehicle. This method is assumed to be useful for quality control (QC) of various forms of pharmaceutical products of the peptide.