Prospective randomized controlled trial comparing treatment efficacy and tolerance of picosecond alexandrite laser with a diffractive lens array and triple combination cream in female asian patients with melasma.

BACKGROUND: Recent evidence suggests melasma to be a photoaging disorder. Triple combination creams (TCC: fluocinolone acetonide 0.01%, hydroquinone 4% and tretinoin 0.05%) remain the gold standard treatment. Picosecond alexandrite laser treatment using a diffractive lens array (DLA) has been identified to be effective for improving photoaging conditions. OBJECTIVE: We aimed to compare the efficacy and tolerance of the picosecond alexandrite laser with those of DLA and TCC in female Asian patients with melasma. METHODS: Twenty-nine patients were randomly assigned to group A1 (3 laser sessions at 4-week intervals), A2 (5 laser sessions at 4-week intervals) or B (TCC daily for at least 8 weeks and then tapered until the final evaluation). The Melasma Area, Severity Index (MASI) score and VISIA were assessed at baseline, week 12 and week 20. By week 20, the follow-up periods for groups A1 and A2 were 3 months and 1 month, respectively. RESULTS: Nine, 11 and 6 participants in groups A1, A2 and B completed the study, respectively. MASI scores were significantly improved in all 3 groups at weeks 12 and 20. In groups A1, A2 and B, the improvement rates at week 20 were 53%, 38% and 50%, respectively. VISIA® analysis additionally revealed a significant improvement in spots, porphyria, pores and brown spots after 3 laser sessions (P < 0.05). Group A2 showed greater improvements than group A1 in terms of spots, wrinkles and pores; however, only red areas were significantly different (P < 0.001). All side-effects in the 3 groups were transient and gradually subsided after 1-3 months. CONCLUSION: Picosecond alexandrite laser treatment using DLA showed comparable efficacy with TCC for the treatment of melasma. Improvements in texture, spots, wrinkles and pores were observed in the laser groups. Patients with melasma lesions that exhibit telangiectasia may benefit from additional laser treatment sessions.

Preliminary feasibility study using a solution of synthetic enzymes to replace the natural enzymes in polyhemoglobin-catalase-superoxide dismutase-carbonic anhydrase: effect on warm ischemic hepatocyte cell culture.

This is a study on a simple solution of chemically prepared small chemical molecules of synthetic enzymes: catalase, superoxide dismutase, and carbonic anhydrase (CAT, SOD, and CA). We carried out a study to see if these synthetic enzymes can replace the natural enzymes (CAT, SOD, and CA) and avoid the need for the complicated cross-linking of natural enzymes to PolyHb to form PolyHb-CAT-SOD-CA. We compared the effect a solution of these three synthetic enzymes has on the viability of warm-ischemic hepatocytes that were exposed to nitrogen for 1 h at 37°C. PolyHb significantly increased the viability. The three synthetic enzymes themselves also significantly increased the viability. The use of both PolyHb and the three synthetic enzymes resulted in an additive effect in the recovery of viability. Increasing the concentration of the synthetic enzymes resulted in further increase in the effect due to the synthetic enzymes. Implications: In addition to PolyHb, there are a number of other HBOC oxygen carriers. However, only Biopure's HBOC product has received regulatory approval, but only in Russia and South Africa. None of the HBOCs has received regulatory approval by other countries. If regulatory agencies require HBOCs to have antioxidant or CO2 transport properties, all that is needed is to add or inject the solution of synthetic enzymes as a separate component.

Microencapsulated genetically engineered live E. coli DH5 cells administered orally to maintain normal plasma urea level in uremic rats.

Safety concerns about introducing genetically engineered cells into the body have prevented their use in medical treatments. To solve this problem, we prepared polymeric membrane artificial cells (semipermeable microcapsules) containing genetically engineered live cells from the bacteria Escherichia coli DH5. When given orally, the cells remain at all times in the microcapsules and are finally excreted in the stool. During their passage through the intestine, small molecules like urea diffuse rapidly into the microcapsules and are acted on by the genetically engineered cells. This lowers the high plasma urea level to normal in uremic rats with induced kidney failure, and has exciting implications for the use of this and many other types of genetically engineered cells in a number of medical applications.

Does conventional early life academic excellence predict later life scientific discovery? An assessment of the lives of great medical innovators.

BACKGROUND: Perhaps, as never before, we need innovators. With our growing population numbers, and with increasing pressures on our education systems, are we in danger of becoming more rigid and formulaic and increasingly inhibiting innovation? When young can we predict who will become the great innovators? For example, in medicine, who will change clinical practice? AIMS: We therefore determined to assess whether the current academic excellence approach to medical school entrance would have captured previous great innovators in medicine, assuming that they should all have well fulfilled current entrance requirements. METHODS: The authors assembled a list of 100 great medical innovators which was then approved, rejected or added to by a jury of 12 MD fellows of the Royal Society of Canada. Two reviewers, who had taken both the past and present Medical College Admission Test as part of North American medical school entrance requirements, independently assessed each innovator's early life educational history in order to predict the innovator's likely success at medical school entry, assuming excellence in all entrance requirements. RESULTS: Thirty-one percent of the great medical innovators possessed no medical degree and 24% would likely be denied entry to medical school by today's standards (e.g. had a history of poor performance, failure, dropout or expulsion) with only 24% being guaranteed entry. Even if excellence in only one topic was required, the figure would only rise to 41% certain of medical school entry. CONCLUSION: These data show that today's medical school entry standards would have barred many great innovators and raise questions about whether we are losing medical innovators as a consequence. Our findings have important implications for promoting flexibility and innovation for medical education, and for promoting an environment for innovation in general.

Structure and dynamics of N,N-diethyl-N-methylammonium triflate ionic liquid, neat and with water, from molecular dynamics simulations.

We investigated by means of molecular dynamics simulations the properties (structure, thermodynamics, ion transport, and dynamics) of the protic ionic liquid N,N-diethyl-N-methylammonium triflate (dema:Tfl) and of selected aqueous mixtures of dema:Tfl. This ionic liquid, a good candidate for a water-free proton exchange membrane, is shown to exhibit high ion mobility and conductivity. The radial distribution functions reveal a significant long-range structural correlation. The ammonium cations [dema](+) are found to diffuse slightly faster than the triflate anions [Tfl](-), and both types of ions exhibit enhanced mobility at higher temperatures, leading to higher ionic conductivity. Analysis of the dynamics of ion pairing clearly points to the existence of long-lived contact ion pairs. We also examined the effects of water through characterization of properties of dema:Tfl-water mixtures. Water molecules replace counterions in the coordination shell of both ions, thus weakening their association. As water concentration increases, water molecules start to connect with each other and then form a large network that percolates through the system. Water influences ion dynamics in the mixtures. As the concentration of water increases, both translational and rotational motions of [dema](+) and [Tfl](-) are significantly enhanced. As a result, higher vehicular ionic conductivity is observed with increased hydration level.

Dual effects include antioxidant and pro-oxidation of ascorbic acid on the redox properties of bovine hemoglobin.

The oxidation reactions have become the main obstacle of development of bovine hemoglobin-derivates products. Herein, the effects of vitamin C (Vc), a easily available natural antioxidant reagent, on the redox reaction of bovine hemoglobin were systematically investigated through methemoglobin (MetHb) formation and spectrophotometric analysis and oxygen affinity monitoring of hemoglobin. The results showed that Vc presented antioxidant effects in the initial stage of reaction and then could accelerated the MetHb content increasing by production of hydrogen peroxide, which can be indirectly characterized by the formation of choleglobin in the following side reactions. The dual effects of Vc include antioxidant and pro-oxidant effects could be confirmed by the spectrophotometric spectrums analysis in this research. The results of this research supplied the novel insight into understanding of redox properties of bovine hemoglobin and also revealed the main obstacle in exploration of Vc application in the future development of bovine hemoglobin-derivates products.

Mechanism of acid-induced release of secretin in rats. Presence of a secretin-releasing peptide.

In fasting rats, intraduodenal infusion of dilute hydrochloric acid results in significant increases in both pancreatic exocrine secretion and plasma concentration of secretin. To test the hypothesis that acid-induced release of secretin is mediated by a secretin-releasing factor (S-RF), anesthetized rats were prepared with pyloric ligation, duodenal and jejunal cannulas, and pancreatic duct cannulas. Donor rats were infused intraduodenally with 0.01 N HCl, 0.15 M NaCl, or a combination of 0.01 N HCl and 0.05 N NaHCO3 at 0.3 ml/min for 1.5 h, and the perfusates were collected via jejunal cannulas. The perfusates with pH adjusted to 6.0 were concentrated threefold and infused into the duodena of recipient rats. The concentrate of acid perfusate (CAP) significantly increased both pancreatic volume flow and bicarbonate output and plasma concentration of secretin, whereas concentrates of the saline perfusate (CSP) or the perfusate of a combination of 0.01 N HCl and 0.05 N NaHCO3 (CABP) did not influence pancreatic secretion or plasma concentration of secretin. The increased pancreatic secretion by CAP was attributed to increased circulating secretin because when secretin was immunoneutralized by a rabbit antisecretin serum, CAP-stimulated pancreatic secretion was abolished. The bioactivity of CAP was trypsin-sensitive and heat stable. The active substance in CAP had a molecular weight of less than 5,000 and greater than 1,000, as determined by ultrafiltration and bioassay. In conclusion, dilute HCl releases an S-RF into the upper small intestinal lumen to stimulate release of secretin. This substance, with molecular weight of less than 5,000, is heat stable and trypsin sensitive. Thus, the acid-stimulated release of secretin is mediated by a secretin-releasing peptide in the upper small intestinal lumen.

Identification of a transcriptional enhancer important for enteroendocrine and pancreatic islet cell-specific expression of the secretin gene.

It is well established that the gene encoding the hormone secretin is expressed in a specific enteroendocrine cell, the S cell. We now show that the secretin gene is transiently expressed in insulin-producing B cells of the developing pancreatic islets in addition to the intestine. Furthermore, secretin is produced by most established islet cell lines. In order to identify and characterize the regulatory elements within the secretin gene that control tissue-specific expression, we have introduced secretin reporter gene constructions into the secretin-producing HIT and STC-1 cell lines as well as the nonexpressing INR1-G9 glucagonoma line. Analysis of deletion mutants revealed that sequences between 174 and 53 bp upstream from the transcriptional start site are required for maximal expression in secretin-producing cells. This positive element functioned independently of position and orientation. Further deletions into the enhancer resulted in a stepwise loss of transcriptional activity, suggesting the presence of several discrete control elements. The sequence CAGCTG within the secretin enhancer closely resembles that of the core of the B-cell-specific enhancer in the insulin gene. Point mutations introduced into this putative element led to greater than 85% reduction in transcriptional activity. Gel mobility shift assays suggested that a factor in B cells closely related or identical to proteins that bind to the insulin enhancer interacts with the CAGCTG motif in the secretin gene.

Polyhemoglobin-superoxide dismutase-catalase as a blood substitute with antioxidant properties.

Polyhemoglobin-superoxide dismutase-catalase is designed to function as an oxygen carrier with antioxidant properties. This is based on cross-linking hemoglobin with superoxide dismutase and catalase (PolyHb-SOD-CAT). This study describes the structural and antioxidant properties of this solution. Our studies show that superoxide dismutase and catalase retain their enzymatic activity following glutaraldehyde polymerization with 8:1 and 16:1 glutaraldehyde:hemoglobin ratio. We have analyzed the optimal SOD/CAT ratios to prevent oxidation of hemoglobin in the presence of oxygen free radicals. The circulation half-life of crosslinked hemoglobin, SOD, and catalase in Sprague-Dawley rats correlates with the degree of polymerization as determined by high-performance molecular weight gel filtration. PolyHb-SOD-CAT decreases the formation of oxygen radicals compared with PolyHb in a rat intestinal ischemia-reperfusion model.

Numerical investigation of non-Newtonian microcirculatory blood flow in hepatic lobule.

The circulation in the liver is unique at macroscopic and microscopic levels. At the macroscopic level, there is an unusual presence of portal and arterial inputs rather than a single arterial input. At the microscopic level, a series of microenvironments in the acinar system is essential in controlling the functional characteristics of hepatic parenchymal cells. Since the hemodynamics is much less studied in the multifunctional liver, an attempt is made to study the hepatic hemodynamics in a segment of a hepatic lobular structure, that is made up of high-pressure oxygenated arteriole, low-pressure nutrient-rich portal venule, fenestrated sinusoidal space and hepatic venule. Our goal is to dispel some of the myths of this complex vascular bed by means of finite volume blood flow simulation. Flow features like high-velocity gradients near the fenestrations, flow reversal and Dean vortices in the sinusoidal space are analyzed within the non-Newtonian framework. Since no distinct exact or numerical solutions are available for this complex vascular bed, the present simulated results are compared with the available clinical observations. Results revealed that the pressure plays a key role in hepatic blood flow.

Temperature stability of Poly-[hemoglobin-superoxide dismutase-catalase-carbonic anhydrase] in the form of a solution or in the lyophilized form during storage at -80 °C, 4 °C, 25 °C and 37 °C or pasteurization at 70 °C.

Polyhemoglobin-superoxide dismutase-catalase-carbonic anhydrase (Poly-[Hb-SOD-CAT-CA]) contains all three major functions of red blood cells (RBCs) at an enhanced level. It transports oxygen, removes oxygen radicals and transports carbon dioxide. Our previous studies in a 90-min 30 mm Hg Mean Arterial Pressure (MAP) sustained hemorrhagic shock rat model shows that it is more effective than blood in the lowering of elevated intracellular pCO2, recovery of ST-elevation and histology of the heart and intestine. This paper is to analyze the storage and temperature stability. Allowable storage time for RBC is about 1 d at room temperature and 42 d at 4 °C. Also, RBC cannot be pasteurized to remove infective agents like HIV and Ebola. PolyHb can be heat sterilized and can be stored for 1 year even at room temperature. However, Poly-[Hb-SOD-CAT-CA] contains both Hb and enzymes and enzymes are particularly sensitive to storage and heat. We thus carried out studies to analyze its storage stability at different temperatures and heat pasteurization stability. Results of storage stability show that lyophilization extends the storage time to 1 year at 4 °C and 40 d at room temperature (compared to respectively, 42 d and 1 d for RBC). After the freeze-dry process, the enzyme activities of Poly-[SFHb-SOD-CAT-CA] was 100 ± 2% for CA, 100 ± 2% for SOD and 93 ± 3.5% for CAT. After heat pasteurization at 70 °C for 2 h, lyophilized Poly-[Hb-SOD-CAT-CA] retained good enzyme activities of CA 97 ± 4%, SOD 100 ± 2.5% and CAT 63.8 ± 4%. More CAT can be added during the crosslinking process to maintain the same enzyme ratio after heat pasteurization. Heat pasteurization is possible only for the lyophilized form of Poly-[Hb-SOD-CAT-CA] and not for the solution. It can be easily reconstituted by dissolving in suitable solutions that continues to have good storage stability though less than that for the lyophilized form. According to the P50 value, Poly-[SFHb-SOD-CAT-CA] retains its oxygen carrying ability before and after long-term storage.

Propentdyopent: the scaffold of a heme metabolite as an electron reservoir in transition metal complexes.

The dipyrrin-1,9-dione scaffold of heme metabolite propendyopent coordinates late transition metals (Co, Ni, Cu, and Zn) forming homoleptic, pseudo-tetrahedral complexes. Electrochemical and spectroscopic studies reveal that the monoanionic, bidentate ligands behave as electron reservoirs as the complexes reversibly host one or two ligand-based radicals.

Hepatic uptake of asialoglycoprotein is different among mammalian species due to different receptor distribution.

Isolated hepatocytes of rat, rabbit and guinea pig were found to take up and degrade 125I-labelled asialoorosomucoid at different rates with the rank order: rabbit greater than rat greater than guinea pig. Measurement of 125I-asialoorosomucoid binding at 4 degrees C to these hepatocytes revealed that all these cells had two classes of receptors with a major difference occurring in the number of high-affinity binding sites. The average binding affinity constants (K) and receptor concentration (N) calculated from a least-square analysis of the Scatchard plots were K1 = 1.15.10(9) M-1, K2 = 0.93.10(7) M-1, N1 = 0.049 pmol/mg cell protein and N2 = 0.27 pmol/mg cell protein for the rat; K2 = 3.16.10(7) M-1, N1 = 0.027 pmol/mg cell protein and N2 = 0.13 pmol/mg cell protein for the guinea pig and K1 = 0.74.10(9) M-1, K2 = 3.85.10(7) M-1, N1 = 0.205 pmol/mg cell protein and N2 = 0.37 pmol/mg cell protein for the rabbit hepatocytes, respectively. Measurement of the total number of cellular receptors after solubilization with Triton X-100 also revealed the same receptor concentration rank order of rabbit (5.8 pmol/mg cell protein) greater than rat (0.55 pmol/mg cell protein) greater than guinea pig (0.18 pmol/mg cell protein). Intravenous injection of 125I-asialoorosomucoid into anesthetized animals of matched body weight also indicated that the rate of plasma clearance and the rate of appearance of the degraded product of the tracer were different among these species with the same rank order as that observed with isolated hepatocytes. Thus there is a fundamental difference in the number of asialoglycoprotein receptors both on the cell surface and inside hepatocytes of these mammalian species.

Phenylalanine ammonia-lyase immobilized in microcapsules for the depletion of phenylalanine in plasma in phenylketonuric rat model.

Microencapsulation of the enzyme phenylalanine ammonia-lyase was developed for in vivo depletion of systemic phenylalanine in phenylketonuric rats. Compared to normal rats, systemic phenylalanine blood levels in phenylketonuric rats was increased by 15-20-fold. Daily oral administration of 1 unit of phenylalanine ammonia-lyase-loaded artificial cells to phenylketonuric rats lowered the systemic phenylalanine level to 58% +/- 18% (mean + S.D.) in 7 days (P less than 0.010), while 5 units lowered the systemic phenylalanine level to 25% +/- 8%. 5 units of the immobilized enzyme lowered the systemic phenylalanine level to normal levels within 6 days. Phenylketonuric treated rats showed no signs of abnormal behavior and weight loss compared to phenylketonuric non-treated rats. The immobilized enzyme within artificial cells is therefore protected against low gastrointestinal pH and proteolytic enzymes.

Diacytosis of asialoglycoprotein in isolated hepatocytes is dependent on the structure of ligand and cellular distribution of the receptors.

Diacytosis, degradation and retention of 125I-labeled asialoorosomucoid (ASOR), its reduced and carboxymethylated N-terminal cyanogen bromide-cleaved fragment (RC-ASCNBr-I) and asialofetuin preloaded into isolated rat or rabbit hepatocytes for various periods of time were compared. In rat hepatocytes preloaded with a saturating concentration (3.10(-8) M) of the ligands, the proportion of the preloaded ligands distributed to degradation and diacytosis was fairly constant during 4 h of preincubation. In addition, a small portion of the preloaded ligands was neither diacytosed nor degraded, but was retained intracellularly. Diacytosis of 125I-ASOR (29%) was greater than that of either 125I-RC-ASCNBr-I (23%) or 125I-asialofetuin (15%). Diacytosis of 125I-ASOR, when preloaded in the presence of 5 microM colchicine, was significantly enhanced by 79% (increasing from 29% to 52%), whereas those of 125I-RC-ASCNBr-I and 125I-asialofetuin were not significantly altered (with average increases of 14% and 19%, respectively). The fraction of the preloaded 125I-asialofetuin (69%) and 125I-RC-ASCNBr-I (68.6%) that was degraded was slightly higher than that of 125I-ASOR (64%) and all was decreased by colchicine. The fraction of all three ligands retained by the cells was increased 2- to 4-fold by colchicine. The extents of retention of 125I-asialofetuin and 125I-ASCNBr-I were greater than that of 125I-ASOR, particularly after preloaded for more than 2 h. Preloading of the cells with ligands at a non-saturating concentration (6.5.10(-10) M) did not change these patterns of ligand distribution. Conjugation of diphtheria toxin fragment A (DTA) to ASOR or RC-ASCNBr-I also did not significantly alter the pattern of ligand distribution. In rabbit hepatocytes containing more asialoglycoprotein receptors than rat cells, 125I-ASOR was diacytosed to a greater extent (50%, -colchicine; 61%, + cholchicine) but degraded to a lesser extent (33%, -colchicine; 13%, + colchicine) than was observed in rat cells. The extent of retention of 125I-ASOR in rabbit cells was also greater than that in rat cells. A similar pattern of differences between rabbit and rat hepatocytes was observed for 125I-DTA-ASOR. These results indicate that intracellular sorting of internalized asialglycoproteins between diacytosis and degradation is dependent on both the structure of the ligand and the distribution of the cellular receptors.

Diacytosis of 125I-asialoorosomucoid by rat hepatocytes. A non-lysosomal pathway insensitive to inhibition by inhibitors of ligand degradation.

Diacytosis of 125I-asialoorosomucoid by rat hepatocytes was studied by preincubating the cells with the labelled ligand at 37 degrees C for 30 min or 18 degrees C for 2 h, washing free of cell surface receptor-bound tracer at 4 degrees C and then reincubating at 37 degrees C. The cells preloaded at 37 degrees C released a maximum of 18% of the total intracellular ligand as undegraded molecules after 1 h of incubation with an apparent first-order rate constant of 0.018 min-1 (t1/2 = 39 min). When the preloaded cells were incubated in the presence of 100 micrograms/ml unlabelled asialoorosomucoid or 5 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, the amount of the released ligand increased to 32 and 37%, respectively, without apparent change in kinetics, indicating that these agents prevented rebinding of the released ligand. In the presence of 5 microM colchicine, 20 microM cytochalasin B, 20 microM chloroquine, 10 mM NH4Cl, 10 microM monensin or 20 microM leupeptin, degradation of the preloaded ligand was inhibited, whereas the release of the ligand was either slightly increased or unchanged. Similar effects of leupeptin, colchicine and asialoorosomucoid were observed with cells preloaded at 18 degrees C. These results indicate that diacytosis of 125I-asialoorosomucoid occurs from a prelysosomal compartment via a route insensitive to inhibition by the inhibitors of ligand degradation.

Epnzymatric recycling of coenzymes by a multi-enzyme system immobilized within semipermeable collodion microcapsules.

Hexokinase (ATP:D-glucose 6-phosphotransferase EC 2.7.1.2) and pyruvate kinase (ATP:pyruvate 2-0-phosphotransferase EC 2.7.1.40) were co-immobilized within semipermeable collodion microcapsules. The resulting microcapsules displayed excellent hexokinase and pyruvate kinase activities, with the measured pyruvate kinase activity considerably greater than that measured for hexokinase. The co-immobilized enzymes, when used sequentially were capable of recycling both ATP and ADP when exposed to the appropriate conditions. Furthermore, when exposed to limiting amounts of coenzyme, the cycles were capable of reusing the total amount of coenzyme supplied at least three times in 90 min. The use of microencapsulation to produce partially "self sufficient" enzyme systems is discussed.

Study on the variables affecting toxicity of hybrid toxins. The effect of different target cell receptor distribution and toxin binding chain.

Disulfide conjugates of diphtheria toxin (DT) and its fragment A (DTA) to asialoorosomucoid (ASOR) were prepared. The toxicity of the conjugates were compared with DT in isolated rat, rabbit and guinea pig hepatocytes containing different concentration of asialoglycoprotein receptors (Biochim. Biophys, Acta 942, 57, 1988). In rat hepatocytes DTA-ASOR was highly toxic with half-maximal inhibitory concentration (IC50) of protein synthesis occurring at 4 +/- 3.10(-11) M (n = 7) which was much lower than that of DT DT (7.8 +/- 9.8.10(-9) M, n = 7). In rabbit hepatocytes toxicity of the conjugate (IC50 = 5.4 +/- 4.9.10(-10) M, n = 7) was higher than that of DT (IC50 = 5 +/- 4.10(-11) M, n = 7). In guinea pig hepatocytes, DTA-ASOR was not toxic at concentration below 10(-8) M, although DT was highly toxic (IC50 = 1.8 +/- 1.4.10(-10), n = 3). In the presence of 5 microM colchicine, the toxicity of DTA-ASOR in rat and rabbit hepatocytes increased by 10-fold, while in guinea pig hepatocytes it became detectable with an IC50 of 1.2 +/- 0.8.10(-9) M (n = 3). The toxicity of DT in the rat cells was also enhanced 10-fold by colchicine, but not at all in either the rabbit or the guinea pig cells. Addition of isolated diphtheria toxin fragment B (DTB) did not affect significantly the toxicity of DTA-ASOR in all three hepatocytes and that of DT in rat hepatocytes, but reduced toxicity of DT more than 20-fold in the rabbit and guinea pig cells. Toxicity of DT-ASOR in rat hepatocytes was the same as DTA-ASOR both in the absence and presence of colchicine, and abolished completely by excess ASOR, but not by DTB. Toxicity of DT-ASOR in rabbit hepatocytes was 40-times higher than DTA-ASOR, enhanced 10-fold by cochicine and reduced more than 30-fold by excess ASOR, but only slightly by DTB. These results indicate that entry of DTA from DTA-ASOR involve a DTB-independent translocation mechanism which can be as efficient as the DTB-dependent mechanism used by DT in the rabbit and guinea pig cells. The entry of both conjugates appeared to be mediated by the asialoglycoprotein receptors. However, the DTB moiety of DT-ASOR could function only in the DT-sensitive cells indicating the lack of a DTB-mediated translocation in the DT-resistant cells.

The anaerobic reaction of bovine hemoglobin with divinyl sulfone: structural changes and functional consequences.

The anaerobic reaction of bovine hemoglobin with divinyl sulfone results in a non-cross-linked intramolecularly-modified new derivative. This chemically-modified hemoglobin is homogeneous with respect to its molecular mass (64 kDa) and electrophoretic properties. The absence of any 32 kDa band from its SDS-PAGE pattern proves the lack of intramolecular cross-linkage, while a single-peak high-performance gel-permeation chromatogram demonstrates the absence of intermolecular cross-linkage. The oxygen binding properties determined at 37 degrees C, 0.15 M Cl- and pH 7.4 display a P50 of 52 mmHg and a Hill coefficient n of 1.9. Under the same experimental conditions the oxygen affinity is not sensitive to chloride anions, suggesting that the covelant modification is in the beta-cleft. The maximum number of Bohr protons released is 0.8/tetramer, which is half that of normal bovine hemoglobin. The retention time in circulation, measured in rats, is similar to that of native bovine hemoglobin. Using a high molar ratio of divinyl sulfone to modified hemoglobin, it is feasible to effect anaerobic intermolecular cross-linkage. The polymerized material, isolated from a 24 h reaction, is a mixture of modified intermolecularly-crosslinked hemoglobins with a molecular mass range from 130 to approx. 500 kDa. The oxygen-transport characteristics of the polymerized material are similar to those of the modified non-cross-linked derivative, whereas its retention time in rats is increased three-fold with respect to native bovine hemoglobin.

Effect of rate of intracellular transport and diacytosis on cytotoxicity of hybrid toxins. Study with hybrids using hepatic asialoglycoprotein receptor-mediated endocytosis.

The effects of diacytosis and intracellular transport rate on cytotoxicity of hybrid toxins were studied with conjugates of diphtheria toxin fragment A (DTA) to asialoorosomucoid (ASOR) and its reduced and carboxymethylated cyanogen bromide fragment I (RC-ASCNBr-I) in cultured rat hepatocytes. In the hepatocytes the kinetics of uptake of the conjugate of asialoorosomucoid (DTA-ASOR) and that of the conjugate of the cyanogen bromide fragment (DTA-RC-ASCNBr-I) were quite similar, but the rate of accumulation of DTA moiety into the lysosomes, as determined by Percoll density gradient centrifugation, was found to be greater for the latter than the former. However, after internalization, DTA-RC-ASCNBr-I was diacytosed to a lesser extent than that of DTA-ASOR, particularly when colchicine was present during internalization. Analysis of the subunits of DTA-ASOR internalized by the hepatocytes indicated that they were accumulated disproportionately in a time-dependent manner so that the glycoprotein moiety was accumulated progressively more than the toxin moiety. Cytotoxicity of DTA-ASOR toward the hepatocytes was 2-times as much as that of DTA-RC-ASCNBr-I. Colchicine enhanced the toxicity of DTA-RC-ASCNBr-I (33-fold) to a greater extent than that of DTA-ASOR (12-fold). The difference in enhancement by colchicine was also observed in the rate of cell intoxication by the conjugates. Both conjugates were more toxic to the hepatocytes after incubation with the cells at 18 degrees C than at 37 degrees C. In the presence of vanadate (0.2 mM), which enhanced diacytosis, toxicity of DTA-ASOR decreased by 5-fold. After incubation with the hepatocytes, a partial dissociation of DTA-ASOR was found to occur independently of the receptor-mediated endocytosis. Taken together, these results indicate that diacytosis, subunit dissociation and rapid transport of conjugate toward lysosomes affect kinetically the rate of accumulation of the conjugate into a yet unidentified compartment of toxin translocation.