Evaluation methods and design for bioartificial liver based on perfusion model

A bioartificial liver (BAL) is a medical device entrapping living hepatocytes or immortalized cells derived from hepatocytes. Many efforts have already been made to maintain the functions of the hepatocytes in a BAL device over a long term. However, there is still some uncertainty as to their efficacy, and their limitations are unclear. Therefore, it is important to quantitatively evaluate the metabolic functions of a BAL. In previous studies onin vitro BAL devices, two test methods, an initial bolus loading and constant-rate infusion plus initial bolus loading, were theoretically carried out to obtain physiologic data on drugs. However, in the current study, the same two methods were used as a perfusion model and derived the same clearance characterized by an interrelationship between the perfusate flow rate and intrinsic clearance. The interrelationship indicated that the CL increased with an increasing perfusate flow rate and approached its maximum value,i.e. intrinsic clearance. In addition, to set up anin vivo BAL system, the toxic plateau levels in the BAL system were calculated for both series and parallel circuit models. The series model had a lower plateau level than the parellel model. The difference in the toxic plateau levels between the parallel and series models increased with an increasing number of BAL cartridges.     

Kinetic constants for intestinal transport of four monosaccharides determined under conditions of variable effective resistance of the unstirred water layer

Summary Theoretical considerations have suggested that variations in the resistance of the unstirred water layer (UWL) have a profound effect on the kinetic constants of intestinal transport. In this study, a previously validatedin vitro technique was employed to determine the unidirectional flux rate of glucose, galactose, 3-O-methyl glucose and fructose into the rabbit jejunum under carefully-defined conditions of stirring of the bulk phase known to yield different values for the effective resistance of the UWL. For each monosaccharide, uptake is much greater when the resistance of the UWL is low than when high. The maximal transport rate,J _d ^m , of glucose was half as large as theJ _d ^m of galactose and 3-O-methyl glucose (3-O-MG), and was twice as great as theJ _d ^m of fructose. The apparent affinity constant,K _m ^* ,of glucose is less than that of fructose, which was lower than theK _m ^* of galactose and 3-O-MG. The use of the Lineweaver-Burk double reciprocal plot is associated with an overestimation of bothJ _d ^m andK _m ^* .This discrepancy between the true and apparent values of the kinetic constants is much greater for lower than for higher values ofJ _d ^m andK _m ^* ;variations in the resistance of the unstirred layer influences the magnitude and direction of the discrepancy. The apparent passive permeability coefficient is similar for each sugar, but because of the different values ofJ _d ^m , passive permeation contributes relatively more to the uptake of glucose and fructose than of galactose or 3-O-MG. Under conditions of high unstirred layer resistance, differences in uptake rates of the sugars are due to differences in theirJ _d ^m rather than theirK _m ^* .Kinetic analysis is compatible with the suggestion that the glucose carriers are predominantly near the tip of the villus, whereas those for galactose and 3-O-MG are located along the entire villus and theK _m ^* of their carriers at the tip is lower than theirK _m ^* towards the base of the villus. It is proposed that there are multiple or heterogeneous intestinal carriers for glucose, galactose and 3-O-methyl glucose in the jejunum of the rabbit.Abbreviations Used in this Paper C ₁ Concentration of the probe molecule in the bulk phase - C ₂ Concentration of the probe molecule at the aqueous-membrane interface - d Effective thickness of the intestinal unstirred water layer - D Free diffusion coefficient of the probe molecule     

Characterization of responding cells in oxidative mitogen stimulation

Ia antigens coded by genes of the murine major histocompatibility complex are expressed on the surface of a population of cells critical to the proliferative response of murine spleen cells to the oxidative mitogen neuraminidase/galactose oxidase. By selective depletion with antiserum and complement, Ia antigens coded (or determined) by theI-A andI-J, E, C subregions of theIr region can be detected on the surface of cells required for the response. In addition, I-A-subregion products have a functional significance in cellular activation which can be demonstrated by blocking experiments with anti-la serum in the absence of complement.     

Evolution of the<Emphasis Type="Italic"> Drosophila broad</Emphasis> locus: the<Emphasis Type="Italic"> Manduca sexta broad</Emphasis> Z4 isoform has biological activity in<Emphasis Type="Italic"> Drosophila</Emphasis>

The Drosophila melanogaster broad locus is essential for normal metamorphic development. Broad encodes three genetically distinct functions (rbp, br, and 2Bc) and a family of four zinc-finger DNA-binding proteins (Z1-Z4). The Z1, Z2, and Z3 protein isoforms are primarily associated with the rbp, br, and 2Bc genetic functions respectively. The Z4 protein isoform also provides some rbp genetic function, however an essential function for the Z4 isoform in metamorphosis has not been identified. To determine the degree of conservation of Z4 function between the tobacco hornworm Manduca sexta and Drosophila we generated transgenic Drosophila expressing the Manduca broad Z4 isoform and used this transgene to rescue rbp mutant lethality during Drosophila metamorphosis. We find that the Manduca Z4 protein has significant biological activity in Drosophila with respect to rescue of rbp-associated lethality. There was also some overlap in effects on cuticle gene expression between the Manduca Z4 and Drosophila Z1 isoforms that was not shared with the Drosophila Z4 isoform. Our findings show that Z4 function has been conserved over the 260-million-year period since the divergence of Diptera and Lepidoptera, and are consistent with the hypothesis that the Drosophila Z4 and Manduca Z4 isoforms have essential roles in metamorphosis.Edited by M. Akam     

A three-state MWC analysis of oxygenation in tench (Tinca tinca) hemoglobin

Summary Oxygen equilibria in tench hemoglobin were analysed according to a three-state MWC model. In addition to theT andR states of the traditionally used two-state model, the three-state model introduces an additional state, theS state, when organic phosphates bind to theT-structure hemoglobin. Under conditions covering natural red cell pH values and nucleoside triphosphate-hemoglobin ratios, it was possible to closely fit experimental data to the three-state equation with constant values of the association constantsK _R ,K _T , andK _S , and with only the allosteric constantsL andM varying with effector conditions. Thus, in contrast to a twostate analysis of oxygen equilibria, the three-state analysis was consistent with the basic assumption of the MWC model, that heterotropic ligands only affect allosteric constants and not association constants. The temperature-dependence of the three-state parameter values showed that in the presence of nucleoside triphosphate the dominance of theS state over theT state was most pronounced at low temperatures. Furthermore, the numerical values of the enthalpy and entropy change of oxygenation were lower in theS state than in theT andR states, and the enthalpy and entropy change for the allostericSR transition were much larger than for theTR transition.Abbreviations Hb hemoglobin - Y fractional O₂ saturation - ATP adenosine triphosphate     

The familial and subfamilial relationships ofApocynaceae andAsclepiadaceae evaluated withrbcL data

Sequence data for therbcL gene from twenty-four taxa of the familiesApocynaceae andAsclepiadaceae were cladistically analysed in order to evaluate the existing familial and subfamilial classification. The taxa sampled represent all described subfamilies and a majority of the described tribes. The cladistic analysis shows that theAsclepiadaceae are nested within theApocynaceae. An amalgamation of the two families is therefore recommended. The subfamilial classification is also in need of revision: the subfamiliesPlumerioideae andApocynoideae of the current classifications are paraphyletic, as are many of the tribes. Potential subfamily candidates and characters traditionally used in the classification are discussed.     

Chloroplast DNA reassociation and grass phylogeny

Sequence variation in chloroplast DNA (cpDNA) as measured by DNA reassociation was examined in 12 grass species to address systematic problems in thePoaceae at the subfamilial and tribal levels. Two species,Petunia (Solanaceae) andGlycine (Leguminosae), were included to determine degrees of sequence divergence in cpDNA between monocots and dicots. The data were analyzed phenetically and phylogenetically. Species were segregated into four major groups that corresponded to the subfamiliesPooideae, Oryzoideae, Chloridoideae, andPanicoideae. Representatives of thePooideae andOryzoideae grouped together as did members of theChloridoideae andPanicoideae. ThePooideae split into two major groups corresponding to the recently recognized supertribesTriticanae andPoanae. Internodes between subfamily branches were short which might indicate a burst of divergence in the family early in its evolution. Sequence similarity values between the monocot grass species and the two dicot taxa ranged from 0.15 to 0.27, representing the highly conserved sequences of the chloroplast genome.     

In vitro effect of prostaglandins,prostaglandin endoperoxides and thromboxane on rat intestinal alkaline phosphatase and (Ca2+-Mg2+) adenosine triphosphatase

The activity of alkaline phosphate and²⁺-Mg²⁺ adenosine triphosphatase, two of the enzymes involved in limpid and calcium uptake across the intestinal membrane, were increased in experimental atherosclerosis. Administration ofAnnapavala sindhooram, an antiatherosclerotic drug, lowers these enzyme levels to near normal values. Prostaglandin E2 stimulated the enzyme activitiesin vitro, while prostaglandin endoperoxide inhibited the activity. Thromboxane and other prostaglandins had no effect on the enzyme activities. Addition of the antiatherosclerotic drug to thein vitro assay system reversed the effect of both prostaglandin E₂ and prostaglandin endoperoxide.     

Modulation of gene expression by the product offitA gene inEscherichia coli

Physiological parameters such as viability, gross RNA synthesis,β-galactosidase induction, development of phages T4, T7 andλ have been studied in temperature-sensitiveEscherichia coli strains harbouring fit A76,fit A24 andfit A76fit A24 mutations in rpoB⁺ andrpoB240 genetic backgrounds. The efficiently of expression of these functions is influenced by thefit A alleles depending upon the medium of growth and/or temperature. Strains harbouring therpoB240 mutation and thefit A76 mutation, either alone or together with thefit A24 mutation, are rifampicin-sensitive even at the perfssive temperature. The results suggest possible interaction between thefit A gene product and RNA polymerase invivo. This paper is dedicated to Proof. S. Krishnaswamy on his Sixty First Birthday.     

Genetic variability within theAspergillus viridinutans species

The intraspecific variability of theAspergillus viridinutans species was examined using various techniques including morphological examinations, carbon-source-utilization tests, restriction-enzyme analysis of the mitochondrial and nuclear DNA, and sequence analysis of part of the β-tubulin gene. Although the genetic distances between the isolates were higher than between,e.g., A. fumigatus andNeosartorya fischeri, mostA. viridinutans isolates, together withN. aureola andN. udagawae strains, were found to belong to a single cluster based on sequence data. Strain FRR 1266, which was earlier classified as a highly divergentA. fumigatus isolate, was found to belong to theA. viridinutans species. The ochratoxin A producingA. viridinutans strain IMI 306 135 was most closely related to an asexual isolate. These two latter strains were more closely related toA. fumigatus andN. fischeri than to anyA. viridinutans strains, and possibly represent a new species in theAspergillus sectionFumigati. The dendrogram based on carbon source utilization data and results of restriction analysis of the mitochondrial and nuclear DNA of the strains supported most of the evolutionary relationships observed on the basis of sequence data. The results indicate that the presence or absence of nodding conidial heads is not an unequivocal morphological character for the identification of species within theAspergillus sectionFumigati.     

Effect of low temperature preservation and cell density on metabolic function in a bioartificial liver

Difficulties associated with bioartificial liver (BAL) preservation limit not only the commercialization of BAL, but also its clinical trials. In this study, the possibility of cold preservation of BAL cartridges containing porcine hepatocytes was examined at 4 °C. In anin vitro perfusion culture system, BAL cartridges maintained cytochrome P450 metabolic function for at least 50 days. However, all BAL cartridges completely lost their ammonia eliminating ability when stored at 4 °C. We also studied the effect of cell density on the maintenance of BAL liver function in a highly differentiated and healthy state. As expected, BALs containing a larger number of hepatocytes demonstrated higher metabolic functions. When metabolic functions were compared per gram of hepatocytes, no large differences were observed between devices containing different densities of hepatocytes. Decreased cell density did not successfully prolong BAL function. The viability and function of isolated hepatocytes highly depend on the culture conditions, such as cell density, substrata, culture media, and additives to the culture media. Perfusion culture of BAL cartridges at 4°C gave a promosing result with respect to the maintenance of P450 activity. However, as indicated by the rapid loss of ammonia metabolic activity, many factors still remain to be optimized for preservation of BAL keeping high metabolic functions for a longer time.     

A minimal physiologically based pharmacokinetic model to investigate FcRn-mediated monoclonal antibody salvage: Effects of Kon,Koff, endosome trafficking,and animal species

ABSTRACT

Manipulation of binding affinity between monoclonal antibodies (mAbs) and the neonatal Fc receptor (FcRn) has been leveraged to extend mAb half-life; however, the steps required for success remain ambiguous and experimental observations are inconsistent. Recent models have considered the time course of endosomal transit a major contributor to the relationship between FcRn affinity and antibody half-life. Our objective was to develop a minimal physiologically based pharmacokinetic model to explain how changes in IgG-FcRn association rate constant (K_on), dissociation rate constant (K_off), and endosomal transit time [T_(w)] translate to improved IgG clearance across mice, monkeys and humans. By simulating mAb clearance across physiological values of K_on, K_off, and T_(w), we found that lowering K_off improves clearance only until the dissociation half-life reaches endosomal transit time. In contrast, K_on influenced clearance independently of T_(w).The model was then applied to fit 66 mAb plasma profiles across species digitized from the literature, and clearance of mAb (CL_IgG) and vascular fluid-phase endocytosis rate (CL_up) were estimated. We found that CL_IgG scaled well with body weight (allometric exponent of 0.90). After accounting for mAbs with significant FcRn binding at physiological pH, CL_up was allometrically scalable (exponent 0.72). For the antibodies surveyed, K_on was more highly correlated with CL_IgG across all species. The relationship between K_off and K_D with CL_IgG was largely inconsistent. Taken together, this model provides a parsimonious approach to evaluate endosomal transit kinetics using only mAb plasma concentrations. These findings reinforce the idea that endosomal transit kinetics should be considered when modeling FcRn salvage.     

Sieve-element plastids,exine sculpturing and the systematic affinities of theBuxaceae

The sieve-element plastids of members of several genera in theBuxaceae (Buxus, Pachysandra andSarcococca) were found to be of the specific subtype PVI, which contains a central globular protein crystal.Simmondsia (Simmondsiaceae) andDaphniphyllum (Daphniphyllaceae), on the other hand, were found to contain S-type sieve-element plastids. The occurrence of the highly restricted PVI plastids in theBuxaceae mitigates against a close relationship between theBuxaceae andSimmondsia, Daphniphyllum andEuphorbiaceae. Exine sculpturing of theBuxaceae andSimmondsiaceae also shows no close similarities. Both of these EM characters are discussed in connection with other available data and with respect to earlier systematic treatment of these families.     

Chromosome numbers and rust susceptibility as taxonomic criteria inRosaceae

BothNeuradoideae andChrysobalanoideae seem rust-free. TheSpiraeoideae andPomoideae are heavily susceptible toGymnosporangium rusts. While thePrunoideae resemble theRosoideae in being vulnerable to attacks ofPuccinia species, they are additionally susceptible toTranzschelia andThekopsora, whereas theRosoideae are characteristically afflicted byPhragmidium.—It is suggested (a) to treat theChrysobalanoideae as a separate family (Chrysobalanaceae), (b) to transferDryas from theRosoideae-Potentilleae-Dryadinae to theRosoideae-Cercocarpeae, and (c) to divide theRosoideae into two main groups of tribes: (i) the rust-freeKerrieae andCercocarpeae with x = 9, and (ii) the rust-susceptiblePotentilleae, Ulmarieae, Roseae andSanguisorbeae with x = 7.     

Genetic control of repair of radiation damage produced under euoxic and anoxic conditions in diploid yeastSaccharomyces cerevisiae

Summary Diploid wild type yeast strains 211, X2180 and the radiation sensitive mutantsrad2, 6, 9, 18, 50–55, and57 were exposed to cobalt-60 gamma radiation, in the presence and absence of oxygen, in order to identify the RAD loci involved in the repair of sublethal damage (SLD), recovery from potentially lethal damage (PLD) and oxygen enhancement ratio (OER). Response of wild type and mutants were compared in terms of survival curve parameters D_q, D₁₀, D₁, and D₀. As compared to wild type the mutants showed increased sensitivity to radiation lethality, both under euoxic and hypoxic conditions, as judged by the reduction in D_q and D₀ values. OER was reduced in therad2, 9, 18, 50, 51, and57 mutants indicating that these genes could be associated with the repair of gamma radiation damage produced under hypoxic condition.Shoulder (D_q) a measure of the ability of the cells to repair SLD, was reduced in therad6, 9, 18, 50, 53, and57 strains and was almost absent in therad51, 52, 54, and55 mutants. The ability to recover from PLD was equal to that of wild type strain in therad2, 6, 9, and18 strains, reduced in therad53, 55, and57 strains and was absent in therad50–52 and54 strains. In the mutants with liquid holding recovery ability, the extent of recovery from PLD produced under euoxic and hypoxic conditions was the same. These observations suggest that different groups of loci are involved in the control of different repair processes and that the expression of therad50–57 loci play a very important role in the repair of ionising radiation damage.On the basis of the liquid holding recovery data presented here and the observations made by others it is suggested that the unrepaired DSB constitute the PLD and that the repair of DSB involves recombination between homologous chromosomes.     

Two distinct subtypes of theHLA-DRw12 haplotypes in the Japanese population detected by nucleotide sequence analysis and oligonucleotide genotyping

We determined the DNA sequence of the enzymatically amplified second exon of theDRB1 gene of theDRw12 haplotypes derived from three Japanese donors and found two distinct subtypes of theDRw12 haplotype. The two subtypes, designatedDRw12a andDrw12b, had single-base substitutions that predicted one amino acid change at residue number 67. The sequence of theDrw12a andDRw12b subtypes differed from those of the otherDR haplotypes, but in the first hypervariable region of theDRB1 gene the sequences were identical to those of theDRw8(Dw8.1) andDRw8(Dw8.3) haplotypes. TheDRw12a andDRw12b subtypes were detected in a wide range of Japanse donors by genotyping with sequence-specific oligonucleotide probes synthesized according to the DNA sequence of the two subtypes. Results of this study demonstrated that theDRw12 haplotypes in the Japanese population are genetically diverse, as many otherDR haplotypes are. The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M27509, M27510, M27511.     

A method to forecast the start of theBetula, Platanus andQuercus pollen seasons in North London

This paper attempts the prediction of the start of theBetula, Quercus andPlatanus pollen seasons in London, UK based on pollen sampling conducted over a 5-year period, 1987–1991. The times at which eight different thresholds of accumulated daily pollen counts (M⁻³) were passed were correlated against heat sums, chill units, accumulated sunshine hours, monthly meteorological parameters and the start dates of earlier pollen seasons to identify significant associations. Few meteorological parameters were significantly correlated with the start dates of the three pollen seasons, the exceptions being significant negative correlations between the average monthly air temperature in the months immediately preceding theBetula andPlatanus pollen season. However, significant relationships were identified between the start dates of theBetula, Quercus andPlatanus pollen seasons and the start of theCorylus, Taxus andPopulus pollen seasons with coefficients of determination as high as 98%. These indicator species were then used as predictors to forecast the start of theBetula, Quercus andPlatanus pollen seasons, both individually and in combination with one another, providing levels of explanation of up to 99%.     

Serologische Untersuchungen zur Gliederung derBrassicaceae

The serological investigations support the opinion ofJanchen (1942) to combine the generaBunias, Isatis, andSisymbrium in the tribeSisymbrieae; Cheiranthus, Erysimum, andMatthiola in the tribeHesperideae; andBrassica, Crambe, Sinapis, andSuccowia in the tribeBrassiceae. They further underline the central position of theSisymbrieae and the isolated position of theHeliophileae. In accordance withEigner (1973) theBrassiceae are placed closer to theSisymbrieae than inJanchen; the same holds for thePringleeae. No serological justification could be found to uniteArabis andBarbarea in the tribeArabideae, andAlyssum andLunaria in theAlysseae. From the antigen-systems used among the representatives ofJanchen's Lepidieae the generaLepidium andNeslia show remarkable correspondence both toCamelina andThlaspi, but not toCochlearia which appears distant fromCamelina andThlaspi also.

Teil 1/Part 1.     

Regulation of expression of mouseC4 andSlp genes by non-H-2-linked genes

C4 (the fourth complement component) and Slp (sexlimited protein) are two homologous plasma proteins encoded by genes in theS-region of theH-2 gene complex. We studied the genetic factors influencing the plasma levels of these proteins and their mRNA levels in liver. Considerable differences in both protein and mRNA levels were found between mouse strains carrying the sameS-region allele on different genetic backgrounds, indicating a pretranslational effect of non-H-2-linked genes on the expression of the twoS-region genes. The expression of Slp is androgen-dependent in the strains tested. However, testosterone treatment cannot increase the low levels of Slp caused by non-H-2-linked regulatory genes. In mice with Slp-negativeS-region alleles we found liver mRNA hybridizing with Slp-specific oligonucleotides, indicating expression of theSlp gene in Slp-negative strains. Our data demonstrate the complexity of the regulation of theC4 andSlp genes and pave the way for the analysis of the regulatory factors involved.