Assessment of liver cirrhosis for patients with Child's A classification before hepatectomy using dynamic contrast-enhanced MRI.

AIM: To determine the feasibility of semi-quantitative haemodynamic parameters derived from dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI) to assess liver fibrosis. MATERIALS AND METHODS: Seventy-five patients with Child's A classification (males/females=24/51; average age, 58 years; range, 30-80 years) received DCE-MRI 3 days prior to hepatectomy. Semi-quantitative haemodynamic parameters, including the wash-in slope, wash-out slope, and time-to-peak, were calculated from DCE-MRI data. Liver fibrosis of the resected non-tumour liver was graded pathologically from F0 (no fibrosis) to F6 (cirrhosis) in the regions corresponding to those assessed by DCE-MRI. RESULTS: The wash-out slope showed higher interobserver and intra-observer reliabilities than the wash-in slope and time-to-peak. There was a significant positive correlation between the wash-out slope and pathological grade of fibrosis (Spearman's correlation coefficient: r=0.5331, p<0.0001). The area under the receiver operating characteristic curve was 0.8066 when using the wash-out slope to differentiate cirrhosis (grade F6) from non-cirrhosis (grades F0-5). Using the cut-off point that maximised specificity, the sensitivity was 62.07%, specificity was 91.30%, positive predictive value was 81.81%, negative predictive value was 79.25%, and accuracy was 80%. CONCLUSIONS: The wash-out slope derived from DCE-MRI might be potentially useful in assessing liver cirrhosis in patients with Child's A classification before hepatectomy.

Trajectories of Nutritional Status and Cognitive Impairment among Older Taiwanese with Hip Fracture.

OBJECTIVE: This paper describes the trajectories of nutritional status and cognitive impairment and their correlation among older Taiwanese over 1 year after hip-fracture surgery. DESIGN: Secondary analysis of data from a clinical trial evaluating the effects of three types of post-discharge care for 292 older hip-fracture patients (age >60 years). MEASUREMENTS: Nutritional status was assessed by the Mini Nutritional Assessment before and 1, 3, 6, 12 months after hospital discharge. Cognitive function was measured by the Mini-Mental State Examination before surgery, at hospital discharge, 6 and 12 months after discharge. Trajectories of nutritional status and cognitive impairment were depicted by latent class growth modeling, whereas linkages between nutritional-status and cognitive-impairment trajectories were assessed by multinomial logistic regression. RESULTS: Nutritional status in general improved significantly, particularly during the first 3 months after discharge. We identified three trajectories of nutritional status: malnourished (15.4%), at risk for malnutrition (38.9%), and well-nourished (45.7%). In contrast, cognitive changes followed four largely linear but distinct trajectories: moderately impaired (12.2%), mildly impaired (27.8%), borderline impaired (21.8%), and cognitively intact (38.2%). Trajectories of nutritional status were significantly associated with cognitive-function trajectories. For instance, relative to malnourished patients, well-nourished patients were 95% less likely (OR=0.05, CI =0.01-0.24) to be moderately cognitively impaired. CONCLUSION: A good nutritional-status trajectory after hip fracture was associated with better cognitive function. To treat and care for elderly hip-fractured patients, specific interventions need to target those who are malnourished or at risk of malnutrition to decrease their risk for cognitive impairment.

A new anti-HBeAg lignan, kadsumarin A, from Kadsura matsudai and Schizandra arisanensis.

A new C18 dibenzocyclooctadiene lignan, kadsumarin A (1) was isolated from Kadsura matsudai Hayata and Schizandra arisanensis Hayata. The anti-HBeAg test revealed that kadsumarin A had activity at a concentration of 40 micrograms/ml (= 90.1 microM). Its structural elucidation by spectral analysis was discussed in this note.

Antitumour and anti-AIDS triterpenes from Celastrus hindsii.

Four new triterpene compounds, celasdin-A, celasdin-C, anti-AIDS celasdin-B and cytotoxic maytenfolone-A, were isolated from Celastrus hindsii. The structural determination of maytenfolone-A, celasdin-A, celasdin-B and celasdin-C, as well as the structure-activity relationships of these new compounds and derivatives, are discussed. Maytenfolone-A was further confirmed by X-ray studies. Biological evaluation showed that Maytenfolone-A demonstrated cytotoxicity against hepatoma (HEPA-2B, ED50 = 2.3 micrograms ml-1) and nasopharynx carcinoma (KB, ED50 = 3.8 micrograms ml-1). Celasdin-B was found to exhibit anti-HIV replication activity in H9 lymphocyte cells with an EC50 of 0.8 microgram ml-1).

Specific domains of beta-amyloid from Alzheimer plaque elicit neuron killing in human microglia.

Alzheimer's disease (AD) is found to have striking brain inflammation characterized by clusters of reactive microglia that surround senile plaques. A recent study has shown that microglia placed in contact with isolated plaque fragments release neurotoxins. To explore further this process of immunoactivation in AD, we fractionated plaque proteins and tested for the ability to stimulate microglia. Three plaque-derived fractions, each containing full-length native A beta 1-40 or A beta 1-42 peptides, elicited neurotoxin release from microglia. Screening of various synthetic peptides (A beta 1-16, A beta 1-28, A beta 12-28, A beta 25-35, A beta 17-43, A beta 1-40, and A beta 1-42) confirmed that microglia killed neurons only after exposure to nanomolar concentrations of human A beta 1-40 or human A beta 1-42, whereas the rodent A beta 1-40 (5Arg-->Gly, 10Tyr-->Phe 13His-->Arg) was not active. These findings suggested that specific portions of human A beta were necessary for microglia-plaque interactions. When coupled to microspheres, N-terminal portions of human A beta (A beta 1-16, A beta 1-28, A beta 12-28) provided anchoring sites for microglial adherence whereas C-terminal regions did not. Although itself not toxic, the 10-16 domain of human A beta was necessary for both microglial binding and activation. Peptide blockade of microglia-plaque interactions that occur in AD might prevent the immune-driven injury to neurons.

Celahinine A, a new sesquiterpene pyridine alkaloid from Celastrus hindsii.

A new sesquiterpene pyridine alkaloid, celahinine A [1], and the related known polyester celahin A, as well as the known cytotoxic sesquiterpene pyridine alkaloid emarginatine A [2], were isolated from Celastrus hindsii. The structure of 1 was determined by 2D nmr techniques and was also confirmed by spectral comparison with the related 2.

Antitumor agents, 112. Emarginatine B, a novel potent cytotoxic sesquiterpene pyridine alkaloid from Maytenus emarginata.

A new sesquiterpene pyridine alkaloid, emarginatine B [2] has been isolated, along with maytansine [3], from Maytenus emarginata. Emarginatine B showed potent cytotoxicity against human KB cells (ED50 = 0.4 micrograms/ml). Spectral correlations established its structure as a 9-benzoate and C-8 epimer of emarginatine A [1]. The conformational aspect of the latter compound was elucidated by nmr studies, including the use of 2D-nmr techniques and difference nOe methods.

Structure-function relationships for the IL-2 receptor system. V. Structure-activity analysis of modified and truncated forms of the Tac receptor protein: site-specific mutagenesis of cysteine residues.

IL-2R on activated lymphocytes contain the Tac protein. As part of an effort to characterize this molecule, we examined the structure-activity relationship for each of its 12 Cys residues. A preliminary map of intramolecular disulfide bonding was derived by analysis of cystine-linked enzymatic fragments of the Tac protein. The results indicated that disulfide bonds linked Cys-3 with Cys-147, Cys-131 with Cys-163, and Cys-28,30 with Cys-59,61. The contribution of the Cys residues to an active protein conformation was tested by site-specific mutagenesis, followed by expression of the modified molecules in murine L cells. The results indicated that Cys-192 and -225 could be replaced without affecting ligand binding. In contrast, modification of any of the other 10 Cys residues, either singly or in combinations corresponding to the predicted disulfide bonds, greatly reduced the ability of the corresponding protein to bind IL-2 or either of two mAb (anti-Tac and 7G7/B6) which recognize the Tac protein. Each of the latter mutations also interfered with the molecule's post-translational modification and cell-surface expression. Consistent with these findings, transfection of the L cells with vectors containing truncated Tac cDNA inserts resulted in secretion of Tac fragments capable of ligand binding when the polypeptide chains terminated after Cys-163 (the 10th Cys residue in the full length molecule), but resulted in inactive fragments of Tac which were poorly secreted when they terminated before Cys-163. These findings emphasize the remarkable sensitivity of the active conformation of the Tac molecule to each of the postulated intramolecular disulfide bonds.

Structure function relationships for the IL 2 receptor system. III. Tac protein missing amino acids 102 to 173 (exon 4) is unable to bind IL 2: detection of spliced protein after L cell transfection.

High affinity receptors for interleukin 2 (IL 2) contain the Tac protein as one ligand-binding subunit. Localization of the IL 2-binding site on this molecule, as well as localization of the complementary site on IL 2, should provide insight into the design of IL 2 analogs. In this report, we examine the ability of normal and modified Tac protein to bind IL 2 and several antibodies that recognize the native Tac molecule. Using a transient L cell expression system, we have determined that transfection with cDNA-missing Tac exon 4 resulted in expression of spliced protein that had no measurable binding to IL 2 or the monoclonal anti-receptor antibodies, anti-Tac, and 7G7/B6. This protein was detected, however, by rabbit polyclonal antibodies prepared against synthetic Tac peptides. Thus, one or more amino acids encoded by exon 4 is important, either for direct ligand contact or for the proper folding of critical segments of the Tac molecule. In addition, insertion of stop codons at a unique restriction enzyme site near the beginning of exon 5 resulted in cellular secretion of truncated Tac molecules that were capable of binding IL 2, anti-Tac, and 7G7/B6. Amino acids encoded by exons 5 to 8 thus play no critical role in IL 2 binding. The ligand association demonstrated for truncated Tac protein produced by exons 2 to 4 should guide attempts to define the IL 2-binding segment of the Tac molecule.

Structure-function relationships for the IL 2-receptor system. I. Localization of a receptor binding site on IL 2.

Interleukin 2 (IL 2) modulates the growth and differentiation of a variety of lymphocyte subclasses through its interaction with a specific cell surface receptor. Although both IL 2 and its receptor have been characterized extensively, the location of interaction sites on the two molecules is unknown. Synthetic peptides based on the IL 2 sequence were used to determine the epitopes seen by a number of antibodies reactive with IL 2, some of which inhibited the receptor binding of the factor and the proliferative response of target cells. The results indicated that inhibitory antibodies bound at either of two spatially distinct sites defined by amino acids 8-27 and 33-54. By inference, these segments may also encode distinct contact sites for receptor association. Alternatively, a single contact site may be located between the antibody epitopes in the three-dimensional configuration of the molecule. Although the data did not directly address the role of the C-terminal portion of IL 2, this preliminary localization of receptor contact sites within the N-terminal half of the molecule should prove useful in correlating structure and function during crystallographic analysis of the factor.

Structure-function relationships for the IL 2-receptor system. II. Localization of an IL 2 binding site on high and low affinity receptors.

The ligand-binding component of high and low affinity IL 2 receptors is a 55,000 m.w. glycoprotein termed Tac. Correlating the structure and function of this molecule should provide insight into the mechanism of IL 2-initiated signal transduction and the structural basis for high and low affinity receptor forms. As a first step in this process, various approaches were used to localize the IL 2 binding region of the Tac molecule. Antibodies prepared to synthetic fragments of Tac were tested for their ability to interfere with IL 2 binding and bioactivity. The results delineated segments in the C-terminal portion of the molecule which appeared to be distal to the ligand binding site. In a more direct approach, radioiodinated IL 2 was cross-linked to high and low affinity receptors, and the resulting complexes were subjected to mild tryptic digestion. Consistent with the antibody data, the IL 2 remained covalently associated with an N-terminal tryptic fragment which apparently consisted of residues 1-83 of the Tac protein. These results suggest that the N-terminal region of the Tac molecule contains important contact sites for ligand-receptor interaction.

Monoclonal antibody to human cytochrome c: effect on electron-transfer reactions.

A monoclonal antibody has been produced to an antigenic site on human cytochrome c which includes amino acid number 58 (isoleucine). This area is on the bottom back of the cytochrome, removed from the postulated binding/reaction sites for oxidase and reductase, but in the area of the molecule where an appreciable change in conformation is seen on oxidation-reduction. In spectrophotometric assays, where binding of cytochrome c to the oxidase or reductase is rate-limiting, the antibody gave stimulation of the reductase reaction under some conditions, where the oxidase reaction was inhibited. Also variation of the pH of the reaction medium resulted in differential effects on the oxidase and reductase reactions. Different effects of the antibody were seen when the oxidase was assayed polarographically, as compared to the spectrophotometric measurements. The data show that the binding/reaction sites on cytochrome c for the oxidase and reductase must be different. They suggest that binding of antibody may affect conformational changes in the whole molecule, distorting the binding/reaction sites. Conformational changes may be involved as a control mechanism in cytochrome c-mediated electron-transfer reactions.

Monoclonal antibodies to cytochrome c from Paracoccus denitrificans: effects on electron transport reactions.

The effect of a monoclonal antibody to a soluble cytochrome c from Paracoccus denitrificans was tested on the membrane-bound electron-transport system of this bacterium. This antibody (F3-10.2) and one previously described (F3-29.4) (Kuo, L.M., Davies, H.C. and Smith, L. (1984) Biochim. Biophys. Acta 766, 472-482) were deduced to bind to the cytochrome c in the area including amino acid residue number 23 on a loop on the side of the heme crevice. In contrast to the observations with the previously tested antibody, the present data show the second antibody to block completely the reaction of the cytochrome c with cytochrome c oxidase but not that with cytochrome c reductase. Neither antibody has an appreciable inhibitory effect on the NADH oxidase of the isolated detergent-treated membranes. The two antibodies bind in different ways, giving insight into the interaction of a soluble protein with membrane-bound enzymes. The data indicate that the reaction sites on the cytochrome c for the oxidase and reductase moieties of P. denitrificans are different. They also argue against the need for a dissociable cytochrome c comparable to that which functions on the mitochondrial inner membrane.

Effects of monoclonal antibodies to bovine and Paracoccus denitrificans cytochromes c on reactions with oxidase, reductase and peroxidase.

The effects of monoclonal antibodies to bovine and Paracoccus denitrificans cytochromes c (Kuo, L.M. and Davies, H.C. (1983) Mol. Immunol. 20, 827-838) in the reactions of the cytochromes c with cytochrome c oxidase, reductase and peroxidase were studied. Spectrophotometric assays were employed, under conditions where binding of cytochrome c to the enzymes appears to be rate-limiting. Less than stoichiometric amounts of antibodies to P. denitrificans cytochrome c added to the cytochrome rendered some of it nonoxidizable or nonreducible by the P. denitrificans membrane-bound electron transport system and decreased the rate constant with the remaining cytochrome c. The antibodies appear to affect both electron transport reactions (blocking effects) with the oxidase and reductase and binding effects (effects on rate constants) and to distinguish between the two. Different ratios of antibody site to cytochrome c gave different extents of blocking of the reductase as compared with the oxidase reaction. Differences were also apparent in the effect of these antibodies on the reaction of yeast peroxidase and the oxidase with the P. denitrificans cytochrome c. Antibodies to bovine and P. denitrificans cytochromes c had considerably less effect on the reactions of the bovine cytochrome with bovine oxidase and reductase. One antibody was inhibitory to the oxidase reaction with bovine cytochrome c, but not to that with the reductase. Also, an antibody which inhibited the oxidase reaction had no effect on the reaction with yeast peroxidase. The data give evidence that the interaction areas on cytochrome c for oxidase and reductase and peroxidase are not identical, although they may be nearby.

Production, isolation and characterization of monoclonal antibodies to cytochromes c of beef heart and Paracoccus denitrificans.

Hybridoma cell lines secreting monoclonal antibodies which bind beef heart cytochrome c or Paracoccus denitrificans cytochrome c have been produced using spleen cells from BALB/c mice immunized with cytochrome c. Immunization was performed with either the native cytochrome c, succinylated hemocyanin-conjugated cytochrome c, or beef heart cytochrome c polymerized with glutaraldehyde. Of 10 such fusions, the hybridization frequency ranged from 0 to 42%. The cell fusion efficiency, the possible factors involved in the cell fusion efficiency and the frequency of antibody producing hybridomas are described. The percentage of hybridomas positive for anti-cytochrome c antibody production as screened for by radioimmunoassay or ELISA was 2%. Of the antibodies from 12 hybridoma cell lines which resulted from 10 fusions, three were specific to beef heart cytochrome c, another three were specific to P. denitrificans cytochrome c, and the remainder reacted with both cytochromes c. These groups of monoclonal antibodies react to different sets of sites on these two cytochromes c. The monoclonal antibodies from ten representative clones have been isolated and characterized by different methods.

Suppression of the formation of polygenotypic recombinant colonies by a maf mutation in mating with HfrH.

W3011, a Cavalli-type Hfr (HfrC), was mated with F-KY9474, maf-1, which cannot maintain F or F-like plasmids, and with F-OU9474, Maf+, a spontaneous revertant of KY9474. The recombinant colonies obtained were 100% monogenotypic from KY9474 and 90% monogenotypic from OU9474. On the other hand, in matings with OU11, a Hayes-type Hfr (HfrH), and these two F- strains, recombinant colonies derived from KY9474 showed only 22% polygenotypic recombinant colonies; whereas, those derived from OU9474 showed a high production rate (57%) of polygenotypic recombinant colonies. Among the polygenotypic recombinant colonies derived from KY9474 maf-1, 50% contained three or more recombinant types. These were probably derived from a small fraction of Maf+ revertants in the KY9474 population, as suggested by the results of mating this strain with M80, an F' strain that contains an amber mutation in traH. These results support the hypothesis that the donor DNA fragments derived from an HfrH can undergo a limited replication in the recipient to produce polygenotypic recombinant colonies, whereas those derived from HfrC cannot.