Role of nucleoside transport and purine release in a rabbit model of myocardial stunning

Previously, we have demonstrated the role of nucleoside transport and purine release in post-ischemic reperfusion injury (myocardial stunning) in several canine models of ischemia. Since rabbits are deficient of xanthine oxidase, it is not known whether selective blockade of purine release is beneficial in a rabbit model of coronary artery occlusion and reperfusion (stunning). Therefore, we determined the hemodynamic and metabolic correlates in response to myocardial stunning in the presence or absence of selective nucleoside transport blocker (p-nitrobenzylthioinosine, NBMPR) and adenosine deaminase inhibitor (erythro-9-(2-hydroxy-3-nonyl)adenine, EHNA).Sixty adult anaesthetized rabbits were surgically prepared for hemodynamic measurements. After stabilization period, the left anterior descending coronary artery was occluded for 15 min and reperfused for 30 min. Transmural myocardial biopsies were obtained from the ischemic LAD area and from the non-ischemic posterior (circumflex, CFX) segment of the myocardium.Rabbits (n = 60) were randomly assigned to either the control or the EHNA/NBMPR-treated group (n = 30 each). Each group was further divided to either functional or metabolic groups (n = 15 each subgroup). Each animal received intravenously 30 ml of either a vehicle solution or 100 M EHNA and 25 M NBMPR 10 min before ischemia.Although administration of EHNA/NBMPR did not affect the heart rate, it did cause mild hypotension (about 20-30%). Fifteen minutes of LAD occlusion resulted in significant ATP depletion and concomitant accumulation of nucleosides in both groups (p < 0.05 vs. baseline and non-ischemic CFX segment). AMP was higher in the LAD compared to the CFX segment. Significant accumulation of adenosine was observed in the treated group compared to the control group.It is concluded that EHNA/NBMPR induced site specific entrapment of adenosine of nucleoside transport in the rabbit heart, in vivo.     

Fas Ligand-Mediated Lysis of Self Bystander Targets by Human Papillomavirus-Specific CD8+ Cytotoxic T Lymphocytes

Mouse cytotoxic T lymphocytes (CTL) reactive with a H-2D^b-presented 9-mer peptide of the human papillomavirus type 16 protein E7^49-57 (RAHYNIVTF) were generated from the spleen cells of wild-type C57BL/6 (B6) or B6 perforin-deficient (B6.P⁰) mice. CD8⁺ B6 CTL displayed peptide-specific perforin- and Fas-mediated lysis of E7-transfected mouse RMA lymphoma cells (RMA-E7), while CD8⁺ CTL from B6.P⁰ mice lysed RMA-E7 cells via Fas ligand (FasL) exclusively. Rapid and efficient lysis of syngeneic bystander B6 blasts or RMA cells by either B6 or B6.P⁰ Ag-activated CTL was mediated by a FasL-Fas mechanism. Fas-resistant bystanders were not lysed, nor were allogeneic Fas-sensitive C3H/HeJ (H-2^k) or BALB/c (H-2^d) bystander blasts. Interestingly, however, phorbol myristate acetate-ionomycin preactivation of B6.P⁰ effectors enabled lysis of allogeneic H-2^k and H-2^d bystanders even in the absence of antigenic stimulation. Lysis of syngeneic bystander cells was always FasL-Fas dependent and required effector-bystander contact and, in particular, an interaction between CTL LFA-1 and bystander ICAM-1. Thus, in the context of major histocompatibility complex class I molecule-peptide ligation of the T-cell receptors of CD8⁺ CTL, neighboring bystander cells that are syngeneic and Fas sensitive and express the adhesion molecule ICAM-1 are potential targets of CTL attack.With the dissection of two basic cytolytic mechanisms of cytotoxic T lymphocytes (CTL) (10, 14, 20, 34), it has become possible to delineate the important criteria that determine direct (Ag-restricted) and bystander cytotoxicity. CTL use complementary cytotoxic mechanisms, one based on the granule exocytosis of a calcium-dependent pore-forming protein, perforin (8, 26), and granzymes (35) and another that depends on a calcium-independent interaction of effector T-cell tumor necrosis factor or Fas ligand (TNF or FasL) and target cell TNF receptor (TNFR) or Fas (22, 33). The function of the granule exocytosis pathway appears to be largely in non-major histocompatibility complex (MHC)-restricted NK lysis of class I molecule-defective tumor cells and in direct CTL-mediated immunity against tumor cells (37) or virus-infected cells (11, 19, 39). By contrast, the FasL-Fas and TNF-TNFR interactions are important for the maintenance of T-cell homeostasis following exposure to foreign Ag (5, 42) and Th-1 FasL-mediated B-cell apoptosis (27, 28). Blockage of both TNF and FasL is required to abrogate T-cell death: TNF mediates the death of most CD8⁺ T cells, whereas FasL mediates the death of most CD4⁺ T cells (42). While FasL-dependent lysis appears to be the primary mechanism used by CD4⁺ Th-1 effectors, CD8⁺ CTL use FasL or TNF secondarily in the absence of perforin-mediated lysis (10, 14, 20).After T-cell activation, a functional role for FasL is not apparent for several days until the T cell becomes Fas sensitive and hence susceptible to autocrine T-cell suicide (1, 5, 38). However, by using alloreactive CTL cultures or clones, it has recently become apparent that in the presence of Ag-bearing target cells (i.e., upon T-cell receptor [TCR] activation) CTL can also lyse Ag-free bystander cells via a FasL-Fas interaction (13, 34). While the specificity of CTL toward Ag-bearing target cells has been considered a hallmark of an efficient immune response, CTL do not appear to spare Ag-free bystander cells during lysis of specific Ag-bearing target cells. In this study, we have generated CD8⁺ CTL from both wild-type and perforin-deficient (P⁰) mice reactive with a high-affinity H-2D^b-binding peptide of human papillomavirus type 16 protein E7. These peptide-specific CTL have been employed to demonstrate the requirements for CD8⁺ CTL-mediated lysis of Ag-free bystander cells and in particular the different properties of CTL activated by antigen versus a nonspecific stimulus.     

Phosphatidylcholine metabolism in ischemic and hypoxic hearts

The rates of phosphatidylcholine biosynthesis in the isolated hamster hearts under ischemic and hypoxic conditions were examined. Global ischemia was produced by perfusion of the heart with a reduced flow, whereas hypoxia was produced by perfusion with a N₂-saturated buffer. A 51% reduction in the biosynthesis of phosphatidylcholine was observed in the ischemic heart. The reduction was caused by a severe decrease in ATP level which resulted in a diminished conversion of choline into phosphocholine. A 22% reduction in the biosynthetic rate of phosphatidylcholine was also detected in the hypoxic heart. The reduction was caused by a diminished level of CTP which resulted in a decreased conversion of phosphocholine to CDP-choline. No compensatory mechanism was triggered during ischemia, but the CTP: phosphocholine cytidylyltransferase activity was enhanced in the hypoxic heart. Our results demonstrate the possible rate-limiting role of choline kinase and reconfirm the regulatory role of the cytidylyltransferase in the biosynthesis of phosphatidylcholine. (Mol Cell Biochem116: 53–58, 1992)     

Infectious molecular clone of a recently transmitted pediatric human immunodeficiency virus clade C isolate from Africa: evidence of intraclade recombination

Although human immunodeficiency virus type 1 (HIV-1) clade C continues to dominate the pandemic, only two infectious clade C proviral DNA clones have been described (N. Mochizuki, N. Otsuka, K. Matsuo, T. Shiino, A. Kojima, T. Kurata, K. Sakai, N. Yamamoto, S. Isomura, T. N. Dhole, Y. Takebe, M. Matsuda, and M. Tatsumi, AIDS Res. Hum. Retrovir. 15:1321-1324, 1999; T. Ndung'u, B. Renjifo, and M. Essex, J. Virol. 75:4964-4972, 2001). We have generated an infectious molecular clone of a pediatric clade C strain, HIV1084i, which was isolated from a Zambian infant infected either intrapartum or through breastfeeding. HIV1084i is an R5, non-syncytium-inducing isolate that bears all known clade C signatures; gag, pol, and env consistently mapped within clade C. Interestingly, gag resembled Asian isolates, whereas pol and env resembled African isolates, indicating that HIV1084i probably arose from an intraclade recombination. As a recently transmitted clade C strain, HIV1084i will be a useful vaccine development tool.     

Biochemical and growth regulatory activities of the HIN-200 family member and putative tumor suppressor protein, AIM2

The human HIN-200 family member AIM2 was originally identified in a screen for suppressors of melanoma tumorigenicity following introduction of chromosome 6 into the UACC903 human melanoma cell line. Although the AIM2 protein contained many of the conserved structural motifs common to other HIN-200 proteins, the biochemical characteristics of AIM2 and the ability of overexpressed AIM2 to phenocopy the effect of introduction of chromosome 6 in the UACC903 cells had not been assessed. Herein we demonstrated that AIM2 was localised within the nucleus of transfected or interferon-treated human cells. In addition, AIM2 could homodimerise via the amino-terminal (PAAD/DAPIN) region and heterodimerise with the related IFI 16 protein. However, overexpressed AIM2 did not significantly affect the growth or survival of UACC903 cells or another human melanoma cell line. These data indicate that AIM2 has many of the biochemical and structural characteristics of HIN-200 proteins, however, its expression is not sufficient to induce a tumor-suppressor-like phenotype.     

Modification patterns in germinating barley--malting II

Modification refers to breaking down of cell walls and the conversion of starch-to-sugars in the endosperm of grains by the action of enzymes released from the aleurone layer and possibly the scutellum during germination. Experimentalists have observed two completely different modification patterns in germinating barley. Based on an enzyme reaction, strongly nonlinear diffusion model developed in Part I of this series of papers on malting we examine simple models which predict modifications patterns similar to both observed patterns. We show that one of the observed patterns represents a natural propagation mode that acts as an attractor for the system. The speed of approach to this mode is strongly effected by initial conditions, a consequence of the enzymic nature of the reaction and the dramatic change of diffusivity brought about by the reaction.     

Actin-binding proteins from Burkholderia mallei and Burkholderia thailandensis can functionally compensate for the actin-based motility defect of a Burkholderia pseudomallei bimA mutant

Recently we identified a bacterial factor (BimA) required for actin-based motility of Burkholderia pseudomallei. Here we report that Burkholderia mallei and Burkholderia thailandensis are capable of actin-based motility in J774.2 cells and that BimA homologs of these bacteria can restore the actin-based motility defect of a B. pseudomallei bimA mutant. While the BimA homologs differ in their amino-terminal sequence, they interact directly with actin in vitro and vary in their ability to bind Arp3.     

Fab-based bispecific antibody formats with robust biophysical properties and biological activity

A myriad of innovative bispecific antibody (BsAb) platforms have been reported. Most require significant protein engineering to be viable from a development and manufacturing perspective. Single-chain variable fragments (scFvs) and diabodies that consist only of antibody variable domains have been used as building blocks for making BsAbs for decades. The drawback with Fv-only moieties is that they lack the native-like interactions with C_H1/C_L domains that make antibody Fab regions stable and soluble. Here, we utilize a redesigned Fab interface to explore 2 novel Fab-based BsAbs platforms. The redesigned Fab interface designs limit heavy and light chain mixing when 2 Fabs are co-expressed simultaneously, thus allowing the use of 2 different Fabs within a BsAb construct without the requirement of one or more scFvs. We describe the stability and activity of a HER2×HER2 IgG-Fab BsAb, and compare its biophysical and activity properties with those of an IgG-scFv that utilizes the variable domains of the same parental antibodies. We also generated an EGFR × CD3 tandem Fab protein with a similar format to a tandem scFv (otherwise known as a bispecific T cell engager or BiTE). We show that the Fab-based BsAbs have superior biophysical properties compared to the scFv-based BsAbs. Additionally, the Fab-based BsAbs do not simply recapitulate the activity of their scFv counterparts, but are shown to possess unique biological activity.     

Isolation of primary human hepatocytes from normal and diseased liver tissue: a one hundred liver experience

Successful and consistent isolation of primary human hepatocytes remains a challenge for both cell-based therapeutics/transplantation and laboratory research. Several centres around the world have extensive experience in the isolation of human hepatocytes from non-diseased livers obtained from donor liver surplus to surgical requirement or at hepatic resection for tumours. These livers are an important but limited source of cells for therapy or research. The capacity to isolate cells from diseased liver tissue removed at transplantation would substantially increase availability of cells for research. However no studies comparing the outcome of human hepatocytes isolation from diseased and non-diseased livers presently exist. Here we report our experience isolating human hepatocytes from organ donors, non-diseased resected liver and cirrhotic tissue. We report the cell yields and functional qualities of cells isolated from the different types of liver and demonstrate that a single rigorous protocol allows the routine harvest of good quality primary hepatocytes from the most commonly accessible human liver tissue samples.