Improved high-performance liquid chromatography determination of methotrexate and its major metabolite in plasma using a poly(styrene-divinylbenzene) column

A sensitive high-performance liquid chromatographic assay has been developed for measuring plasma concentrations of methotrexate and its major metabolite, 7-hydroxymethotrexate. Methotrexate and metabolite were extracted from plasma using solid-phase extraction. An internal standard, aminopterin was used. Chromatographic separation was achieved using a 15-cm poly(styrene-divinylbenzene) (PRP-1(R)) column. This column is more robust than a silica-based stationary phase. Post column, the eluent was irradiated with UV light, producing fluorescent photolytic degradation products of methotrexate and the metabolite. The excitation and emission wavelengths of fluorescence detection were at 350 and 435 nm, respectively. The mobile phase consisted of 0.1 M phosphate buffer (pH 6.5), with 6% N,N-dimethylformamide and 0.2% of 30% hydrogen peroxide. The absolute recoveries for methotrexate and 7-hydroxymethotrexate were greater than 86%. Precision, expressed as a coefficient of variation (n=6), was

Determination of chemical shielding tensor of an indole carbon and application of tryptophan orientation of a membrane peptide

Using tryptophan C-13-enriched at the C-4 (C epsilon(3)) of the indole, the orientation of the C epsilon(3) chemical shift tensor relative to the C epsilon(3)-H dipolar axis was determined from the C-13 chemical shift/C-13-H-1 dipolar 2D NMR powder pattern. The principal values obtained were 208, 137 and 15 ppm with sigma(33) perpendicular to the indole plane, and sigma(11) (least shielded direction) 5 degrees off the C epsilon(3)-H bond toward C xi(3). The side off the C epsilon(3)-H bond was determined by comparing the reduced chemical shift anisotropies obtained by solid-state NMR and from molecular dynamics calculations of [4-C-13] tryptophans in gramicidin A aligned in phospholipid membranes. (C) 1999 Elsevier Science B.V. All rights reserved.

Ca2+ sensitivity of phospholipid scrambling in human red cell ghosts

The phospholipids in plasma membranes of erythrocytes, as well as platelets, lymphocytes and other cells are asymmetrically distributed, with sphingomyelin and phosphatidylcholine residing predominantly in the outer leaflet of the bilayer, and phosphatidylserine and phosphatidylethanolamine in the inner leaflet. It is known that Ca2+ can disrupt the phospholipid asymmetry by activation of a protein known as phospholipid scramblase, which affects bidirectional phospholipid movement in a largely non-selective manner. As Ca2+ also inhibits aminophospholipid translocase, whose Mg2+-ATPase activity is responsible for active translocation of aminophospholipids from the outer to the inner leaflet, it is important to accurately determine the sensitivity of scramblase to intracellular free Ca2+. In the present study we have utilized the favourable K-d, of Mag-fura-2 for calcium in the high micromolar range to determine free Ca2+ levels associated with lipid scrambling in resealed human red cell ghosts. The Ca2+ sensitivity was measured in parallel to the translocation of a fluorescent-labelled lipid incorporated into the ghost bilayer. The phospholipid scrambling was found to be half-maximally activated at 63-88 mu M free intracellular Ca2+. The wider applicability of the method and the physiological implications of the calcium sensitivity determined is discussed.

Ontological changes in the crystallin composition of the eye lenses of the territorial damselfish Parma microlepis and their possible effects on trace-metal accumulation

The eye lenses of Parma microlepis from the rocky barrens of Sydney (New South Wales, Australia) were found to contain Ba, Hg, Rb, and Sr at concentrations above the quantitative detection limits of solution-based inductively-coupled plasma-mass spectrometry (ICP-MS). Lenses were separated into the hard central nucleus and the softer surrounding cortex. Nuclei contained lower (equal for Ba) concentrations of these metals. Biochemical analysis of the protein composition of these lenses revealed differences in the ratio of gamma-crystallin to beta-crystallin in the lens nucleus and cortex. These changes were shown to be attributable both to protein degradation and changes in protein synthesis as fish age. Such changes may lead to the loss of sequestered metals from older cell layers, or change the affinity of new layers for particular trace metals. Differential binding affinities of these crystallins may, therefore, partially account for trace-metal differences observed in the lens nucleus and cortex.

A role for protein kinase B beta/Akt2 in insulin-stimulated GLUT4 translocation in adipocytes

Insulin stimulates glucose uptake into muscle and fat cells by promoting the translocation of glucose transporter 4 (GLUT4) to the cell surface. Phosphatidylinositide 3-kinase (PI3K) has been implicated in this process. However, the involvement of protein kinase B (PKB)/Akt, a downstream target of PI3K in regulation of GLUT4 translocation, has been controversial. Here we report that microinjection of a PKB substrate peptide or an antibody to PKB inhibited insulin-stimulated GLUT4 translocation to the plasma membrane by 66 or 56%, respectively. We further examined the activation of PKB isoforms following treatment of cells with insulin or platelet-derived growth factor (PDGF) and found that PKB beta is preferentially expressed in both rat and 3T3-L1 adipocytes, whereas PKB alpha expression is down-regulated in 3T3-L1 adipocytes. A switch in growth factor response was also observed when 3T3-L1 fibroblasts were differentiated into adipocytes. While PDGF was more efficacious than insulin in stimulating PKB phosphorylation in fibroblasts, PDGF did not stimulate PKB beta phosphorylation to any significant extent in adipocytes, as assessed by several methods. Moreover, insulin, but not PDGF, stimulated the translocation of PKB beta to the plasma membrane and high-density microsome fractions of 3T3-L1 adipocytes. These results support a role for PKB beta in insulin-stimulated glucose transport in adipocytes.

Altered adhesion, proliferation and death in neural cultures from adults with schizophrenia

The causes of schizophrenia are unknown, but there is evidence linking subtle deviations in neural development with schizophrenia. Embryonic brain development cannot be studied in an adult with schizophrenia, but neurogenesis and early events in neuronal differentiation can be investigated throughout adult life in the human olfactory epithelium. Our past research has demonstrated that neuronal cultures can be derived from biopsy of the human adult olfactory epithelium. In the present study, we examined mechanisms related to neurogenesis and neuronal differentiation in adults with schizophrenia versus well controls. Forty biopsies were collected under local anaesthesia from ten individuals with DSM III-R schizophrenia and ten age- and sex-matched well controls. All patients, except one, were receiving antipsychotic medication at the time of the biopsy, Immunostaining for neuronal markers indicated that neurogenesis occurred in the biopsies from both patients and controls since all contained cells expressing tubulin and/or olfactory marker protein. The major findings of this study are: 1. biopsies from patients with schizophrenia showed a significantly reduced ability to attach to the culture slide: 29.9% of patient biopsies attached compared to 73.5% of control biopsies; 2. biopsies from patients with schizophrenia had a significantly greater proportion of cells undergoing mitosis: 0.69% in the patients compared to 0.29% in the controls; and 3. dopamine (10 mu M) significantly increased the proportion of apoptotic cells in the control cultures but significantly decreased the proportion in patients' cultures. (C) 1999 Elsevier Science B.V. All rights reserved.

Thylakoid membrane architecture

Confocal scanning laser microscopic observations were made on live chloroplasts in intact cells and on mechanically isolated, intact chloroplasts. Chlorophyll fluorescence was imaged to observe thylakoid membrane architecture. C-3 plant species studied included Spinacia oleracea L., Spathiphyllum sp. Schott, cv. 'Mauna Loa', and Pisum sativum L. C-4 plants were also investigated: Saccharum officinarum L., Sorghum bicolor L. Moench, Zea mays L. and Panicum miliaceum L. Some Spinacia chloroplasts were treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) to enhance or sodium dithionite (SD) to reduce the photosystem II fluorescence signal. Confocal microscopy images of C-3 chloroplasts differed from electron microscopy pictures because they showed discrete spots of bright fluorescence with black regions between them. There was no evidence of fluorescence from stroma thylakoids. The thylakoid membrane system at times appeared to be string-like, with brightly fluorescing grana lined up like beads. C-4 bundle sheath chloroplasts were imaged from three different types of C-4 plants. Saccharum and Sorghum bundle sheath chloroplasts showed homogeneous fluorescence and were much dimmer than mesophyll chloroplasts. Zea had rudimentary grana, and dim, homogeneous intergrana fluorescence was visualised. Panicum contained thylakoids similar in appearance and string-like arrangement to mesophyll chloroplasts. Isolated Pisum chloroplasts, treated with a drop of 5 mM MgCl2 showed a thylakoid membrane system which appeared to be unravelling. Spongy mesophyll chloroplasts of Spinacia treated with 5 mM sodium dithionite showed a granal thylakoid system with distinct regions of no fluorescence. A time-series experiment provided evidence of dynamic membrane rearrangements over a period of half an hour.

Evolutionary conservation of the membrane fusion machine

Recent structural studies of proteins mediating membrane fusion reveal intriguing similarities between diverse viral and mammalian systems. Particularly striking is the close similarity between the transmembrane envelope glycoproteins from the retrovirus HTLV-1 and the filovirus Ebola. These similarities suggest similar mechanisms of membrane fusion. The model that fits most currently available data suggests fusion activation in viral systems is driven by a symmetrical conformational change triggered by an activation event such as receptor binding or a pH change. The mammalian vesicle fusion mediated by the SNARE protein complex most likely occurs by a similar mechanism but without symmetry constraints.

Structure of the tegument and ectocommensal microorganisms of Gyliauchen nahaensis (Digenea : Gyliauchenidae), an inhabitant of herbivorous fish of the Great Barrier Reef, Australia

The ultrastructure of the tegument and tegument-associated microorganisms of the gyliauchenid digenean Gyliauchen nahaensis is described by transmission and scanning electron microscopy. The tegument is devoid of surface spines and is characterized by a moderately folded apical membrane, abundant vesicles, basal mitochondria, a folded basal plasma membrane, and a thick basal matrix. Microorganisms form a dense biofilm on the tegument of the posterodorsal surface and the excretory papilla. At least 7 microbial morphotypes were identified, including eubacteria, spirochaetes, and nanobacteria.

E2F-1 induces proliferation-specific genes and suppresses squamous differentiation-specific genes in human epidermal keratinocytes

Squamous differentiation of keratinocytes is associated with decreases in E2F-1 mRNA expression and E2F activity, and these processes are disrupted in squamous cell carcinoma cell lines. We now show that E2F-1 mRNA expression is increased in primary squamous cell carcinomas of the skin relative to normal epidermis, To explore the relationship between E2F-1 and squamous differentiation further, we examined the effect of altering E2F activity in primary human keratinocytes induced to differentiate. Promoter activity for the proliferation-associated genes, cdc2 and keratin 14, are inhibited during squamous differentiation. This inhibition can be inhibited by overexpression of E2F-1 in keratinocytes, Overexpression of E2F-1 also suppressed the expression of differentiation markers (transglutaminase type 1 and keratin 10) in differentiated keratinocytes, Blocking E2F activity by transfecting proliferating keratinocytes with dominant negative E2F-1 constructs inhibited the expression of cdc2 and E2F-1, but did not induce differentiation. Furthermore, expression of the dominant negative construct in epithelial carcinoma cell lines and normal keratinocytes decreased expression from the cdc2 promoter. These data indicate that E2F-1 promotes keratinocyte proliferation-specific marker genes and suppresses squamous differentiation-specific marker genes. Moreover, these data indicate that targeted disruption of E2F-1 activity may have therapeutic potential for the treatment of squamous carcinomas.

Chopper, a new death domain of the p75 neurotrophin receptor that mediates rapid neuronal cell death

The cytoplasmic juxtamembrane region of the p75 neurotrophin receptor (p75(NTR)) has been found to be necessary and sufficient to initiate neural cell death. The region was named Chopper to distinguish it from CD95-like death domains. A 29-amino acid peptide corresponding to the Chopper region induced caspase- and calpain-mediated death in a variety of neural and nonneural cell types and was not inhibited by signaling through Trk (unlike killing by full-length p75(NTR)). Chopper triggered cell death only when bound to the plasma membrane by a lipid anchor, whereas non-anchored Chopper acted in a dominant-negative manner, blocking p75(NTR)-mediated death both in vitro and in vivo. Removal of the ectodomain of p75(NTR) increased the potency of Chopper activity, suggesting that it regulates the association of Chopper with downstream signaling proteins.

PMCA1 mRNA expression in rat aortic myocytes: A real-time RT-PCR study

The plasmalemmal Ca2+ adenosine triphosphatase (PMCA) is a key regulator of Ca2+ efflux in vascular smooth muscle. In these studies are developed a realtime reverse transcriptase-polymerase chain reaction (real-time RT-PCR) assay for assessing PMCA1 mRNA levels in rat primary cultured aortic myocytes. This assay detected fetal bovine serum-induced increases in PMCA1 mRNA (relative to 18S rRNA) 4, 8, and 24 h after stimulation. Early fetal bovine serum-induced increases in PMCA1 mRNA were insensitive to the Ca2+ channel blockers nifedipine, flunarizine, and SKF-96365. These studies demonstrate the feasibility of real-time RT-PCR to assess mRNA levels of PMCA1 and illustrate dynamic regulation of this Ca2+ pump isoform in rat primary cultured aortic myocytes, (C) 2000 Academic Press.

An economic evaluation of plasma production via erythroplasmapheresis and whole blood collection

This paper presents cost-effectiveness analyses (CEAs) of plasma collection via two alternative methods: whole blood collection (WBC) and erythroplasmapheresis collection (EPC). The objective of the study is to provide an answer to the question 'What is the least-cost method of plasma production'. This question is answered, both from the viewpoint of the blood collection agency (using financial CEA) and from that of 'society' as a whole (using economic CEA). We employ detailed financial data and economic survey data for collections made by a blood collection agency and to WBC and EPC donors in Brisbane, Australia. The results indicate that, despite the superior yield provided by EPC, WBC is actually more cost-effective. This result is robust to thorough sensitivity analysis and arises regardless of whether an economic or financial perspective is taken. We conclude that, ceteris paribus, the cost of recruiting new plasma donors would need to be quite substantial for marginal investments in EPC to be considered cost-effective. Crown Copyright (C) 2002 Published by Elsevier Science Ltd. All rights reserved.