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Herpes simplex virus (HSV) infection triggers apoptosis in infected cells. However, proteins synthesized later in infected cells prevent apoptotic cell death from ensuing. In vivo data showing that apoptosis accompanies herpes stromal keratitis and encephalitis suggest that apoptotic modulation plays a role in the development of herpetic disease. Tremendous progress has been made toward identifying the viral factors that are responsible for inducing and inhibiting apoptosis during infection. However, the mechanisms whereby they act are still largely unknown. Recent studies have illustrated a wide diversity in the cellular response to HSV-triggered apoptosis, emphasizing the importance of host factors in this process. Together, these findings indicate that apoptosis during HSV infection represents an important virus-host interaction process, which likely influences viral pathogenesis.

During herpes simplex virus 1 (HSV-1) infection, apoptosis is initiated by immediate early gene transcription and is later modulated by proteins synthesized in infected cells. We have previously shown that procaspase 3 levels are reduced during HSV-1 replication. We now demonstrate that a replication-defective HSV-1 recombinant virus which is incapable of packaging viral DNA into capsids activated caspase 3 but retained the ability to prevent the apoptotic process from killing the infected cells. This implies that HSV-1-dependent apoptosis is not merely a response to abortive infection. Maximum accumulation of the active form of caspase 3 accompanied complete HSV-1-dependent apoptosis. Additionally, caspase 7 was found to be activated during HSV-1-dependent apoptosis. Infected MCF-7 cells which ectopically express caspase 3 underwent more efficient apoptosis than their caspase 3-null parental counterparts, confirming that caspase 3 contributes to HSV-1-dependent apoptosis. However, caspase 3 reconstitution did not make the MCF-7 cells as sensitive as HEp-2 cells to HSV-1-dependent apoptosis, suggesting that other cellular factors may be involved in conferring resistance to this process. These results indicate that caspase 3 activation is a consequence of HSV-1 infection and have important implications in our understanding of the interactions of the virus with host cells.

Telomere length and function are crucial factors that determine the capacity for cell proliferation and survival, mediate cellular senescence, and play a role in malignant transformation in eukaryotic systems. The telomere length of a specific mammalian species is maintained within a given range by the action of telomerase and telomere-associated proteins. TRF1 is a telomere-associated protein that inhibits telomere elongation by its binding to telomere repeats, preventing access to telomerase. Human TRF1 interacts with tankyrase 1 and tankyrase 2 proteins, two related members of the tankyrase family shown to have poly(ADP-ribose) polymerase activity. Human tankyrase 1 is reported to ADP-ribosylate TRF1 and to down-regulate the telomeric repeat binding activity of TRF1, resulting in telomerase-dependent telomere elongation. Human tankyrase 2 is proposed to have activity similar to that of tankyrase 1, although tankyrase 2 function has been less extensively characterized. In the present study, we have assessed the in vivo function of mouse tankyrase 2 by germ line gene inactivation and show that inactivation of tankyrase 2 does not result in detectable alteration in telomere length when monitored through multiple generations of breeding. This finding suggests that either mouse tankyrases 1 and 2 have redundant functions in telomere length maintenance or that mouse tankyrase 2 differs from human tankyrase 2 in its role in telomere length maintenance. Tankyrase 2 deficiency did result in a significant decrease in body weight sustained through at least the first year of life, most marked in male mice, suggesting that tankyrase 2 functions in potentially telomerase-independent pathways to affect overall development and/or metabolism.

During HSV-1 infection, IE gene expression triggers apoptosis, but subsequent synthesis of infected cell proteins blocks apoptotic death from ensuing. This "HSV-1-dependent" apoptosis was identified in HEp-2/HeLa cells infected with wild-type HSV-1 in the presence of an inhibitor of protein synthesis or a virus lacking ICP27 {HSV-1(vBSDelta27)}. Unlike HEp-2/HeLa cells, vBSDelta27-infected Vero cells fail to exhibit dramatic apoptotic morphologies at times prior to 24 hpi. Here, we examined the basis of these different apoptotic responses to HSV-1. We found that infected Vero cells take substantially longer than HEp-2/HeLa cells to display membrane blebbing, chromatin condensation, DNA laddering, and PARP cleavage. Vero, but not HEp-2/HeLa, cells required de novo protein synthesis to exhibit efficient HSV-1-dependent apoptosis, which included changes in mitochondrial membrane potential, and these factors were produced prior to 3 hpi. Vero cells infected with recombinant viruses devoid of the ICP27 and ICP4 proteins alone or both the ICP27 and ICP22 proteins were apoptotic. These results indicate a requirement for cellular or other viral protein synthesis in Vero cells and provide insight into cell type differences in HSV-1-dependent apoptosis.

The plasmodial surface anion channel (PSAC) is an unusual ion channel induced on the human red blood cell membrane after infection with the malaria parasite, Plasmodium falciparum. Because PSAC is permeant to small metabolic precursors essential for parasite growth and is present on red blood cells infected with geographically divergent parasite isolates, it may be an ideal target for future antimalarial development. Here, we used chemically induced mutagenesis and known PSAC antagonists that inhibit in vitro parasite growth to examine whether resistance mutations in PSAC can be readily induced. Stable mutants resistant to phloridzin were generated and selected within 3 weeks after treatment with 1-methyl-3-nitro-1-nitrosoguanidine. These mutants were evaluated with osmotic lysis and electrophysiological transport assays, which indicate that PSAC inhibition by phloridzin is complex with at least two different modes of inhibition. Mutants resistant to the growth inhibitory effects of phloridzin expressed PSAC activity indistinguishable from that on sensitive parasites, indicating selection of resistance via mutations in one or more other parasite targets. Failure to induce mutations in PSAC activity is consistent with a highly constrained channel protein less susceptible to resistance mutations; whether this protein is parasite- or host-encoded remains to be determined.

Nitrogen removal performance is reported for constructed wetlands treating subsurface drainage from irrigated and rain-fed dairy pastures in North Island, New Zealand. Flow-proportional sampling of inflow and outflow concentrations were combined with continuous flow records to calculate mass balances for the wetlands. Drainage flows from the irrigated catchment were 2.5-4 fold higher and N exports up to 5 fold higher per unit area than for the rain-fed catchment. Hydraulic and associated N loadings to the wetlands were highly pulsed, associated with rainfall, soil water status, and irrigation events. Transient pulses of organic nitrogen were an important form of N loss from the rain-fed landscape in the first year, and were very effectively removed in the wetland (> 90%). Median nitrate concentrations of approximately 10 g m(-3) in the drainage inflows were reduced by 15-67% during passage through the wetlands and annual nitrate-N loads by 16-61% (38-31 7 g N m(-2)y(-1)). Generation in the wetlands of net ammoniacal-N and organic-N (irrigated site) partially negated reduction in nitrate-N loads. The results show that constructed wetlands comprising 1-2% of catchment area can provide moderate reductions in TN export via pastoral drainage, but performance is markedly influenced by variations in seasonal loading and establishment/maturation factors.

Phosphorus (P) is one of the most important mineral nutrients in agricultural systems, and along with nitrogen (N), is generally the most limiting nutrient for plant production. Farming systems have intensified greatly over time, and in recent years it has become apparent that the concomitant increase in losses of N and P from agricultural land is having a serious detrimental effect on water quality and the environment. The last two decades have seen a marked increase in research into the issues surrounding diffuse losses of P to surface and ground water. This paper reviews this research, examining the issue of P forms in runoff, and highlighting the exceptions to some generally held assumptions about land use and P transport. In particular the review focuses on P losses associated with recent P fertilizer application, as opposed to organic manures, both on the amounts and the forms of P in runoff water. The effects of the physicochemical characteristics of different forms of P fertilizer are explored, particularly in relation to water solubility. Various means of mitigating the risk of loss of P are discussed. It is argued that the influence of recent fertilizer applications is an under-researched area, yet may offer the most readily applicable opportunity to mitigate P losses by land users. This review highlights and discusses some options that have recently become available that may make a significant contribution to the task of sustainable management of nutrient losses from agriculture.

Background: The amount of dietary fat required for optimal bioavailability of carotenoids in plant matrices is not clearly defined., Objective: The objective was to quantify the appearance of carotenoids in plasma chylomicrons after subjects ingested fresh vegetable salads with fat-free, reduced-fat, or full-fat salad dressings., Design: The subjects (n = 7) each consumed 3 salads consisting of equivalent amounts of spinach, romaine lettuce, cherry tomatoes, and carrots with salad dressings containing 0, 6, or 28 g canola oil. The salads were consumed in random order separated by washout periods of > or =2 wk. Blood samples were collected hourly from 0 to 12 h. Chylomicrons were isolated by ultracentrifugation, and carotenoid absorption was analyzed by HPLC with coulometric array detection., Results: After ingestion of the salads with fat-free salad dressing, the appearance of alpha-carotene, beta-carotene, and lycopene in chylomicrons was negligible. After ingestion of the salads with reduced-fat salad dressing, the appearance of the carotenoids in plasma chylomicrons increased relative to that after ingestion of the salads with fat-free salad dressing (P < 0.04). Similarly, the appearance of the carotenoids in plasma chylomicrons was higher after the ingestion of salads with full-fat than with reduced-fat salad dressing (P < 0.02)., Conclusions: High-sensitivity HPLC with coulometric array detection enabled us to quantify the intestinal absorption of carotenoids ingested from a single vegetable salad. Essentially no absorption of carotenoids was observed when salads with fat-free salad dressing were consumed. A substantially greater absorption of carotenoids was observed when salads were consumed with full-fat than with reduced-fat salad dressing.

Riparian wetlands containing springs are thought to be ineffective at removing nitrate because contact times between the upwelled ground water and the underlying microbially active soils are short. Tracer experiments using lithium bromide (LiBr) and nitrate (NO3-N) injected at the surface were used to quantify residence times and NO3-N removal in a riparian swale characteristic of New Zealand hill-country pasture. An experimental enclosure was used with collecting trays at the downstream end to measure flow and concentration, shallow wells to measure subsurface concentrations, and an array of logging conductivity probes to monitor tracer continuously. The majority of added tracer reached the outlet more slowly than could be explained by surface flow, but more quickly than could be explained by Darcy seepage flow. There was evidence from the wells of tracer diffusing vertically to a depth of at least 5 cm into the surface soil layer, which was permanently saturated and highly porous. During dry weather 24 +/- 9% of added NO3-N was removed over a distance of 1.5 m largely by denitrification. The net uptake length coefficient for this wetland (K = 0.08 +/- 0.03 m(-1)) is slightly higher than the range (K = 0.01-0.07 m(-1)) measured in a small stream channel infested with macrophytes. Nitrate removal is expected to decrease with increasing flow. Seepage flow is estimated to have removed only 7 +/- 4% of the added NO3-N and we hypothesize that vertical diffusion substantially increases NO3-N removal in this type of wetland. Riparian wetlands with springs and surface flows should not be dismissed as having low NO3-N removal potential without checking whether there is significant vertical mixing.

The roles of PDZ domain-containing proteins such as Dlg and Scrib have been well described for Drosophila; however, their requirement for mammalian development is poorly understood. Here we show that Dlg, Scrib, MAGI1, MAGI3, and MPDZ are expressed in the mouse ocular lens. We demonstrate that the increase in proliferation and defects in cellular adhesion and differentiation observed in epithelia of lenses that express E6, a viral oncoprotein that can bind to several PDZ proteins, including the human homologs of Dlg and Scrib, is dependent on E6's ability to bind these proteins via their PDZ domains. Analyses of lenses from mice carrying an insertional mutation in Dlg (dlg(gt)) show increased proliferation and proliferation in spatially inappropriate regions of the lens, a phenotype similar to that of lenses expressing E6. The results from this study indicate that multiple PDZ domain-containing proteins, including Dlg and Scrib, may be required for maintaining the normal pattern of growth and differentiation in the lens. Furthermore, the phenotypic similarities among the Drosophila dlg mutant, the lenses of dlg(gt) mice, and the lenses of E6 transgenic mice suggest that Dlg may have a conserved function in regulating epithelial cell growth and differentiation across species.

Human papillomaviruses (HPVs) are the causative agent of warts. Infections with high-risk HPVs are associated with anogenital and head and neck cancers. One of the viral genes responsible for HPV's oncogenic activity is E6. Mice expressing the HPV-16 E6 protein in their epidermis (K14E6(WT)) develop epithelial hyperplasia and squamous carcinomas. Numerous cellular proteins interact with E6, some of which can be grouped based on common amino acid motifs in their E6-binding domains. One such group, the PDZ partners, including hDLG, hSCRIBBLE, MUPP1, and MAGI, bind to the carboxy-terminal four amino acids of E6 through their PDZ domains. E6's interaction with the PDZ partners leads to their degradation. Additionally, E6's binding to PDZ proteins has been correlated with its ability to transform baby rat kidney cells in tissue culture and to confer tumorigenicity onto cells in xenograft experiments. To address whether the ability of E6 to bind PDZ domain partners is necessary for E6 to confer epithelial hyperproliferation in vivo, we generated transgenic mice that express in stratified squamous epithelia a mutant of E6 lacking the last six amino acids at its carboxyl terminus, E6(Delta 146-151), from the human keratin 14 (K14) promoter. The K14E6(Delta 146-151) mice exhibit a radiation response similar to that of the K14E6(WT) mice, demonstrating that this protein, as predicted, retains an ability to inactivate p53. However, the K14E6(Delta 146-151) mice fail to display epithelial hyperplasia. These results indicate that an interaction of E6 with PDZ partners is necessary for its induction of epithelial hyperplasia.

Performance data, during the start-up period, are presented for constructed wetlands treating subsurface drainage from dairy pastures in Waikato (rain-fed) and Northland (irrigated), North Island, New Zealand. The wetlands comprised an estimated 1 and 2% of the drained catchment areas, respectively. Nitrate concentrations were high in the drainage inflows at both sites (medians 10 g m(-3) at Waikato and 6.5 g m(-3) at Northland), but organic N was also an important form of N at Waikato (37% of TN). Comparison of wetland inflow and outflow nutrient concentrations showed overall nutrient reductions during passage through the wetlands for NO3-N (34 and 94% for medians, respectively), TN (56 and 33%, respectively), and DRP (80%, Northland only). Median NH4-N (both sites) and DRP (Waikato) concentrations showed apparent increases between the wetland inlets and outlets. However, a mass balance calculated for the 3 month preliminary monitoring periods showed substantial mass removal of DRP (80%) and all measured forms of N (NO3-N 78%, NH4-N 41%, Org-N 99.8% and TN 96%) in the Waikato wetland. Monitoring of these systems needs to be continued through a range of seasons and years to fully assess their long-term performance.

Background: The toxicity outcome of cancer patients receiving chemotherapy is difficult to predict. In this study the influence of malnutrition and inflammation on acute haematological toxicity was investigated., Patients and Methods: Between January 1999 and January 2000, 48 consecutive cancer patients experienced severe haematological toxicity (SHT), either neutropenic fever or severe thrombocytopenia, following various chemotherapy regimens. Their baseline characteristics were compared with those of 59 control patients. Previous chemotherapy regimens, type of chemotherapy, performance status (PS), calculated creatinine clearance, bilirubin, C-reactive protein (1), alpha-1 acid glycoprotein (2), albumin (3), pre-albumin (4) and the nutritional and inflammatory status (NIS) ratio [NIS = (1 x 2)/(3 x 4)] were studied. Statistical analysis was carried out using either a t-test or a chi-square test. A receiver operating characteristic (ROC) curve determined the cut-off value for NIS., Results: Patients experiencing SHT had a higher PS (P <0.001), inflammatory serum protein levels (P <0.001) and NIS ratio (P <0.0001), but lower haemoglobin (P <0.05) and serum-albumin levels (P <0.0001). Using a cut-off of 0 or 1 for PS and 1 for NIS, sensitivity was 98%, 43% and 89%; specificity was 38%, 90% and 66%, respectively. In 37 patients treated with topotecan as single agent, the determinants for SHT were PS (P <0.0001) and NIS (P <0.0001)., Conclusions: Altered nutritional and inflammatory status correlates with increased risk of severe haematological toxicity following anticancer chemotherapy.

Methods of analysis for determining low quantities of lycopene cis-trans isomers in biological tissues are needed. Development of two liquid chromatography (LC) methods based on the polymeric C30 stationary phase equipped with coulometric electrochemical array detection (ED) is described. Separation of 13 lycopene isomers including prolycopene, (a novel tetra-cis-lycopene found in Tangerine tomatoes) was accomplished with both isocratic and gradient methods using different proportions of methanol, methyl tert.-butyl ether, water and 1 M ammonium acetate buffer. Carotenoids were detected at potential settings between 200 and 620 mV. Differences in generated current-voltage curves aided in tentative identification of trans carotenoid species and select cis isomers of lycopene. These methods were successfully applied in the analysis of small quantities of plasma, buccal mucosal cells, prostate and cervical tissues. Limits of detection for trans-lycopene by ED were found to be 50 fmol representing a 10- to 100-fold increase over conventional UV-Vis absorbance methods.

Gold immunolabeling combined with negative staining (GINS) provides a valuable immunocytochemical approach that allows a direct ultrastructural definition of all viral vaccine constituents that share common antigenic features with pathogenic viral particles. These results have implications for the development of viral vaccines since it has been demonstrated that incomplete viral particles such as natural empty capsides and Rotavirus-like particles lacking the infective genome are potential candidates for the production of neutralizing antibodies. Furthermore comparative results of the application of GINS to either inactivated vaccines or unfixed samples provide direct evidence that even after inactivation specific antigenic sites are still available for gold immunolabeling.

We investigate theoretically how the stress propagation characteristics of granular materials evolve as they are subjected to increasing pressures, comparing the results of a two-dimensional scalar lattice model to those of a molecular dynamics simulation of slightly polydisperse disks. We characterize the statistical properties of the forces using the force histogram and a two-point spatial correlation function of the forces. For the lattice model, in the granular limit the force histogram has an exponential tail at large forces, while in the elastic regime the force histogram is much narrower, and has a form that depends on the realization of disorder in the model. The behavior of the force histogram in the molecular dynamics simulations as the pressure is increased is very similar to that displayed by the lattice model. In contrast, the spatial correlations evolve qualitatively differently in the lattice model and in the molecular dynamics simulations. For the lattice model, in the granular limit there are no in-plane stress-stress correlations, whereas in the molecular dynamics simulation significant in-plane correlations persist to the lowest pressures studied.

Protoporphyrin IX, induced by the exogenous addition of delta-aminolevulinic acid, reaches different levels in different tumor cells. Because many of the steps in heme biosynthesis, of which protoporphyrin IX is penultimate, are located in the mitochondria, we surmised that the mitochondrial content of cells may relate to the amount of protoporphyrin IX synthesized in response to excess delta-aminolevulinic acid. We observed that accumulation of MitoTracker, a fluorescent mitochondrial probe, delta-aminolevulinic acid-induced protoporphyrin IX levels, and porphobilinogen deaminase activity all presented with the same cell-line-dependent rank order among the four different neoplastic cells. This rank order, however, differed for cytochrome c oxidase activity, the final enzyme in mitochondrial electron transport, and for accumulation of radioactive label from [(14)C]delta-aminolevulinic acid. The data demonstrate that enzymes involved in heme biosynthesis, in general, display a rank order associated with mitochondrial content. These data imply that such parameters may have value as prognosticators of cells to produce delta-aminolevulinic acid-induced protoporphyrin IX, a photosensitizer for photodynamic therapy of cancer., (Copyright 1999 Academic Press.)

Recently, considerable interest has been given to photodynamic therapy of cancer using delta-aminolaevulinic acid to induce protoporphyrin IX as the cell photosensitizer. One advantage of this modality is that protoporphyrin IX is cleared from tissue within 24 h after delta-aminolaevulinic acid administration. This could allow for multiple treatment regimens because of little concern regarding the accumulation of the photosensitizer in normal tissues. However, the haem biosynthetic pathway would have to be fully functional after the first course of therapy to allow for subsequent treatments. Photosensitization of cultured R3230AC rat mammary adenocarcinoma cells with delta-aminolaevulinic acid-induced protoporphyrin IX resulted in the inhibition of porphobilinogen deaminase, an enzyme in the haem biosynthetic pathway, and a concomitant decrease in protoporphyrin IX levels. Cultured R3230AC cells exposed to 0.5 mM delta-aminolaevulinic acid for 27 h accumulated 6.07 x 10(-16) mol of protoporphyrin IX per cell and had a porphobilinogen deaminase activity of 0.046 fmol uroporphyrin per 30 min per cell. Cells cultured under the same incubation conditions but exposed to 30 mJ cm(-2) irradiation after a 3-h incubation with delta-aminolaevulinic acid showed a significant reduction in protoporphyrin IX, 2.28 x 10(-16) mol per cell, and an 80% reduction in porphobilinogen deaminase activity to 0.0088 fmol uroporphyrin per 30 min per cell. Similar effects were evident in irradiated cells incubated with delta-aminolaevulinic acid immediately after, or following a 24 h interval, post-irradiation. There was little gain in efficacy from a second treatment regimen applied within 24 h of the initial treatment, probably a result of initial metabolic damage leading to reduced levels of protoporphyrin IX. These findings suggest that a correlation may exist between the delta-aminolaevulinic acid induction of porphobilinogen deaminase activity and the increase in intracellular protoporphyrin IX accumulation.

We attempt to describe the stress distributions of granular packings using lattice-based layer-by-layer stochastic models that satisfy the constraints of force and torque balance and nontensile forces at each site. The inherent asymmetry in the layer-by-layer approach appears to lead to an asymmetric force distribution, in disagreement with both experiments and general symmetry considerations. The vertical force component probability distribution is robust and in agreement with predictions of the scalar q model of Liu et al. [Science 269, 513 (1995)] and Coppersmith et al. [Phys. Rev. E 53, 4673 (1996)] while the distribution of horizontal force components is qualitatively different and depends on the details of implementation.

The vesicular acetylcholine transporter (VAChT) mediates ACh storage in synaptic vesicles by exchanging cytoplasmic ACh with vesicular protons. This study sought to determine the stoichiometry of exchange by analysis of ligand binding and transport kinetics. The effects of different pH values inside and outside, external ACh concentrations, and electrical potential gradients on ACh transport by vesicles isolated from the electric organ of Torpedo were determined using a pH-jump protocol. The equilibrium binding of a high-affinity analogue of ACh is inhibited by protonation with a pKa of 7.4 +/- 0.3. A two-proton model fits the transport data much better than a one-proton model does, and uptake increases at more positive internal electrical potential, as expected for the two-proton model. Thus, the results support the two-proton model. The transport cycle begins with binding of external ACh to outwardly oriented site 2 (KACho = 20 mM) and protonation of inwardly oriented site 1 (pKa1 = 4.73 +/- 0.05). Loaded VAChT reorients quickly (73 000 min-1) and releases ACh to the inside (KAChi = 44 000 mM) and the proton to the outside. Unloaded, internally oriented site 2 binds a proton (pKa2 = 7.0), after which VAChT reorients (150 +/- 20 min-1) in the rate-limiting step and releases the proton to the outside to complete the cycle. Rate constants for the reverse direction also were estimated. Two protons provide a thermodynamic driving force beyond that utilized in vivo, which suggests that vesicular filling is regulated. Other phenomena related to VAChT, namely the time required to fill synaptic vesicles, the fractional orientation of the ACh binding site toward cytoplasm, orientational lifetimes, and the rate of nonquantal release of ACh from cholinergic nerve terminals, were computer-simulated, and the results are compared with physiological observations.

Accumulating epidemiological evidence continues to show that lycopene, found in tomatoes, grapefruits and watermelons, is associated with a reduced risk of developing certain chronic diseases and cancers. With respect to lycopene in tomato products, the effect of thermal processing on its stability has not yet been rigorously addressed. This paper assesses the effect of several different heat treatments on lycopene's isomeric distribution in a variety of tomato products, as well as in organic solvent mixtures containing all-trans lycopene. Experimental results indicate that in contrast to beta-carotene, lycopene remained relatively resistant to heat-induced geometrical conversion during typical food processing of tomatoes and related products. The presence of fat, the change in percentage of solids, and the severity of heat treatment were not contributing factors in the formation of lycopene isomers in tomato products, except at extreme conditions not regularly employed in the food industry or during food preparation. However, lycopene in organic solvent isomerized readily as a function of time even in the absence of light and the presence of antioxidants. These findings suggest that while lycopene is stable in the tomato matrix, sample handling techniques should be carefully evaluated to minimize the formation of lycopene cis isomers in organic solutions.

High titers of Ag-specific human IgG were consistently achieved in SCID mice reconstituted with human splenocytes that had been primed with Ag in vitro and then boosted with Ag after engraftment into SCID mice. Specific human IgG titers in the hu-SPL-SCID mice reached approximately 1:4 x 10(5) when the mice were immunized with a neo-antigen, whereas titers reached 1:2 x 10(6) when recall responses were induced. Booster immunizations with Ag 21 days after the initial in vivo boost further enhanced this response, and specific human IgG titers of 1:17 x 10(6) were achieved. This represented an essentially monospecific IgG population. These responses were CD4+ T cell dependent. In addition, affinity maturation of the human Ab responses was observed. Spleens of hu-SPL-SCID mice with Ag-specific titers < or = 1:1 x 10(6) were often significantly enlarged and often displayed visible tumors. Fourteen of sixteen B cell tumors removed from spleens of five such hu-SPL-SCID mice, produced Abs that were specific for the immunizing Ags. From such tumor, cloned cell lines were established. One such mAb, MLN-7 (gamma1, kappa), was raised to tetanus toxoid and had no identified cross-reactivity.

As an initial approach to optimize delta-aminolaevulinic acid (delta-ALA)-induced photosensitization of tumours, we examined the response of three enzymes of the haem biosynthetic pathway: delta-ALA dehydratase, porphobilinogen deaminase (PBGD) and ferrochelatase. Only PBGD activity displayed a time- and dose-related increase in tumours after intravenous administration of 300 mg kg(-1) delta-ALA. The time course for porphyrin fluorescence changes, reflecting increased production of the penultimate porphyrin, protoporphyrin IX (PPIX), showed a similar pattern to PBGD. This apparent correlation between PBGD activity and porphyrin fluorescence was also observed in four cultured tumour cell lines exposed to 0.1-2.0 mM delta-ALA in vitro. The increase in PBGD activity and PPIX fluorescence was prevented by the protein synthesis inhibitor cycloheximide. As the apparent Km for PBGD was similar before and after delta-ALA, the increase in PBGD activity was attributed to induction of enzyme de novo. These observations of an associated response of PBGD and PPIX imply that PBGD may be a rate-limiting determinant for the efficacy of delta-ALA-induced photosensitization when used in photodynamic therapy.

Many adaptive changes occur in response to chronic or repeated stress, involving complex regulatory interactions between central stress-related afferents and the central components of the hypothalamo-pituitary-adrenal (HPA) axis. One change associated with chronic stress is an attenuation of corticosteroid receptor-mediated feedback inhibition of the HPA axis, a process thought to involve corticosteroid receptors in the hippocampus. A prominent stress-related central afferent that innervates the hippocampus and that may participate in the regulation of the HPA axis is the central noradrenergic system. Previous evidence suggests that alpha1 adrenergic receptors may down-regulate hippocampal corticosteroid receptors, and may thus contribute to stress-induced facilitation of HPA responses. In the present study, we used combined nonisotopic and radioisotopic in situ hybridization to examine the overlapping expression and co-localization of mRNA encoding the post-synaptic alpha1D adrenergic receptor subtype, the major alpha1 subtype expressed in hippocampus, with mRNA for the two corticosteroid receptor subtypes, type I (mineralocorticoid receptor, MR) and type II (glucocorticoid receptor, GR) in rat hippocampal neurons. We observed overlapping distributions and an extensive degree of co-localization of alpha1D receptor mRNA with both corticosteroid receptor subtype messages, establishing an anatomical substrate by which these two receptor systems may directly regulate each other. The potential interaction between co-localized adrenergic and corticosteroid receptors in hippocampus may contribute to stress-induced alterations in the HPA response to subsequent stress.

Objectives: To assess the changing epidemiology of candidemia in the 1990s, to evaluate the clinical implications for the presence of non-Candida albicans in blood, and to evaluate the presence of antifungal resistance in relation to prior antifungal administration., Design: Multicenter prospective observational study of patients with positive blood cultures for Candida species or Torulopsis glabrata., Setting: Four tertiary care medical centers., Results: Four hundred twenty-seven consecutive patients were enrolled. The frequency of candidemia due to non-C. albicans species significantly increased in each hospital throughout the 3.5-year study period (P = 0.01). Thirteen percent of candidemias occurred in patients who were already receiving systemic antifungal agents. Candidemias developing while receiving antifungal therapy were more likely caused by non-C. albicans species than by C. albicans species (P = 0.0005). C. parapsilosis and C. krusei were more commonly seen with prior fluconazole therapy, whereas T. glabrata was more commonly seen with prior amphotericin B therapy. Candida species isolated during episodes of breakthrough candidemia exhibited a significantly higher MIC to the antifungal agent being administered (P < 0.001)., Conclusion: In this large scale study, the non-C. albicans species, especially T. glabrata, emerged as important and frequent pathogens causing fungemia. This finding has major clinical implications given the higher complication and mortality rate associated with the non-C. albicans species. The change in the pattern of candidemia might be partly attributed to the increase in number of immunocompromised hosts and the widespread use of prophylactic or empiric antifungal therapy. This is an ominous sign given the in vitro resistance of the non-C. albicans species to currently available antifungal agents.

Candida prosthetic valve endocarditis (PVE) is a rare entity; however, its incidence is expected to increase given the recent increase in incidence of nosocomial bloodstream candida infection. This report reviews six cases of candida PVE studied prospectively plus 12 cases previously reported in the literature. Transesophageal echocardiography was more sensitive than transthoracic echocardiography in detecting vegetations. Valvular replacement combined with antifungal therapy has been the standard treatment. However, successful therapy with long-term administration of oral fluconazole has been reported for five patients. The mortality due to candida PVE was high, especially when PVE was complicated by congestive heart failure and persistent fungemia. For uncomplicated PVE, the mortality rate for patients receiving antifungal therapy alone (40%) was no worse than for those receiving combined medical and surgical therapy (33%).

Uptake of acetylcholine (ACh) by synaptic vesicles isolated from the electric organ of Torpedo was induced with an artificially imposed proton gradient. The gradient was formed by hyposmotic lysis and resealing of vesicles in a low pH buffer to form vesicular ghosts followed by sudden elevation of the pH of the ghost suspension. [3H]ACh accumulated rapidly, the proton gradient collapsed spontaneously within 5 min as monitored by [14C]methylamine uptake, and the accumulated ACh leaked out of the ghosts after 5 min. Vesamicol blocked both uptake and efflux of the [3H]ACh, demonstrating that both processes are mediated by the ACh transporter. The protonophore nigericin also blocked uptake very potently. Specific uptake was titrated with variable concentrations of [3H]ACh. It exhibited Km and Vmax values of approximately 200-500 microM and 7-30 nmol [3H]ACh/mg at 5 min, respectively, which are values close to those commonly observed for ATP-dependent uptake by intact vesicles. Specific uptake by ghosts was titrated with variable internal pH and constant external pH. It exhibited maximal uptake between internal pH 4.5 and 5.5. The dependence was very steep and could be fit best by assuming that the active form of the transporter requires protonation of two internal sites of apparent pK value of 5.3 +/- 0.2. A similar result was obtained when the uptake was titrated with variable internal pH with a constant thermodynamic driving force maintained by keeping the external pH approximately 2.6 units higher. The origin of the transport inhibition that sets in at very low internal pH values is not clear.(ABSTRACT TRUNCATED AT 250 WORDS)

Veratridine produces a marked elevation in spontaneous quantal release from nerve endings through its ability to enhance sodium-channel activity, leading to sustained membrane depolarization. In the course of an electrophysiological investigation into the effects of vesamicol, an inhibitor of the synaptic vesicle acetylcholine transporter, on veratridine-induced acetylcholine release from rat motor nerve terminals we observed that veratridine itself has an effect on miniature endplate current amplitude distributions suggestive of an effect of the compound on the filling of cholinergic synaptic vesicles with acetylcholine. This effect of veratridine is release-dependent, being inhibited by either removal of extracellular calcium ions or by the addition of the sodium channel blocking toxin, tetrodotoxin. Biochemical studies using synaptic vesicles isolated from Torpedo electroplaque confirmed the ability of veratridine to directly inhibit the vesicular transport of acetylcholine. This appears to be a consequence of its ability to dissipate the trans-vesicular membrane proton gradient, which normally drives the active transport of acetylcholine into synaptic vesicles. We discuss how such an action of veratridine could lead to the observed release-dependent effects of the compound on electrophysiologically monitored spontaneous quantal acetylcholine release. The action of veratridine on cholinergic synaptic vesicles could be of considerable import when using this agent to elicit neurotransmitter release from either peripheral or central nerve endings.

Photodynamic therapy has demonstrated efficacy toward primary, metastatic and recurrent human tumors. Here, we investigated the ability of photodynamic therapy, using Photofrin, to inhibit growth of R3230AC mammary adenocarcinomas when tumors were treated as original implants and again as lesions recurring at the initial treatment site. The results demonstrate that both initial implants and lesions recurring after the first photodynamic treatment respond similarly to the same photodynamic therapy protocol, with mean tumor volume doubling times of approximately 11 days in both cases. Cells cultured from original tumor implants or tumors that recurred after photodynamic treatment accumulate equivalent amounts of [14C]polyhematoporphyrin. Single cell suspensions prepared from either original or recurrent tumors from animals administered 5 mg/kg Photofrin and exposed to light in vitro displayed comparable phototoxicity. Additionally, examination of tumors by light microscopy revealed no morphological differences between the original tumor implants and the recurrent lesions. Taken together, these data indicate that lesions which recurred at the site of the initial photodynamic treatment were not resistant to a second identical course of photodynamic therapy.

From a structurally distinct set of o-substituted tetraphenylporphyrins, the picket fence porphyrin (PFP), 3,1-meso-tetrakis(o-propionamidophenyl)porphyrin (3,1-TPro) has been selected as a potential candidate for use in the photodynamic therapy (PDT) of cancer. In this report, the time-dependent tissue distribution of 14C-labeled 3,1-TPro is described along with the results of various treatment regimens. The tissue distribution of radiolabeled 3,1-TPro is comparable to that of other porphyrin photosensitizers with the advantage of being most effective at 4 h and being cleared rapidly from most tissues. The results of the various treatment regimen experiments, as well as other studies, indicate that the 3,1-TPro mechanism of action is similar to that of other photosensitizers, but may include some minor differences. The conclusion is that 3,1-TPro and other PFP offer a class of effective photosensitizers that may be exploited for their structural versatility, straightforward synthesis leading to a compound of high purity and known structure, and stability (both in terms of shelf-life and in vivo metabolism) as potential candidates for PDT.

Murine monoclonal antibodies (mAbs) were raised against human, polyclonal, anti-gp120 antibodies (Ab1) and were selected for binding to broadly neutralizing anti-gp120 antibodies in sera positive for human immunodeficiency virus (HIV). One anti-idiotype mAb (Ab2), 3C9, was found to be specific for human anti-gp120 antibodies directed against an epitope around the conserved CD4 attachment site of gp120. The 3C9 reactive human anti-gp120 antibodies (3C9+ Ab) neutralized MN, IIIB, RF, and four primary isolates of HIV type 1 (HIV-1). Cynomolgus monkeys were immunized with 3C9 in adjuvant to test whether this anti-idiotype mAb could induce neutralizing anti-gp120 antibodies. The results show that purified anti-anti-idiotype antibodies (Ab3) from 3C9 immune sera bind to an epitope around the CD4 attachment site of gp120SF and gp120IIIB. Furthermore, purified gp120-specific Ab3 neutralize MN, IIIB, and RF isolates. These results demonstrate that primates immunized with an anti-idiotype mAb produce broadly neutralizing anti-HIV-1 antibodies. Since this anti-idiotype mAb was selected by identifying a clonotypic marker, its biological activity can be explained as the results of clonotypic B-cell stimulation.

A 67-year-old woman who had underlying rheumatoid arthritis and diabetes mellitus had an 8-year history of recurrent hemorrhagic cystitis. During her most recent episode of cystitis, a specimen of urine yielded herpes simplex virus type 2 in culture. A biopsy of the bladder mucosa revealed intranuclear inclusions in multinucleated and mononuclear giant cells that were positive for herpes simplex virus type 2 by immunoperoxidase staining. She had no evidence of infection with herpes simplex virus outside her bladder.

In developing therapeutic reagents for the control of HIV infection, it is necessary to screen candidate products in vitro for their ability to reduce or neutralize viral infection. Although the current literature describes numerous neutralization assays, no universally accepted standards have been adopted. In this article, we briefly review the available neutralization assays and describe in detail the methods we have selected in our laboratory for the screening and characterization of reagents with potential anti-HIV properties. After evaluating many different technical protocols and experimental procedures, we have found the syncytium inhibition and syncytial focus assays to be particularly useful and have found p24 gag antigen production to be an excellent objective measure of HIV infection under a variety of conditions. These assays proved reproducible and sensitive and are suitable for use in the majority of laboratories.

As specialized laboratory tests became more widely available, Legionella species were found to be common causes of nosocomial and community-acquired pneumonia. Patients with chronic lung disease and organ transplants are at greatest risk. Clinical manifestations are non-specific, although fever greater than 39 degrees C and diarrhea are common. Erythromycin remains the antibiotic of choice, although many alternative agents are available. Once cases are discovered, a search for the organism in water distribution systems and respiratory equipment can be fruitful. Disinfection of water distribution systems by superheating and flushing or by hyperchlorination is feasible.

Viral pneumonitis can affect all age groups and normal as well as compromised hosts. This article discusses salient features of pneumonitis caused by respiratory syncytial virus, adenoviruses, varicella-zoster virus, herpes simplex virus, influenza A and B viruses, and cytomegalovirus. The clinical picture, diagnosis, treatment and prevention for each agent are discussed.

Preparations from livers of fed and fasted genetically diabetic and nondiabetic mice (C57BL/KsJ db/db, db/+, or +/+) were used to determine whether changes in pantothenate kinase activity and/or properties corresponded to hormonally directed changes in liver total CoA content. Livers of fasted, nondiabetic mice had ratios of pantothenate kinase (PAK) to lactate dehydrogenase (LDH) activity 1.6 times values for fed nondiabetic controls, and they had a total CoA content per milligram of DNA that was 1.8 times control values. Livers of fed genetically diabetic mice had values for PAK/LDH and total CoA per milligram of DNA that were 1.5 and 2.8 times, respectively, those of nondiabetic controls. Liver PAK from genetically diabetic mice was inhibited by acetyl-CoA to the same extent as enzyme from nondiabetic mice and by CoASH to nearly the same extent. Rat hepatocytes in primary culture incubated with dibutyryl cAMP + theophylline + dexamethasone had PAK/LDH levels 1.5 times those of cells not treated with hormonal effectors, and PAK was inhibited to the same extent by acetyl-CoA and nearly the same extent by CoASH. The data show an increase in extractable hepatic PAK activity under conditions in which the total CoA content is elevated, and they suggest that glucocorticoids and cAMP levels contribute to the increased PAK activity.