Morphometric and molecular characterisation of specimens of Lepidapedon Stafford, 1904 (Digenea: Lepidapedidae) from the deep-sea fish Mora moro (Risso) (Teleostei: Moridae) in the western Mediterranean

In a study of the parasites of the deep-sea fish Mora moro (Risso) (Gadiformes: Moridae) off the Mediterranean coasts of Catalonia and the Balearic Islands (Spain), we were able to distinguish two morphs of specimens belonging to Lepidapedon Stafford, 1904 (Digenea: Lepidapedidae). This material is herein described and illustrated. Comparative sequence analyses using partial mitochondrial nad1 sequences revealed that the material assigned to one of these morphs can be considered conspecific with the material identified as Lepidapedon desclersae Bray & Gibson, 1995 from the same host. However, the published nad1 sequence for L. desclersae was generated from a specimen ex M. moro from the North East Atlantic. Examination of the voucher specimens associated with this sequence revealed that both the North East Atlantic and the Mediterranean specimens ex M. moro differ from L. desclersae as described from its type-host, Lepidion eques (Günther), in the anterior extent of the vitelline fields which is further posterior, reaching only to the posterior margin of the external seminal vesicle in L. desclersae, versus being at the mid-level of this organ and reaching the posterior margin of the ventral sucker. Therefore, we have tentatively assigned the material characterised here, both morphologically and molecularly as Lepidapedon sp. Acquisition of additional sequences for both nad1 mitochondrial and 28S rRNA genes of L. desclersae from material ex Lepidion spp. is required in order to determine whether the observed morphometric variation reflects host-related or inter-specific differences. The second morph of Lepidapedon from M. moro is described and distinguished on morphometric grounds, such as the position of the most anterior vitelline follicles, which reach to the anterior margin of the ventral sucker. Its identity is commented upon, but, in view of the fact that there were few specimens and no molecular data available, it is not named.     

Plasmodium falciparum‐encoded exported hsp70/hsp40 chaperone/co‐chaperone complexes within the host erythrocyte

Malaria parasites modify their host cell, the mature human erythrocyte. We are interested in the molecules mediating these processes, and have recently described a family of parasite‐encoded heat shock proteins (PfHsp40s) that are targeted to the host cell, and implicated in host cell modification. Hsp40s generally function as co‐chaperones of members of the Hsp70 family, and until now it was thought that human Hsp70 acts as the PfHsp40 interaction partner within the host cell. Here we revise this hypothesis, and identify and characterize an exported parasite‐encoded Hsp70, referred to as PfHsp70‐x. PfHsp70‐x is exported to the host erythrocyte where it forms a complex with PfHsp40s in structures known as J‐dots, and is closely associated with PfEMP1. Interestingly, Hsp70‐x is encoded only by parasite species that export the major virulence factor EMP1, implying a possible role for Hsp70‐x in EMP1 presentation at the surface of the infected erythrocyte. Our data strongly support the presence of parasite‐encoded chaperone/co‐chaperone complexes within the host erythrocyte, which are involved in protein traffic through the host cell. The host–pathogen interaction within the infected erythrocyte is more complex than previously thought, and is driven notonly by parasite co‐chaperones, but also by the parasite‐encoded chaperone Hsp70‐x itself.     

Human cytomegalovirus capsid assembly protein precursor (pUL80.5) interacts with itself and with the major capsid protein (pUL86) through two different domains.

We have used the yeast GAL4 two-hybrid system to examine interactions between the human cytomegalovirus (HCMV) major capsid protein (MCP, encoded by UL86) and the precursor assembly protein (pAP, encoded by UL80.5 and cleaved at its carboxyl end to yield AP) and found that (i) the pAP interacts with the MCP through residues located within the carboxy-terminal 21 amino acids of the pAP, called the carboxyl conserved domain (CCD); (ii) the pAP interacts with itself through a separate region, called the amino conserved domain (ACD), located between amino acids His34 and Arg52 near the amino end of the molecule; (iii) the simian CMV (SCMV) pAP and AP can interact with or replace their HCMV counterparts in these interactions, whereas the herpes simplex virus pAP and AP homologs cannot; and (iv) the HCMV and SCMV maturational proteinase precursors (ACpra, encoded by UL80a and APNG1, respectively) can interact with the pAP and MCP. The ACD and CCD amino acid sequences are highly conserved among members of the betaherpesvirus group and appear to have counterparts in the alpha- and gammaherpesvirus pAP homologs. Deleting the ACD from the HCMV pAP, or substituting Ala for a conserved Leu in the ACD, eliminated detectable pAP self-interaction and also substantially reduced MCP binding in the two-hybrid assay. This finding indicates that the pAP self-interaction influences the pAP-MCP interaction. Immunofluorescence studies corroborated the pAP-MCP interaction detected in the GAL4 two-hybrid experiments and showed that nuclear transport of the MCP was mediated by pAP but not AP. We conclude that the pAP interacts with the MCP, that this interaction is mediated by the CCD and is influenced by pAP self-interaction, and that one function of the pAP-MCP interaction may be to provide a controlled mechanism for transporting the MCP into the nucleus.     

ALVAC-SIV-gag-pol-env-based vaccination and macaque major histocompatibility complex class I (A*01) delay simian immunodeficiency virus SIVmac-induced immunodeficiency.

T-cell-mediated immune effector mechanisms play an important role in the containment of human immunodeficiency virus/simian immunodeficiency virus (HIV/SIV) replication after infection. Both vaccination- and infection-induced T-cell responses are dependent on the host major histocompatibility complex classes I and II (MHC-I and MHC-II) antigens. Here we report that both inherent, host-dependent immune responses to SIVmac251 infection and vaccination-induced immune responses to viral antigens were able to reduce virus replication and/or CD4+ T-cell loss. Both the presence of the MHC-I Mamu-A*01 genotype and vaccination of rhesus macaques with ALVAC-SIV-gag-pol-env (ALVAC-SIV-gpe) contributed to the restriction of SIVmac251 replication during primary infection, preservation of CD4+ T cells, and delayed disease progression following intrarectal challenge exposure of the animals to SIV(mac251 (561)). ALVAC-SIV-gpe immunization induced cytotoxic T-lymphocyte (CTL) responses cumulatively in 67% of the immunized animals. Following viral challenge, a significant secondary virus-specific CD8+ T-cell response was observed in the vaccinated macaques. In the same immunized macaques, a decrease in virus load during primary infection (P = 0.0078) and protection from CD4 loss during both acute and chronic phases of infection (P = 0.0099 and P = 0.03, respectively) were observed. A trend for enhanced survival of the vaccinated macaques was also observed. Neither boosting the ALVAC-SIV-gpe with gp120 immunizations nor administering the vaccine by the combination of mucosal and systemic immunization routes increased significantly the protective effect of the ALVAC-SIV-gpe vaccine. While assessing the role of MHC-I Mamu-A*01 alone in the restriction of viremia following challenge of nonvaccinated animals with other SIV isolates, we observed that the virus load was not significantly lower in Mamu-A*01-positive macaques following intravenous challenge with either SIV(mac251 (561)) or SIV(SME660). However, a significant delay in CD4+ T-cell loss was observed in Mamu-A*01-positive macaques in each group. Of interest, in the case of intravenous or intrarectal challenge with the chimeric SIV/HIV strains SHIV(89.6P) or SHIV(KU2), respectively, MHC-I Mamu-A*01-positive macaques did not significantly restrict primary viremia. The finding of the protective effect of the Mamu-A*01 molecule parallels the protective effect of the B*5701 HLA allele in HIV-1-infected humans and needs to be accounted for in the evaluation of vaccine efficacy against SIV challenge models.     

Effect of subculture and elicitation on instability of taxol production in Taxus sp. suspension cultures

The production of secondary metabolites through plant cell suspension cultures is challenging because the level and pattern of production is often unstable and unpredictable. To investigate the factors affecting instability of secondary metabolite production, high Taxol (paclitaxel)-producing Taxus cultures induced by methyl jasmonate elicitation and their low Taxol-producing counterparts were compared with respect to growth and Taxol production kinetics. With Taxus subcultures we observe alternating states of high and low productivity. Parental cultures and their subcultures from five different cell lines were used to test whether a high-producing culture grows more slowly or dies more rapidly than a low-producing one. These cell lines were of three types: (1) Taxol-producing with and without methyl jasmonate, (2) Taxol-producing only upon elicitation, and (3) nonproducing. High-producing cultures show growth inhibition upon subculture, whereas nonproducing elicited cultures show little growth inhibition. Thus, growth inhibition is primarily due to Taxol or taxane accumulation and not a direct result of methyl jasmonate treatment. Through media exchange between high- and low-producing cultures, it appears that culture components generated by cells alter culture properties. To assess variability as a function of culture lineage, two groups of replicate cultures were generated either with a mixing of the parental flasks or segregation of parental flasks at each subculture. Although parental culture mixing did not reduce flask-to-flask variation, the production level of Taxol in subcultures resulting from mixing inocula was sustained at a higher level relative to segregated subcultures. The results are consistent with the possibility of cell signaling within the population that can induce Taxol production.     

The kinetics of taxoid accumulation in cell suspension cultures of Taxus following elicitation with methyl jasmonate

Cell suspension cultures of Taxus canadensis and Taxus cuspidata rapidly produced paclitaxel (Taxol) and other taxoids in response to elicitation with methyl jasmonate. By optimizing the concentration of the elicitor, and the timing of elicitation, we have achieved the most rapid accumulation of paclitaxel in a plant cell culture, yet reported. The greatest accumulation of paclitaxel occurred when methyl jasmonate was added to cultures at a final concentration of 200 microM on day 7 of the culture cycle. The concentration of paclitaxel increased in the extracellular (cell-free) medium to 117 mg/day within 5 days following elicitation, equivalent to a rate of 23.4 mg/L per day. Paclitaxel was only one of many taxoids whose concentrations increased significantly in response to elicitation. Despite the rapid accumulation and high concentration of paclitaxel, its concentration never exceeded 20% of the total taxoids produced in the elicited culture. Two other taxoids, 13-acetyl-9-dihydrobaccatin III and baccatin VI, accounted for 39% to 62% of the total taxoids in elicited cultures. The accumulation of baccatin III did not parallel the pattern of accumulation for paclitaxel. Baccatin III continued to accumulate until the end of the culture cycle, at which point most of the cells in the culture were dead, implying a possible role as a degradation product of taxoid biosynthesis, rather than as a precursor.     

Isolation of the carotenoid-containing cell wall of three unicellular cyanobacteria.

A carotenoid-containing membrane fraction devoid of chlorophyll and phycobiliproteins was isolated from three unicellular cyanobacteria, Synechococcus sp., Synechococcus leopoliensis UTEX 625, and Anacystis nidulans R-2, by aqueous-phase separation, hydrophobic chromatography, and differential centrifugation. The presence of 2-keto-3-deoxyoctonate, muramic acid, and diaminopimelic acid suggests that the preparation is highly enriched in cell wall. Electron micrographs of thin sections of this material showed C-shaped membrane profiles similar to those seen in other gram-negative cell wall preparations. The inactivation of cyanophage AS-1 by this fraction confirmed its identity as cell wall. The cell wall contained approximately equal weights of total carbohydrate and protein. Absorption maxima at 434, 452, and 488 nm indicated the presence of carotenoids. These were in the outer membrane and were not due to contaminating cytoplasmic or thylakoid membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the preparations showed a broad band of approximately 50,000 molecular weight which contained 35% of the total outer membrane protein. This band was resolved into at least two components running at approximately 50,000 and 52,000 molecular weight. The smaller of these polypeptides was a glycoprotein. The polypeptide components were unaffected by protease or detergent treatment in either whole cells or isolated cell wall preparations, indicating that the polypeptide components were not exposed to the surface or easily removed from the hydrophobic environment.     

Identifying and managing adverse environmental health effects: 5. Persistent organic pollutants

CONCERN AND AWARENESS IS GROWING about the health effects of exposures to environmental contaminants, including those found in food. Most primary care physicians lack knowledge and training in the clinical recognition and management of the health effects of environmental exposures. We have found that the use of a simple history-taking tool — the CH²OPD² mnemonic (Community, Home, Hobbies, Occupation, Personal habits, Diet and Drugs) — can help physicians identify patients at risk of such health effects. We present an illustrative case of a mother who is concerned about eating fish and wild game because her 7-year-old son has been found to have learning difficulties and she is planning another pregnancy. Potential exposures to persistent organic pollutants (POPs) and mercury are considered. The neurodevelopmental effects of POPs on the fetus are reviewed. We provide advice to limit a patient''s exposure to these contaminants and discuss the relevance of these exposures to the learning difficulties of the 7-year-old child and to the planning of future pregnancies.CaseA 27-year-old woman who lives in a town on the shore of Lake Huron wants to have a second child but has concerns. Her 7-year-old son is being assessed by the school psychologist for a learning disorder. She tells her family physician that she saw something on television about contaminants in fish affecting children''s intelligence. She is worried that her diet may have caused her son''s learning disorder and wants advice on how to protect her second child against environmental contaminants that may cause learning problems. Her past medical history is unremarkable. She is taking no medications other than folate (0.4 mg/d). She has had only the one pregnancy. Her pregnancy and delivery of her son were uncomplicated, and the boy met the developmental milestones. The concern about a learning disorder is recent. There is no family history of congenital anomalies, early deafness or twins. Her maternal grandmother had type 2 diabetes, and her father-in-law has hypertension; the rest of the family is healthy. Because the woman is worried about environmental exposures, you take an exposure history using the CH²OPD² mnemonic (Community, Home, Hobbies, Occupation, Personal habits, Diet and Drugs)¹ to identify possible sources of environmental contaminants (Open in a separate windowThe environmental contaminants that can affect the neurobehavioural development of the fetus include metals (lead, mercury and manganese), nicotine, pesticides (e.g., organophosphates), dioxins, polychlorinated biphenyls (PCBs) and solvents (e.g., alcohol).^2,3 In this article we focus on persistent organic pollutants (POPs) and mercury. These are the contaminants identified in the environmental exposure history of the case subject (4POPs are carbon-containing chemicals that share several properties. They are lipophilic, accumulating in the fat of living organisms, and increase in quantity up the food chain. Most are semivolatile, which means that they can travel in the air thousands of miles from their source before they settle. They resist photolytic, biological and chemical degradation and persist in the environment, taking as long as a century to degrade.⁵ Twelve POPs, including 9 pesticides, have been identified by the United Nations Environment Programme as powerful threats to the health of humans and wildlife and have been targeted for elimination (6 In the 1970s many countries banned or severely restricted the use of the 9 pesticides and PCBs and implemented pollution control strategies to reduce the amount of dioxin and furan released in the environment. However, it is thought that all 9 pesticides and PCBs are still used in many countries today.Table 2Open in a separate windowDespite significant achievements in reducing the production and use of POPs, these pollutants remain ubiquitous, as evident by the global distribution of PCBs and organochlorine pesticides in butter samples from around the world.⁷ Most human exposure comes from dietary sources. POPs are ingested, stored in fatty tissue and excreted in feces and breast milk. The concentration of certain chemical contaminants in breast milk serves as an indicator of population exposure. From 1967 to 1992, there was a downward trend in the concentrations of organochlorine pesticides and PCB hydrocarbons in samples of Canadian breast milk during the phase-out of these chemicals.⁸ The estimated daily intake of PCBs from the current diet of the average Canadian is less than 1 μg/d.⁹Although everyone is exposed to a background level of POPs, certain people may have higher levels of POPs exposure because of their eating habits. Some people eat more fish than the general population. Southeast Asian Canadians, Native Americans, sport anglers and hunters who regularly eat large amounts of Great Lakes fatty fish or wildlife from the top of the food chain, such as waterfowl and waterfowl eggs, turtles and turtle eggs, muskrat, otter, moose and deer, may be at risk of high exposure.¹⁰ Northern Aboriginals, such as the Inuit of Nunavik, who consume the fat of seals and beluga and narwhal whales, have been found to have higher body burdens of POPs.^11,12