Grace's Insect Medium Research Articles

Development of an animal-derived component-free medium for Spodoptera frugiperda (Sf9) cells using response surface methodology.

To develop an animal-derived component-free medium for Spodoptera frugiperda (Sf9) growth and green fluorescent protein (GFP) expression. OSF9-ADCFM contained optimum concentrations of CDLC, YE and ST at 0.5% (v/v), 11.0g/L, and 3.0g/L, respectively. A mean viable cell concentration of 1.71 ± 0.14 × 105 cells/mL was obtained from 5 passages (P1-P5). The use of both peptones after 10kDa ultrafiltration had a significant effect on Sf9 cell growth. Grace's insect medium with 10% FBS gave higher un-infected cell number than SF-900II and OSF9-ADCFM for 4.29 and 5.38 times, respectively. The average cell number of un-infected cells and GFP-fluorescent cells of SF-900II were higher than OSF9-ADCFM 1.25 and 7 times, respectively. In-house OSF9-ADCFM could support growth and GFP expression in Sf9 less than commercial SF-900II. However, it could lower the production cost at least 50% comparing to commercial SF-900II. The development of in- house OSF9-ADCFM would be continued to increase both cell numbers and protein expression in the next step.

Establishing and characterising a new cell line from Calliphora vicina (diptera: calliphoridae) fly embryonic tissues

Insect cell lines represent a promising and expanding field as they have several research applications including biotechnology, virology, immunity, toxicology, cell signalling mechanisms and evolution. They constitute a powerful tool having a direct impact on human and veterinary medicine and agriculture. Although more than 1000 cell lines have currently been established from various insect species, Calliphora vicina-derived fly cell lines are lacking. This study was aimed at establishing a new C. vicina embryonic tissue-derived cell line. Adult flies were collected and embryonated eggs were mechanically homogenised and seeded in four types of culture media (L15, Grace's insect medium, Grace's/L15 and DMEM). Cell growth and morphological characteristics were recorded and cytogenetic and molecular patterns were determined. The CV-062020-PPB cell line was established and was shown to have optimal growth in Grace's/L15 medium. CV-062020-PPB cell monolayers that had been sub-cultured over 16 times consisted of firmly adhering cells having different morphologies; a fibroblast-like shape dominated and the karyotype had a 12-chromosome diploid number. RAPD-PCR analysis of the CV-062020-PPB cell line revealed a high similarity index and strong intraspecific relationship with C. vicina adult flies and a weaker relationship with the Lutzomyia longipalpis-derived cell line (Lulo). The CV-062020-PPB cell line constitutes the first cell line obtained from C. vicina embryonic tissues and represents an important basic and applied research tool.

Seminal fluid and sperm diluent affect sperm metabolism in an insect: Evidence from NAD(P)H and flavin adenine dinucleotide autofluorescence lifetime imaging.

Sperm metabolism is fundamental to sperm motility and male fertility. Its measurement is still in its infancy, and recommendations do not exist as to whether or how to standardize laboratory procedures. Here, using the sperm of an insect, the common bedbug, Cimex lectularius, we demonstrate that standardization of sperm metabolism is required with respect to the artificial sperm storage medium and a natural medium, the seminal fluid. We used fluorescence lifetime imaging microscopy (FLIM) in combination with time-correlated single-photon counting (TCSPC) to quantify sperm metabolism based on the fluorescent properties of autofluorescent coenzymes, NAD(P)H and flavin adenine dinucleotide. Autofluorescence lifetimes (decay times) differ for the free and protein-bound state of the co-enzymes, and their relative contributions to the lifetime signal serve to characterize the metabolic state of cells. We found that artificial storage medium and seminal fluid separately, and additively, affected sperm metabolism. In a medium containing sugars and amino acids (Grace's Insect medium), sperm showed increased glycolysis compared with a commonly used storage medium, phosphate-buffered saline (PBS). Adding seminal fluid to the sperm additionally increased oxidative phosphorylation, likely reflecting increased energy production of sperm during activation. Our study provides a protocol to measure sperm metabolism independently from motility, stresses that protocol standardizations for sperm measurements should be implemented and, for the first time, demonstrates that seminal fluid alters sperm metabolism. Equivalent protocol standardizations should be imposed on metabolic investigations of human sperm samples.

The Development and Characterization of a Cell Culture System from Indian Mud Crabs Scylla serrata

Commercially available culture media and supplements were tested for their potential to produce primary cell cultures from tissues of Indian mud crabs Scylla serrata. Eight commercially available culture media from Sigma-Aldrich (Leibovitz's L-15, Medium 199, Grace's InsectMedium, Minimal Essential Medium, Dulbecco's Modified Eagle Medium, TC-100 Insect Medium, IPL-41 Insect Medium, and Roswell Park Memorial Institute) were examined. Three different supplements (amino acid and sugar [AS], crab muscle extract [CME], and natural seawater [NSW]) were also examined. The hemocyte culture appeared to grow well for a maximum period of 21d in 2×L-15 medium supplemented with AS and 15% fetal bovine serum (FBS). Partial amplification and sequencing of the cytochrome oxidase subunit I (COI) gene confirmed that the primary hemocytes originated from Indian mud crabs. The effects of four metals on hemocyte viability were evaluated using the MTT assay. Of the four metals examined (arsenic, lead, cobalt, and nickel), cobalt and nickel were more toxic to the crab cells than the other metals. Both acridine orange/ethidium bromide and Hoechst staining showed the presence of apoptosis and necrosis in metal-treated groups, which suggests that metals in an aquatic environment induce death of the Indian mud crab's hemocytes. The hemocyte primary cell culture was also used to study the cytotoxicity effect of bacterial extracellular products from Vibrio harveyi and white spot syndrome virus. This study demonstrates that hemocyte primary cell culture can be used as a tool to study viral and bacterial pathogenesis and to assess the cytotoxicity of pollutants present in aquatic environments.

Axenic amastigote cultivation and in vitro development of Leishmania orientalis

Leishmania (Mundinia) orientalis is a recently described new species that causes leishmaniasis in Thailand. To facilitate characterization of this new species, an in vitro culture system to generate L. orientalis axenic amastigotes was developed. In vitro culture conditions of the axenic culture-derived amastigotes were optimized by manipulation of temperature and pH. Four criteria were used to evaluate the resulting L. orientalis axenic amastigotes, i.e., morphology, zymographic analysis of nucleases, cyclic transformation, and infectivity to the human monocytic cell line (THP-1) cells. Results revealed that the best culture condition for L. orientalis axenic amastigotes was Grace's insect medium supplemented with FCS 20%, 2% human urine, 1% BME vitamins, and 25μg/ml gentamicin sulfate, pH5.5 at 35°C. For promastigotes, the condition was M199 medium, 10% FCS supplemented with 2% human urine, 1% BME vitamins, and 25μg/ml gentamicin sulfate, pH6.8 at 26°C. Morphological characterization revealed six main stages of the parasites including amastigotes, procyclic promastigotes, nectomonad promastigotes, leptomonad promastigotes, metacyclic promastigotes, and paramastigotes. Also, changes in morphology during the cycle were accompanied by changes in zymographic profiles of nucleases. The developmental cycle of L. orientalis in vitro was complete in 12days using both culture systems. The infectivity to THP-1 macrophages and intracellular growth of the axenic amastigotes was similar to that of THP-1 derived intracellular amastigotes. These results confirmed the successful axenic cultivation of L. orientalis amastigotes. The axenic amastigotes and promastigotes can be used for further study on infection in permissive vectors and animals.

REVIEW ON IN-VITRO AMEBOCYTE CULTURE–A LESSON LEARNED FROM PAST

Limulus Amebocyte Lysate (LAL/TAL) assay is the only standard test approved by the Food and Drug Administration (FDA) to quantify bacterial endotoxin in injectable drugs and implantable medical devices. At present, horseshoe crabs are the sole source of LAL/TAL. Biomedical companies harvest and bleed horseshoe crabs which lead to ≤30% post bleeding mortality. The continuity of this practices will eventually deplete wild stock and threat horseshoe crab’s population. Though, many alternative biosensors were developed to quantify endotoxins, they are not without limitations and some of them even need LAL/TAL as source detectors. Hence, the LAL/TAL industry in its current form has ethical, ecological, commercial and technical issues that make it unideal standard test. An alternative method of culturing amebocyte in tissue culture medium has been addressed in literature since last 4 decades. This paper will address the issues in amebocyte culture in-vitro based on the published literatures. The paper will also suggest the best source of amebocyte cells (gill, gill flaps, blood), culture mode (monolayer or suspension), culture media (Grace's Insect Medium, Leibovitz’s L-15 Medium, Modified Essential medium, Shields and Sang insect medium, TNM-FH Medium, RPMI 1640 medium) and best culture conditions (pH, temperature, osmolarity). This review will also emphasize on key elements in establishing a cell line for the continuous harvesting of amebocyte in-vitro.

A Novel Method for Inducing Amastigote-To-Trypomastigote Transformation In Vitro in Trypanosoma cruzi Reveals the Importance of Inositol 1,4,5-Trisphosphate Receptor.

Background Trypanosoma cruzi is a parasitic protist that causes Chagas disease, which is prevalent in Latin America. Because of the unavailability of an effective drug or vaccine, and because about 8 million people are infected with the parasite worldwide, the development of novel drugs demands urgent attention. T. cruzi infects a wide variety of mammalian nucleated cells, with a preference for myocardial cells. Non-dividing trypomastigotes in the bloodstream infect host cells where they are transformed into replication-capable amastigotes. The amastigotes revert to trypomastigotes (trypomastigogenesis) before being shed out of the host cells. Although trypomastigote transformation is an essential process for the parasite, the molecular mechanisms underlying this process have not yet been clarified, mainly because of the lack of an assay system to induce trypomastigogenesis in vitro.Methodology/Principal FindingsCultivation of amastigotes in a transformation medium composed of 80% RPMI-1640 and 20% Grace’s Insect Medium mediated their transformation into trypomastigotes. Grace’s Insect Medium alone also induced trypomastigogenesis. Furthermore, trypomastigogenesis was induced more efficiently in the presence of fetal bovine serum. Trypomastigotes derived from in vitro trypomastigogenesis were able to infect mammalian host cells as efficiently as tissue-culture-derived trypomastigotes (TCT) and expressed a marker protein for TCT. Using this assay system, we demonstrated that T. cruzi inositol 1,4,5-trisphosphate receptor (TcIP3R)—an intracellular Ca2+ channel and a key molecule involved in Ca2+ signaling in the parasite—is important for the transformation process.Conclusion/SignificanceOur findings provide a new tool to identify the molecular mechanisms of the amastigote-to-trypomastigote transformation, leading to a new strategy for drug development against Chagas disease.

Role of secondary metabolites in establishment of the mutualistic partnership between Xenorhabdus nematophila and the entomopathogenic nematode Steinernema carpocapsae.

Xenorhabdus nematophila engages in a mutualistic partnership with the nematode Steinernema carpocapsae, which invades insects, migrates through the gut, and penetrates into the hemocoel (body cavity). We showed previously that during invasion of Manduca sexta, the gut microbe Staphylococcus saprophyticus appeared transiently in the hemocoel, while Enterococcus faecalis proliferated as X. nematophila became dominant. X. nematophila produces diverse secondary metabolites, including the major water-soluble antimicrobial xenocoumacin. Here, we study the role of X. nematophila antimicrobials in interspecies competition under biologically relevant conditions using strains lacking either xenocoumacin (ΔxcnKL strain), xenocoumacin and the newly discovered antibiotic F (ΔxcnKL:F strain), or all ngrA-derived secondary metabolites (ngrA strain). Competition experiments were performed in Grace's insect medium, which is based on lepidopteran hemolymph. S. saprophyticus was eliminated when inoculated into growing cultures of either the ΔxcnKL strain or ΔxcnKL:F strain but grew in the presence of the ngrA strain, indicating that ngrA-derived antimicrobials, excluding xenocoumacin or antibiotic F, were required to eliminate the competitor. In contrast, S. saprophyticus was eliminated when coinjected into M. sexta with either the ΔxcnKL or ngrA strain, indicating that ngrA-derived antimicrobials were not required to eliminate the competitor in vivo. E. faecalis growth was facilitated when coinjected with either of the mutant strains. Furthermore, nematode reproduction in M. sexta naturally infected with infective juveniles colonized with the ngrA strain was markedly reduced relative to the level of reproduction when infective juveniles were colonized with the wild-type strain. These findings provide new insights into interspecies competition in a host environment and suggest that ngrA-derived compounds serve as signals for in vivo nematode reproduction.

The potential of trehalose to replace insect hemolymph in artificial media for Trichogramma dendrolimi (Hymenoptera: Trichogrammatidae)

Insect additives have been shown to improve the value of artificial media for Trichogramma species, but at the same time maintain dependence on parallel cultures of host insects. In the present study, Trichogramma dendrolimi Matsumura was reared in vitro from egg to adult on artificial media with different contents of pupal hemolymph of Chinese oak silkmoth Antheraea pernyi (Guérin-Méneville) and with supplements of distilled water, or of trehalose dissolved in water or in Grace's insect medium. The results indicated insect hemolymph was the key component of artificial medium. Developmental parameters, including rates of parasitism, pupation, adult emergence and normal adults, and numbers of produced adults, were increased on media supplemented with trehalose even when the proportion of pupal hemolymph was reduced. Two artificial media, the first containing 30% hemolymph and 10% trehalose in water with 98.9% parasitism rate, 77.7.0% pupation rate, 77.2% emergence rate, 80.0% normal adult rate and 333 produced adults, and the second containing 25% hemolymph and 15% trehalose in Grace's insect medium with 97.8% parasitism rate, 91.0% pupation rate, 85.2% emergence rate, 76.1% normal adult rate and 757 produced adults, were believed to hold potential to mass produce T. dendrolimi. The use of trehalose to partially replace pupal hemolymph in artificial medium of this and other Trichogramma species may contribute to a significant reduction in their production cost and may as such help to evade problems related to short supplies of lepidopteran eggs, which currently constitute the main factitious host for the mass rearing of the parasitoids.

A novel medium for the development of in vitro cell culture system from Penaeus monodon

Lack of a valid shrimp cell line has been hampering the progress of research on shrimp viruses. One of the reasons identified was the absence of an appropriate medium which would satisfy the requirements of the cells in vitro. We report the first attempt to formulate an exclusive shrimp cell culture medium (SCCM) based on the haemolymph components of Penaeus monodon prepared in isosmotic seawater having 27‰ salinity. The SCCM is composed of 22 amino acids, 4 sugars, 6 vitamins, cholesterol, FBS, phenol red, three antibiotics, potassium dihydrogen phosphate and di-sodium hydrogen phosphate at pH 6.8-7.2. Osmolality was adjusted to 720±10mOsmkg(-1) and temperature of incubation was 25 ºC. The most appropriate composition was finally selected based on the extent of attachment of cells and their proliferation by visual observation. Metabolic activity of cultured cells was measured by MTT assay and compared with that in L-15 (2×), modified L-15 and Grace's insect medium, and found better performance in SCCM especially for lymphoid cells with 107% increase in activity and 85±9days of longevity. The cells from ovary and lymphoid organs were passaged twice using the newly designed shrimp cell dissociation "cocktail".

Classification of circulating hemocytes from the red swamp crayfish Procambarus clarkii and their susceptibility to the novel pathogen Spiroplasma eriocheiris in vitro

Hemocytes from the red swap crayfish Procambarus clarkii were classified into three subpopulations: hyalinocytes (H), semigranulocytes (SG) and granulocytes (G) by morphologic observation and flow cytometry (FCM). The primary hemocyte culture was then established for studies on the in vitro propagation of Spiroplasma eriocheiris isolated from naturally infected P. clarkii. Grace's insect medium supplemented with 15% fetal bovine serum (FBS), along with 100Uml−1 penicillin, 100μgml−1 streptomycin, with a final pH of 7.2–7.4, incubated at 28°C, supported the best survival in primary cultures. However, the granulocytes dehisced rapidly in culture medium, potentially impacting the survival of the other cell types. A two-step density gradient centrifugation with Percoll was developed to separate the hemocytes. When cultured separately, hyalinocytes and semigranulocytes maintained higher viability (>75%) after 16days of incubation compared with granulocytes, which degraded over 2–6days. After a challenge with S. eriocheiris, cytopathic effects (CPE) of the cultured hemocytes were observed as early as 48h post-inoculation, and as the infection progressed, CPE became more apparent, with cell debris and cellular exudates in inoculated cultures. Cell lysis was noticeable within 60h after challenging. A quantitative real-time RT-PCR was conducted to detect the immune responses during the challenging process at 2h, 4h, 8h, 10h, 24h, 48h and 60h, respectively. Clear time-dependent expression patterns of the peroxinectin gene (referred as Pcpxin), recently isolated from the crayfish hemocytes, and heat shock protein 70 (HSP70) gene were observed after S. eriocheiris challenge. The results above should be helpful in promoting research with S. eriocheiris, including elucidation of pathogenesis, host pathogen interaction and the defense mechanisms, and ultimately lead to prevention of this crustacean disease.

An improved method of cell culture system from eye stalk, hepatopancreas, muscle, ovary, and hemocytes of Penaeus vannamei

Improved methods of cell culture from eye stalk, hepatopancreas, muscle, ovary, and hemocytes of shrimp (Penaeus vannamei) were established using synthetic media and shrimp muscle extract (SME). For hemocytes and ovarian cell cultures, Grace's insect medium supplemented with 10% (v/v) fetal bovine serum and 10% SME (v/v) showed enhanced attachment and proliferation of the cells. The hemocyte and ovarian cell cultures could be maintained for 48 and 66 days, respectively, and have been sub-cultured four and six times, respectively. Both ovary and hemocyte cell cultures contained primarily epithelial-like cells. Cells derived from ovary tissue grew preferably between 26°C and 28°C with 5% CO(2). Although the temperature preference of hemocyte cells was the same as ovarian cells, CO(2) supplementation did not show any difference in the growth of hemocyte cells. When the shrimp were injected with lipopolysaccharide (8 μg/g of shrimp) and hemolymph was drawn 24 h post-injection, the in vitro multiplicity of hemocytes dramatically improved. The growth of eye stalk, hepatopancreas, and muscle-derived cells was much less compared to ovarian cells and hemocytes under the conditions described above. The optimal culture conditions for ovarian cells and hemocytes were also different from that for eye stalk, hepatopancreas, and muscle cell culture. The proliferation efficiencies of primary cultures of hepatopancreas, eyestalk, and muscle cells were about 30, 12, and <7 d, respectively. The improved culture conditions described here, particularly for hemocytes and ovary, will be very useful for in vitro studies involving viruses infecting shrimp and in shrimp genomic studies.

Leishmania spp. parasite isolation through inoculation of patient biopsy macerates in interferon gamma knockout mice

Isolation of Leishmania parasite and species identification are important for confirmation and to help define the epidemiology of the leishmaniasis. Mice are often used to isolate pathogens, but the most common mouse strains are resistant to infection with parasites from the Leishmania (Viannia) subgenus. In this study we tested the inoculation of interferon gamma knockout (IFNgamma KO) mice with biopsy macerates from Leishmania-infected patients to increase the possibility of isolating parasites. Biopsies from twenty five patients with clinical signs of leishmaniasis were taken and tested for the presence of parasites. Immunohistochemical assay (IHC) and conventional histopathology detected the parasite in 88% and 83% of the patients, respectively. Leishmania sp. were isolated in biopsy macerates from 52% of the patients by culture in Grace's insect medium, but 13% of isolates were lost due to contamination. Inoculation of macerates in IFNgamma KO mice provides isolation of parasites in 31.8% of the biopsies. Most isolates belong to L. (Viannia) subgenus, as confirmed by PCR, except one that belongs to L. (Leishmania) subgenus. Our preliminary results support the use of IFNgamma KO mice to improve the possibility to isolate New World Leishmania species.

Csm3, Tof1, and Mrc1 Form a Heterotrimeric Mediator Complex That Associates with DNA Replication Forks

Mrc1 (mediator of replication checkpoint), Tof1 (topoisomerase I interacting factor), and Csm3 (chromosome segregation in meiosis) are checkpoint-mediator proteins that function during DNA replication and activate the effector kinase Rad53. We reported previously that Mrc1 and Tof1 are constituents of the replication machinery and that both proteins are required for the proper arrest and stabilization of replication forks in the presence of hydroxyurea. In our current study, we show that Csm3 is a component of moving replication forks and that both Tof1 and Csm3 are specifically required for the association of Mrc1 with these structures. In contrast, the deletion of mrc1 did not affect the association of Tof1 and Csm3 with the replication fork complex. In agreement with previous observations in yeast cells, the results of a baculovirus coexpression system showed that these three proteins interact directly with each other to form a mediator complex in the absence of replication forks.

Leishmania sp. isolated from human cases of cutaneous leishmaniasis in Brazil characterized as Leishmaniamajor-like

The identification and characterization of Leishmania are relevant to diagnosis, treatment, eco-epidemiology studies, prophylactic measures and control of the disease. Two strains of Leishmania (MHOM/BR/1971/BH49 and MHOM/BR/1971/BH121), isolated from human cutaneous leishmaniasis, were studied using biological and molecular characteristics, in comparison with WHO reference strains. These studies are important because both strains were incorporated in a vaccine against American cutaneous leishmaniasis, and one of these strains has been used to prepare specific and sensitive antigen for serodiagnosis of visceral leishmaniasis in Brazil. Studies were made on the growth rates of promastigotes in Grace's insect medium, infectivity to C57BL/6 and BALB/c mice, electrophoresic mobility patterns of isoenzymes, random amplification of polymorphic DNA (RAPD), simple sequence repeat-anchored PCR amplification (SSR-PCR) and DNA fingerprinting profiles, infectivity to murine macrophages and cellular immune response. Infections of mice and macrophages were significantly different among the strains studied. Attempts to infect mice with culture promastigotes were unsuccessful with BH121, but BH49 infected BALB/c and C57BL/6 mice. Isoenzyme electrophoretic mobility patterns, RAPD and SSR-PCR using DNA amplified by polymerase chain reaction (PCR) with nine arbitrary primers, as well as DNA fingerprinting studies with a biotin-labeled 33.15 fingerprinting probe showed similar profiles to those of the Leishmania major WHO reference strain.

Effects of diapause and cold-acclimation on the avoidance of freezing injury in fat body tissue of the rice stem borer, Chilo suppressalis Walker

Overwintering freeze-tolerant larvae of Chilo suppressalis can survive at −25 °C, but non-diapausing larvae cannot. We reported earlier that to prevent intracellular freezing, which causes death in overwintering larvae of the Saigoku ecotype distributed in southwestern Japan, water leaves and glycerol enters fat body cells through water channels during freezing. However, it is still unclear how diapause and low-temperature exposure are related to the acquisition of freeze tolerance. We compared the extent of tissue damage, accumulation of glycerol, and transport of glycerol and water in fat body tissues between cold-acclimated and non-acclimated non-diapausing and diapausing larvae. The tissue from cold-acclimated diapausing larvae could survive only when frozen in Grace's insect medium with 0.25 M glycerol at −20 °C. The protection provided by glycerol was offset by mercuric chloride, which is a water-channel inhibitor. Fat body tissue isolated from non-acclimated diapausing larvae was injured by freezing even though glycerol was added to the medium, but the level of freezing injury was significantly lower than in non-diapausing larvae. Radiotracer assays in cold-acclimated diapausing larvae showed that during freezing, water left the cells into the medium and glycerol entered the cells from the medium at the same time. Therefore, in Saigoku ecotype larvae of the rice stem borer, both diapause and cold-acclimation are essential to accumulate glycerol and activate aquaporin for the avoidance of freezing injury.

Catalytic Site Modifications of TAP1 and TAP2 and Their Functional Consequences

The transporter associated with antigen processing (TAP), a member of the ATP binding cassette (ABC) family of transmembrane transporters, transports peptides across the endoplasmic reticulum membrane for assembly of major histocompatibility complex class I molecules. Two subunits, TAP1 and TAP2, are required for peptide transport, and ATP hydrolysis by TAP1.TAP2 complexes is important for transport activity. Two nucleotide binding sites are present in TAP1.TAP2 complexes. Compared with other ABC transporters, the first nucleotide binding site contains non-consensus catalytic site residues, including Asp(668) in the Walker B region of TAP1 (in place of a highly conserved glutamic acid), and Gln(701) in the switch region of TAP1 (in place of a highly conserved histidine). At the second nucleotide binding site, a glutamic acid (TAP2 Glu(632)) follows the Walker B motif, and the switch region contains a histidine (TAP2 His(661)). We found that alterations at Glu(632) and His(661) of TAP2 significantly reduced peptide translocation and/or TAP-induced major histocompatibility complex class I surface expression. Alterations of TAP1 Asp(668) alone or in combination with TAP1 Gln(701) had only small effects on TAP activity. Thus, the naturally occurring Asp(668) and Gln(701) alterations of TAP1 are likely to contribute to attenuated catalytic activity at the first nucleotide binding site (the TAP1 site) of TAP complexes. Due to its enhanced catalytic activity, the second nucleotide binding site (the TAP2 site) appears to be the main site driving peptide transport. A mechanistic model involving one main active site is likely to apply to other ABC transporters that have an asymmetric distribution of catalytic site residues within the two nucleotide binding sites.

Refinement of a cell line based artificial diet for rearing the parasitoid wasp, Trichogramma pretiosum

An artificial diet incorporating insect cells originally developed for Trichogramma australicum Girault (Hymenoptera: Tricho-grammatidae) was successfully used to rear Trichogramm pretiosum Riley (Hymenoptera: Trichogrammatidae). To refine the diet, individual components were removed. Chicken egg yolk and the insect cells were identified as the most important components for T. pretiosum development. Their removal resulted in few pupae and no adults. Removal of Grace's insect medium, a common component of artificial diets, was found to markedly improve the development of T pretiosum, producing 60% larva to pupa transition and 19% pupa to adult transition. There was no significant difference in T pretiosum development on diets in which milk powder, malt powder or infant formula were interchanged, despite differences in nutrient composition. The use of yeast extract resulted in significantly higher survival to the adult stage when compared with yeast hydrolysate enzymatic and a combination of yeast extract and yeast hydrolysate enzymatic. Comparison of four antimicrobial agents showed the antibacterial agent Gentamycin and the antifungal agent Nystatin had the least detrimental effect on T pretiosum development. The use of insect cell line diets has the potential to simplify artificial diet production and significantly reduce T pretiosum production costs in Australia compared to diets using insect hemolymph or the use of natural or factitious hosts. (c) 2005 Elsevier Inc. All rights reserved.

Structural and Functional Features in Human PDE5A1 Regulatory Domain That Provide for Allosteric cGMP Binding, Dimerization, and Regulation

The cGMP-binding cGMP-specific phosphodiesterase (PDE5) contains a catalytic domain that hydrolyzes cGMP and a regulatory (R) domain that contains two GAFs (a and b; GAF is derived from the proteins mammalian cGMP-binding PDEs, Anabaena adenylyl cyclases, and Escherichia coli (FhlA)). The R domain binds cGMP allosterically, provides for dimerization, and is phosphorylated at a site regulated by allosteric cGMP binding. Quaternary structures and cGMP-binding properties of 10 human PDE5A1 constructs containing one or both GAFs were characterized. Results reveal that: 1) high affinity homo-dimerization occurs between GAF a modules (K(D) < 30 nM) and between GAF b modules (K(D) = 1-20 pM), and the sequence between the GAFs (Thr322-Asp403) contributes to dimer stability; 2) 176 amino acids (Val156-Gln331) in GAF a are adequate for cGMP binding; 3) GAF a has higher affinity for cGMP (K(D) < 40 nM) than does the isolated R domain (K(D) = 110 nM) or holoenzyme (K(D) = 200 nM), suggesting that the sequence containing GAF b and its flanking amino acids autoinhibits GAF a cGMP-binding affinity in intact R domain; 4) a mutant (Met1-Glu321) containing only GAF a has high affinity, biphasic cGMP-binding kinetics consistent with structural heterogeneity of GAF a, suggesting that the presence of GAF b is not required for biphasic cGMP-dissociation kinetics observed in holoenzyme or isolated R domain; 5) significant cGMP binding by GAF b was not detected; and 6) the sequence containing GAF b and its flanking amino acids is critical for cGMP stimulation of Ser102 phosphorylation by cyclic nucleotide-dependent protein kinases. Results yield new insights into PDE5 functions, further define boundaries that provide for allosteric cGMP binding, and identify regions that contribute to dimerization.

Morphological and cytochemical characterization of female Anopheles albimanus (Diptera: Culicidae) hemocytes.

Hemocytes of 2- to 3-d-old female Anopheles albimanus Wiedemann are described by morphology, cytochemistry, and functional criteria. Supplemented Grace's insect medium in a modified Foley's perfusion method was used to obtain hemolymph from An. albimanus. Morphological analysis indicated 3 types of hemocytes were present, prohemocytes, plasmatocytes, and granular cells. Prohemocytes were small round cells with a high nuclear/cytoplasmic ratio. Plasmatocytes were the most abundant cell types in the hemolymph, and appeared as small to large and spindle-shaped cells with round or elongate nucleus, variable number of vacuoles, small granules, and pseudopodia. Granular cells were small to large and round with a large number of cytoplasmic granules, vacuoles, and numerous filopodia. Ultrastructurally, prohemocytes were undifferentiated with abundant free ribosomes and with few small electron-dense granules. Plasmatocytes were rich in mitochondria, rough endoplasmic reticulum, free ribosomes, small electron-dense granules, numerous peripheral vacuoles and with an important organelle polarization. Granular cells contained numerous large electron-dense granular inclusions and vacuoles. Cytochemical studies showed that plasmatocytes and granular cells have cationic bactericidal proteins. Only granular cells showed phenoloxidase and probably lysosomal activities. In vitro functional studies demonstrated that both plasmatocytes and granular cells were able to attach to glass slides, and only plasmatocyte had phagocytic activity and motility. These results characterize the hemocytes of An. albimanus and suggest that plasmatocytes and granular cells may have a role in defense responses to foreign organisms.