Cereal Leaf Beetle (Coleoptera: Chrysomelidae) Regional Dispersion and Relationship With Wheat Stand Denseness.

Cereal leaf beetle, Oulema melanopus L., is a pest of small grains and the literature conflicts on whether it is more abundant in sparse or dense stands of wheat. Our objectives were to determine the impact of stand denseness on cereal leaf beetle abundance and to investigate the regional dispersion of cereal leaf beetles across North Carolina and Virginia. One-hundred twenty fields were sampled across North Carolina and Virginia during 2011 for stand denseness, and cereal leaf beetle eggs, larvae, and adults. Two small-plot wheat experiments were planted in North Carolina using a low and a high seeding rate. Main plots were split, with one receiving a single nitrogen application and one receiving two. Egg density, but not larva or adult density, was positively correlated with stand denseness in the regional survey. Furthermore, regional spatial patterns of aggregation were noted for both stand denseness and egg number. In the small-plot experiments, seeding rate influenced stand denseness, but not nitrogen application. In one experiment, egg densities per unit area were higher in denser wheat, while in the other experiment, egg densities per tiller were lower in denser wheat. Larvae were not influenced by any factor. Overall, there were more cereal leaf beetle eggs in denser wheat stands. Previous observations that sparse stands of wheat are more prone to cereal leaf beetle infestation can be attributed to the fact that sparser stands have fewer tillers, which increases the cereal leaf beetle to tiller ratio compared with denser stands.

Cocaine produces conditioned place aversion in mice with a cocaine-insensitive dopamine transporter.

Cocaine is an inhibitor of the dopamine, norepinephrine and serotonin reuptake transporters. Because its administration would elevate signaling of all these three neurotransmitters, many studies have been aimed at attributing individual effects of cocaine to specific transmitter systems. Using mice with a cocaine-insensitive dopamine transporter (DAT-CI mice), we previously showed that cocaine-induced dopamine elevations were necessary for its rewarding and stimulating effects. In this study, we observe that DAT-CI mice exhibit cocaine-conditioned place aversion (CPA), and that its expression depends on their genetic background. Specifically, DAT-CI mice backcrossed to the C57Bl/6J strain background did not display a preference or an aversion to cocaine, whereas DAT-CI mice that were on a mixed 129S1/SvImJ × C57Bl/6J (129B6) background had a robust CPA to cocaine. These results indicate that while inhibition of the DAT is necessary for cocaine reward, other cocaine targets and neurotransmitter systems may mediate the aversive properties of cocaine. Furthermore, the aversive effect of cocaine can be observed in the absence of a DAT-mediated rewarding effect, and it is affected by genomic differences between these two mouse strains.

Use of electron microscopic and immunogold labeling techniques to determine polyomavirus recombinant VP1 capsid-like particles entry into mouse 3T6 cell nucleus.

Murine polyomavirus major structural protein VP1 could assemble into capsid-like particles when expressed in the baculovirus system. The recombinant capsid-like particles that were purified by CsCl density gradient centrifugation were capable of packaging host DNA. Electron microscopic and immunogold labeling techniques were used to study the entry of these VP1 recombinant capsid-like particles into mouse 3T6 cells. It was found that these VP1 recombinant capsid-like particles, which lack polyomavirus minor structural proteins (VP2 and VP3), use the same mechanism to enter mouse 3T6 cell cytoplasm and nucleus as that used by native polyomavirus virions.

Use of the confocal microscope to determine polyomavirus recombinant capsid-like particle entry into mouse 3T6 cells.

The structural protein genes of polyomavirus were expressed in the baculovirus system, and the proteins were found to assemble into capsid-like particles capable of packaging insect cell DNA. Recombinant capsid-like particles could be produced that were composed of the various structural proteins (VP1, VP1/2, VP1/3 and VP1/2 + VP3). Laser scanning confocal microscopy was used to determine if the various capsid-like particles could infect (enter) mouse 3T6 cells. Each of the various capsid-like particles was equally capable of cell entry as determined by indirect immunofluorescence confocal microscopy.

Sequence of the gene encoding hsp90e from Cryptosporidium parvum.

A composite 2364 nt DNA sequence with an open reading frame (ORF) encoding an endoplasmic reticulum-associated heat shock protein 90 (CpHsp90e) was determined from clones isolated from genomic libraries constructed from the KSU-1 isolate of Cryptosporidium parvum. Transcription was verified by isolation of a clone from a cDNA library with a similar restriction map to that observed with genomic DNA. The predicted protein consists of 787 amino acids, has a predicted molecular size of 89.2 kDa, and was found to share strong homology with other endoplasmic reticulum-associated hsp90 proteins.

A case report. Chondromatosis of the temporomandibular joint.

Synovial chondromatosis is a rare pathological condition that usually affects large joints but can affect the temporomandibular joint. The disease typically manifests itself with signs and symptoms similar to internal derangement. The disease is characterized by free-floating or attached cartilaginous bodies in the joint space. In this article, the authors present a case of synovial chondromatosis and discuss its pathological process. They also discuss diagnostic approaches and current treatment.

Toxoplasma gondii sporozoites form a transient parasitophorous vacuole that is impermeable and contains only a subset of dense-granule proteins.

Toxoplasma gondii sporozoites form two parasitophorous vacuoles during development within host cells, the first (PV1) during host cell invasion and the second (PV2) 18 to 24 h postinoculation. PV1 is structurally distinctive due to its large size, yet it lacks a tubulovesicular network (C. A. Speer, M. Tilley, M. Temple, J. A. Blixt, J. P. Dubey, and M. W. White, Mol. Biochem. Parasitol. 75:75-86, 1995). Confirming the finding that sporozoites have a different electron-dense-granule composition, we have now found that sporozoites within oocysts lack the mRNAs encoding the 5' nucleoside triphosphate hydrolases (NTPase). NTPase first appears 12 h postinfection. Other tachyzoite dense-granule proteins, GRA1, GRA2, GRA4, GRA5, and GRA6, were detected in oocyst extracts, and antibodies against these proteins stained granules in the sporozoite cytoplasm. In contrast to tachyzoite invasion of host cells, however, sporozoites did not exocytose the dense-granule proteins GRA1, GRA2, or GRA4 during PV1 formation. Even after NTPase induction, these proteins were retained within cytoplasmic granules rather than being secreted into PV1. Only GRA5 was secreted by the sporozoite during host cell invasion, becoming associated with the membrane surrounding PV1. Microinjection of sporozoite-infected cells with fluorescent dyes showed that PV1 is impermeable to fluorescent dyes with molecular masses as small as 330 Da, indicating that PV1 lacks channels through which molecules can pass from the host cytoplasm into the vacuole. By contrast, lucifer yellow rapidly diffused into PV2, demonstrating the presence of molecular channels. These studies indicate that PV1 and PV2 are morphologically, immunologically, and functionally distinct, and that PV2 appears to be identical to the tachyzoite vacuole. The inaccessibility of PV1 to host cell nutrients may explain why parasite replication does not occur in this vacuole.

Sporozoites of Toxoplasma gondii lack dense-granule protein GRA3 and form a unique parasitophorous vacuole.

The invasion of host cells by sporozoites of Toxoplasma gondii leads to the formation of parasitophorous vacuoles that are distinctly different from those surrounding tachyzoites. In sporozoite-infected cells, the fluid-filled space surrounding the sporozoite is many times larger in volume than the sporozoite, essentially lacks granular or tubular structures, and has no detectable continuous parasitophorous vacuolar membrane when prepared by conventional electron microscopic methods. Consistent with the ultrastructural differences, dense-granule protein GRA3, which associates with the parasitophorous vacuolar membrane of tachyzoites, was not detected by indirect immunofluorescence in sporozoite-infected cells 2-12 h post-inoculation or by Western blot analysis of sporozoite extracts. Western blots incubated with the alpha ROP/DG antiserum, which recognizes tachyzoite rhoptry and dense-granule proteins, revealed numerous other antigenic differences between sporozoites and tachyzoites. Cell cultures inoculated with sporozoites were monitored at various intervals for the expression of GRA3 and the developmentally-regulated tachyzoite surface protein SAG1. Expression of SAG1 and GRA3 was first observed in 30% of the sporozoite-infected cells at 12 and 15 h post-inoculation, respectively, and in all intracellular parasites at 24 h. Parasite replication was only observed in sporozoite-infected cells that were positive for GRA3 and SAG1. Thus, these data indicate that sporozoites and their interaction with host cells differ substantially from tachyzoites and the expression of tachyzoite-specific proteins is likely required for parasite replication.

Resolution of cryptosporidial infection in mice correlates with parasite-specific lymphocyte proliferation associated with both Th1 and Th2 cytokine secretion.

This study was designed to investigate and characterize T-cell responses which lead to elimination of a primary infection of Cryptosporidium muris in BALB/c mice. The proliferative response of spleen cells to parasite antigen was measured by uptake of 3H-thymidine and, in parallel, supernatants were removed from cells to measure levels of IFN-gamma, TNF, IL-2 and IL-4 by ELISA. Oocyst excretion in faeces was first detected on day 10 post infection (p.i.); the level of shedding subsequently increased until day 14 and then declined until no oocysts were detected by day 25. The proliferative response of spleen cells from infected animals was similar to control levels up to day 14 p.i. but increased significantly on day 21 and was even greater on day 26. IFN-gamma and IL-2 were detected initially on day 14 p.i. and significantly higher concentrations were found on days 21 and 26. IL-4 secretion was also detected, but not until day 21 p.i., and production of TNF was not found at any time. Depletion of T-cells or CD4+ cells from spleen cells cultured with antigen resulted in a significant decrease in the levels of cytokine detected. These results indicated, therefore, that in BALB/c mice there was a correlation between the development of immunity to C. muris infection and both a parasite antigen-specific proliferative response and Th1 and Th2 cytokine production by spleen cells.

Cloning and analysis of a Cryptosporidium parvum gene encoding a protein with homology to cytoplasmic form Hsp70.

An intronless gene encoding a protein of 674 amino acid residues with a molecular mass of 73,403 Da showing homology to the cytoplasmic form of the 70 kDa heat shock proteins has been cloned and sequenced from the intestinal pathogen Cryptosporidium parvum. Monospecific polyclonal antibodies obtained to recombinant protein recognized a single band with an approximate molecular mass of 70 kDa on a Western blot of C. parvum proteins, as well as the 70 kDa heat shock protein from bovine brain. Southern blot analysis suggested the gene was single copy in the C. parvum genome. Eleven perfect repeats of the sequence GGMP were found in the predicted protein near the carboxyl terminus.

Effects of select medium supplements on in vitro development of Cryptosporidium parvum in HCT-8 cells.

Surface-sterilized oocysts of Cryptosporidium parvum were applied to subconfluent monolayers of human adenocarcinoma (HCT-8) cells grown on coverslips in six-well cluster plates. Parasite-infected cultures were then incubated in RPMI 1640 with 10% fetal bovine serum, 15 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer, and antibiotics at 37 degrees C in a 5% CO2-95% air incubator for 2 h to allow sporozoites to excyst and enter cells. After cultures were washed free of debris, fresh cell culture media containing select supplements were added and cultures were reincubated. Parasite growth was assessed 66 h later by counting the number of parasite developmental stages in 25 random x 100 oil fields by Nomarski interference-contrast microscopy. Four vitamin supplements, calcium pantothenate, L-ascorbic acid, folic acid, and 4-(para)-aminobenzoic acid, each resulted in a significant increase in parasite numbers in vitro. The addition of insulin and the sugars glucose, galactose, and maltose also had a positive effect on parasite growth, although the effect was less pronounced than with any of the vitamins. Using the above information, we developed a supplemental medium formulation consisting of RPMI 1640 with 10% fetal bovine serum, 15 mM HEPES, 50 mM glucose, and 35 micrograms of ascorbic acid, 1.0 micrograms of folic acid, 4.0 micrograms of 4-aminobenzoic acid, 2.0 micrograms of calcium pantothenate, 0.1 U of insulin, 100 U of penicillin G, 100 micrograms of streptomycin, and 0.25 microgram of amphotericin B (Fungizone) per ml (pH 7.4). The growth of c. parvum in this medium was found to be enhanced approximately 10-fold compared with that in control medium without additional glucose, insulin, or vitamins.

A metallo-dependent cysteine proteinase of Cryptosporidium parvum associated with the surface of sporozoites.

A proteinase of 24 kD was found associated with sporozoites of Cryptosporidium parvum. Optimal hydrolysis of azocasein, casein, bovine serum albumin, and gelatin occurred at a pH of 6.5-7.0. Activity against azocasein was inhibited by ethylenediaminotetraacetic acid (EDTA), iodoacetic acid (IAA), trans-epoxysuccinyl-L-leucylamido(4-guanido) butane (E-64), and phosphoramidon, suggesting that the enzyme was a metallo-dependent cysteine proteinase. Both serine and aspartate protease inhibitors failed to inhibit enzyme activity. The enzyme was partially purified by preparative isoelectric focusing of parasite membrane proteins. Polyclonal antiserum to parasite membrane proteins was generated in rats. The enzyme-containing fraction was subjected to SDS-PAGE and probed with antiserum, and the antibodies against the protease were eluted directly from nitrocellulose blots. An indirect immunofluorescence assay using these monospecific antibodies revealed that the protease occurred on the surface of sporozoites, but was not associated with oocyst walls, rhoptries, or micronemes.

Major antigens of Cryptosporidium parvum recognised by serum antibodies from different infected animal species and man.

Serum humoral immune response to Cryptosporidium parvum was evaluated in six species: mouse, rabbit, lamb, calf, pig and man. Electrophoretic and immunoblot analysis showed that specific animal antibody response appeared between Day 4 and Day 15 post inoculation. The two main target antigens had apparent molecular weights of 15-17 and 23 kDa. They were recognised by each species studied. Serum IgA intensively recognised the 15-17 kDa antigen, except in rabbit. This study demonstrates that these two antigens are consistent targets of humoral immune response and can therefore be of great interest in studies of therapy/prophylaxis.

Both CP15 and CP25 are left as trails behind gliding sporozoites of Cryptosporidium parvum (Apicomplexa).

Sporozoites of Cryptosporidium parvum were examined after gliding upon glass microscope slides using monoclonal antibodies to the 15 and 25 kDa surface molecules and immunogold-silver enhancement. Both antibodies bound to surface antigen deposited as trials behind parasites, suggesting that both surface molecules are involved in substrate attachment.

A simple and reliable method of producing in vitro infections of Cryptosporidium parvum (Apicomplexa).

A variety of techniques have been used to infect cell monolayers in culture with the protozoan, Cryptosporidium parvum. However, most of these methods rely on the use of trypsin and/or bile salts to excyst sporozoites in vitro, followed by washing sporozoites free of excystation solution prior to their addition to subconfluent monolayers. This method not only increases the amount of time required to establish infections in vitro, but also results in prolonged exposure of free sporozoites to environmental conditions. Here we report a simple, fast, and efficient method of obtaining consistent infections of C. parvum in cell monolayers. This technique relies on the ability of the parasite to excyst at 37 degrees C but not at room temperature following pretreatment with sodium hypochlorite. By adding surface-sterilized oocysts directly to monolayers, sporozoites have access to host cells immediately upon excystation.

Comparative development of Cryptosporidium parvum (Apicomplexa) in 11 continuous host cell lines.

Using standardized media, incubation, and parasite inoculating procedures, we compared development of Cryptosporidium parvum between Madin-Darby bovine kidney (MDBK) cells and 10 additional host cell lines available through the American Type Culture Collection. Parasite development was assessed by counting parasite numbers atop monolayers in 25 random oil fields 68 h post-infection using Nomarski interference-contrast optics. Results revealed that the human ileocecal adenocarcinoma (HCT-8) cell line supported nearly twice the number of parasite developmental stages than MDBK cells or any of the other host cell types.

A simple modification of Blum's silver stain method allows for 30 minute detection of proteins in polyacrylamide gels.

A simple and rapid protocol for silver staining of proteins following electrophoresis in polyacrylamide gels (PAGE) is described. We have reduced the number of steps in the procedure of Blum et al. (Electrophoresis (1987) 8, 93-99), and shortened fixation and washing times so that efficient detection of proteins can be achieved within 30 min. In common with more time-consuming silver-staining methods, the present protocol is capable of detecting nanogram quantities of proteins on a colorless background and is suitable for rapid screening of large numbers of samples.

Metallothionein and zinc homeostasis during tumor progression. Effect of methotrexate treatment.

Zinc homeostasis was studied during the induction, growth, and methotrexate (MTX) treatment of Dark Agouti rat mammary adenocarcinomas (DAMA). A progressive fall in plasma Zn concentration (pZn), significant at a tumor burden of less than 1% body weight (bw), was sustained during tumor enlargement to give a 54% reduction in pZn at 16.3% bw (n = 6/group). The hypozincemia was attributed to the increasing Zn demand for tumor growth. Zn content of the 16.3% bw tumors equaled that of muscle (normally 60% of total body Zn). Tumor metallothionein (tMT) was sufficient to bind < 3% of total tumor Zn, and hepatic MT (hMT) remained at basal concentrations during early tumor growth, doubling only in the presence of significant necrosis in large tumors. Methotrexate (MTX, 0.5 mg/Kg im x 2 d) at respective tumor burdens of 5 and 10% bw (n = 9, 10/group) gave 2 therapeutic effects, dependent on tumor size: 1.5% bw tumors in 7 rats remained close to their original size until experiment end when pZn, hMT, and tMT were typical of 5% bw untreated tumors. 2. Tumors in 5 rats given MTX at 10% bw had marked subcapsular necrosis and regression to a size similar to those in group 1; pZn returned toward normal, whereas hMT was 6 times its 5% bw counterpart. Host weight loss was significantly reduced, as were tumor-associated changes in plasma glucose and calcium. In summary, neither tMT nor hMT appears to play a role in the hypozincemia that follows DAMA Zn sequestration and growth. Critically timed MTX can result in tumor regression and return of plasma Zn, Ca, and glucose toward normal. This is associated with an increase in hMT and reduction in host weight loss, suggesting a flow of Zn from the resorbing tumor to the host, enabling the synthesis of hMT and retention of host structural proteins.

Routine use of lamotrigine, a new anti-epileptic medication, and the value of measuring its blood levels.

Lamotrigine (LTG) has recently been approved for marketing in Australia as add-on therapy in resistant partial seizure disorders. Early reports cited a therapeutic blood level for LTG of 1-3 mg/L (4-12 mumol/L). Aspects of routine patient care with LTG, devoid of the restrictions of trial protocols, are discussed. Forty-five patients commenced therapy but 15 discontinued LTG. Of the remaining 30 patients, 9 became seizure free, 3 from the de novo trial in focal epilepsy and 6 with generalised epilepsy. Global evaluation of patients showed mild to moderate improvement for those with focal epilepsy and moderate to marked improvement for those with generalised epilepsy. Blood levels of LTG did not provide clinically useful information.

Clinical trials of antiepileptic drugs performed in the private practice setting.

Clinical trials of antiepileptic drugs (AEDs) are usually performed only in major teaching centers, thus ignoring the very real contribution available from private practice. The relation between the consultant and the referring family physician is often much closer in private practice, allowing much earlier recruitment of de novo patients into clinical trials. Between October 1988 and February 1992, 50 subjects were entered into various clinical trials including gabapentin (both an open add-on trial and parallel-arm add-on study in generalized epilepsies), vigabatrin (placebo-controlled cross-over study in focal epilepsies), and lamotrigine (both placebo-controlled cross-over add-on studies and parallel-arm comparative monotherapy study in newly diagnosed patients with epilepsy). All these trials were coordinated in private neurologic practice in Sydney, Australia, as part of either national or international multicenter AED studies. The experience demonstrated the improved ease of recruitment and patient liaison available in the private sector. It also highlighted the very real logistic problems, such as the need for a noninstitutional-based ethics committee, the training of support staff and a modified primary information resource system. Inclusion of private practice centers in clinical trials demonstrates the potential for improved patient recruitment and administrative management, especially in studies requiring newly diagnosed patients.