Instrumentation and methodology for quantifying GFP fluorescence in intact plant organs.

The General Fluorescence Plant Meter (GFP-Meter) is a portable spectrofluorometer that utilizes a fiber-optic cable and a leaf clip to gather spectrofluorescence data. In contrast to traditional analytical systems, this instrument allows for the rapid detection and fluorescence measurement of proteins under field conditions with no damage to plant tissue. Here we discuss the methodology of gathering and standardizing spectrofluorescence data from tobacco and canola plants expressing GFP. Furthermore, we demonstrate the accuracy and effectiveness of the GFP-Meter. We first compared GFP fluorescence measurements taken by the GFP-Meter to those taken by a standard laboratory-based spectrofluorometer, the FluoroMax-2. Spectrofluorescence measurements were taken from the same location on intact leaves. When these measurements were tested by simple linear regression analysis, we found that there was a positive functional relationship between instruments. Finally, to exhibit that the GFP-Meter recorded accurate measurements over a span of time, we completed a time-course analysis of GFP fluorescence measurements. We found that only initial measurements were accurate; however, subsequent measurements could be used for qualitative purposes.

A previous infection with Toxoplasma gondii does not protect against a challenge with Neospora caninum in pregnant sheep.

Sheep immunized with Toxoplasma gondii (Toxovax) prior to pregnancy were tested for their ability to withstand a challenge at 90 days gestation with 107 Neospora caninum (NC1) tachyzoites. The antibody responses in sheep following immunization with T. gondii were specific for T. gondii whereas peripheral blood mononuclear cells responded to both T. gondii and N. caninum antigen in vitro. This suggested that there was induction of crossreactive immune recognition in the sheep, at least at the cellular level. Following challenge of sheep at mid-gestation with N. caninum, no febrile responses were recorded in the group of sheep which had previously received Toxovax while significant febrile responses were recorded in the group of sheep which received N. caninum challenge alone. Antibody responses to N. caninum developed in all sheep following N. caninum challenge and antibody responses to T. gondii were boosted in the group of sheep which had previously been immunized with Toxovax. No antibodies to T. gondii were observed in the sheep which received the N. caninum challenge alone. Peripheral blood mononuclear cells from both groups of sheep responded to T. gondii and N. caninum antigen in vitro and interferon gamma was present in the cell-free supernatant from activated cells. However despite evidence of the induction of crossreactive immunity between T. gondii and N. caninum, this was not sufficient to prevent foetal death. The group of sheep which had received Toxovax prior to pregnancy and the group of sheep which only received the N. caninum challenge experienced 100% foetal death compared with 0% in the unchallenged control group. Vaccination prior to pregnancy with Toxovax did protect against foetal death following oral challenge at 90 days with 2000 T. gondii oocysts which caused 100% foetal death in a control challenge group.

Initial resuscitation after burn injury: therapies, strategies, and controversies.

Thermal injury disrupts normal hemostasis and sets off a cascade of events: cellular alterations and release of inflammatory mediators cause hypovolemic and cellular shock. Fluid resuscitation in burn injuries has been in use for more than a century, and much research has been devoted to development of resuscitation formulas and appropriate choice of fluid. Parameters for adequate monitoring of resuscitation are greatly debated. Current research efforts focus on minimizing burn edema through mediator modulation and on development of definitive endpoints of resuscitation monitoring.

Western blot analysis of the IgG responses of ruminants infected with Neospora caninum and with Toxoplasma gondii.

The IgG antibody responses of sheep, goats and cattle inoculated subcutaneously with live Neospora caninum tachyzoites of the NC1 isolate were analysed by Western blotting. Antibodies were detected against a wide range of NC1 tachyzoite antigens (6.5 to 80 kDa). The dominant antibody responses were directed against proteins at 36.5-38, 45.5-48.5, 52-53.5, 58, 58.5, 59.5, 60.5, 62, 63.5, 64, 66.5, 67, 67.5, 68.5 and 69.5 kDa, with sera from all three species. These sera were also used to probe blots of Toxoplasma gondii antigen and, while a number of protein bands were recognized, there was no consistency within or between animal species. The IgG antibody responses of sheep, goats and cattle orally infected with T. gondii oocysts of the M3 isolate were analysed by the same methods. Antibodies were detected to a range of S48 toxoplasma tachyzoite antigens (11 to 83 kDa). The dominant antibody responses were directed against proteins at 11, 16-17, 21.5, 22.5-23.5, 26-28.5, 32-35, 49.5, 50.5, 53, 54.5, 60.5 and 61 kDa, with sera from all three species. These sera were also used to probe blots of N. caninum antigen; antibody responses to numerous antigens were detected but showed little consistency within or between animal species.

Identification of Neospora antigens recognized by CD4+ T cells and immune sera from experimentally infected cattle.

Neospora caninum is recognized as a major cause of infectious abortion in cattle. Very little is known about immunity to Neospora. Cell mediated responses have previously been shown to be important in the development of protective immunity to the closely related parasite Toxoplasma gondii, and may therefore be an important component in the immune response to Neospora. In this paper we report that a group of low molecular weight NCI strain tachyzoite antigens (< or = 30 kDa) separated by SDS PAGE and bound to nitrocellulose membrane stimulated proliferation in vitro of CD4+ T cells from calves experimentally infected with N. caninum. Proliferation was accompanied by production of high concentrations of IFN-gamma. Several of these antigens were also recognized by antibody produced in these animals. As the most effective vaccines require the stimulation of both humoral and cell mediated immune responses, these antigens may be important in the development of a vaccine against neosporosis.

Cesarean delivery for the second twin in the vertex-vertex presentation: operative indications and predictability.

BACKGROUND: The incidence of second twin delivery by cesarean section and whether obstetric parameters are predictive should be studied. METHODS: In this retrospective study, all vertex-vertex twin deliveries during a 48-month period were reviewed. RESULTS: During the study period, 106 sets of vertex-vertex twins were identified. In 68 pairs, both were delivered vaginally (group 1), and 21 pairs were born via cesarean section. In the 17 pairs in group 2, the first infant was delivered vaginally and the second by cesarean section because of fetal distress in 6 cases (35%), cord prolapse in 6 (35%), abnormal/unstable lie in 4 (23%), and abruptio placenta in 1 case (6%). The only maternal factor associated with abdominal delivery for twin B was greater maternal age. When cesarean delivery was required for twin B, a longer interval between deliveries and a lower Apgar score were noted. CONCLUSION: Cesarean birth of twin B is a more common clinical event than previously reported but is not easily predicted by obstetric parameters.

Application of RT-PCR for Indexing Avocado Sunblotch Viroid.

A method for the routine detection of avocado sunblotch viroid (ASBVd) in nucleic acid extracts of infected avocado tissues by reverse transcription-polymerase chain reaction (RT-PCR) was developed using ASBVd-specific primers. Amplified cDNA products were analyzed by electrophoresis on nondenaturing 6% polyacrylamide slab gels. The size of the major RT-PCR product from ASBVd-infected tissue was estimated to be 250 bp. This product was absent from amplified extracts of uninfected tissue. The amplification product from ASBVd was sequenced by the dideoxynucleotide chain termination method, and the sequence was over 97% identical to the published sequence. The RT-PCR assay is sensitive enough to allow viroid detection without requiring large amounts of tissue, highly purified ASBVd, or molecular hybridization.

Eosinophilic gastro-enteritis associated with protein-losing enteropathy and protein C deficiency.

Reported below is a case of eosinophilic gastro-enteritis involving the colon, stomach and duodenum in a patient who was also found to have marked hypoalbuminaemia and protein C deficiency due to severe protein-losing enteropathy. The most recent literature is reviewed and the challenging differential diagnosis of the disease is discussed.

Kinetics of the local and systemic antibody response to primary and secondary infection with S48 Toxoplasma gondii in sheep.

Vaccination of sheep with live tachyzoites of Toxoplasma gondii, strain S48, affords protection against subsequent challenge with the parasite, but the mechanisms of immunity have not been fully determined. To understand better the nature of the antibody response the kinetics of both local and systemic antibody production were monitored in vaccinated sheep by means of an enzyme-linked immunosorbent assay and Western blotting. Local specific IgG production was analysed in efferent lymph obtained from the cannulated pre-femoral lymph node draining the site of infection. Antibody in efferent lymph plasma and peripheral blood serum from animals vaccinated with S48 tachyzoites was monitored and compared with IgG production in vaccinated sheep given a secondary tachyzoite challenge. Secondary challenge resulted in a clear immunological memory response, antibody being detected in the lymph 3 to 4 days after infection as compared with 7 to 8 days after a primary infection. IgG production was dominated by antibody recognizing a protein with an apparent molecular weight of 30 kDa, but other antigens (32, 24 and 11 kDa) were also readily detected.

Western blot analysis of the IgG response of sheep vaccinated with S48 Toxoplasma gondii (Toxovax).

The IgG antibody responses of sheep vaccinated by the subcutaneous injection of live tachyzoites of 'incomplete' strain S48 toxoplasma (Toxovax) were analysed by Western blotting. Antibodies corresponding to a range of tachyzoite antigens (13 to 48 kD) were detected, but the response was dominated by antibody recognising a 30 to 32 kD band. Unvaccinated ewes challenged orally with oocysts of the 'complete' M3 toxoplasma strain had a more complex IgG response that recognised antigens in six dominant bands of similar intensity as those in sheep vaccinated with S48 tachyzoites and then challenged with M3 oocysts. No differences were detected between the antigenic structures of the S48 tachyzoites and RH strain tachyzoites when the antigens were probed with immune ovine sera. Many of the anitgens of the S48 tachyzoites that were recognised had molecular weights similar to those of antigens that have been identified in other strains of toxoplasma.

Sex differences in parental influences on children's story-telling skills.

Acquisition of story-telling skills is an aspect of language development that is influenced by hearing parental story telling. Because discourse between parent and child is affected by the sex of both parent and child, we hypothesized that sex differences would affect parental story telling and children's acquisition of story-telling skills. For this study, 24 children (12 girls, 12 boys) told a story from a picture book both before and after hearing a parent (12 mothers or 12 fathers) tell the story. Children's stories improved after hearing a parent tell the story. Sons told stories containing more evaluatives (linguistic forms believed to promote better comprehension and memory of stories) after hearing mother's rather than father's story telling, and daughters told stories containing more evaluatives after hearing father's, rather than mother's, story telling. There were no differences in mothers' and fathers' use of evaluatives when telling stories to sons as compared with daughters.

Identification and localization of an iron-regulated 35 kDa protein of Pasteurella haemolytica serotype A2.

Iodination of intact Pasteurella haemolytica serotype A2 cells labelled a sub-set of total cellular proteins. Comparison of the autoradiographic patterns obtained from iodinated cells grown on complete medium and on iron-depleted medium showed that expression of three proteins, of 100, 70 and 35 kDa, respectively, was increased by growth under iron-depleted conditions. Of these proteins, that of 35 kDa had not been reported previously. Like the 100 and 70 kDa proteins, the 35 kDa protein was expressed in natural infections, since it was recognized by antiserum from sheep that had recovered from an experimental infection with P. haemolytica A2. The 35 kDa protein was partially purified by reverse-phase HPLC and was found to be antigenic in both sheep and mice. A monoclonal antibody that was specific for the 35 kDa protein was used to identify the cellular location of the protein by immunoblotting of cell fractions enriched for particular cellular components. This demonstrated that the 35 kDa protein was located mainly in the periplasm.

Regional metabolic differences in the rat diaphragm.

This study characterized the biochemical properties of the rat diaphragm by measuring the activities of selected citric acid cycle and glycolytic enzymes. The diaphragm was removed from 10 female Sprague-Dawley rats (180 days old) and dissected into five discrete anatomic regions: crural (region 1), left posterior costal (region 2), left anterior costal (region 3), right anterior costal (region 4), and right posterior costal (region 5). Sections were assayed for total protein concentration and the activities of succinate dehydrogenase (SDH) and lactate dehydrogenase (LDH). The SDH activity in the crural region was approximately 18% lower (P less than 0.05) than that in any costal region. Furthermore, protein concentration was significantly lower (P less than 0.05) in the crural region compared with all costal regions. In contrast, costal regions 2-5 did not significantly differ from each other in protein concentration or SDH activity. LDH activity did not differ significantly (P greater than 0.05) between regions. Finally, the LDH-to-SDH activity ratio was significantly higher (P less than 0.05) in the crural diaphragm compared with all costal regions. We conclude that the crural region of the rat diaphragm is significantly lower in oxidative capacity than all the costal regions. Investigators who use a rodent model to study diaphragmatic function and plasticity should consider the oxidative heterogeneity of the diaphragm when designing experiments.

Evidence for a maternal effect on infant hand-use preferences.

Left-handed offspring occur more frequently when one or both parents are left-handed. Among parental pairs with discordant handedness, left-handed mothers have more left-handed offspring than do left-handed fathers. No previous study has looked for this maternal effect in the hand-use preferences of infants. Handedness of 42 infants (21 females) 6-13 months of age (M = 10 months) was assessed by a reliable and valid procedure that provides hand-use preference scores separately for reaching and unimanual manipulation of objects. Equal numbers of age-matched male and female infants were formed into three groups representing different patterns of parental handedness: neither parent left-handed, father left-handed, mother left-handed. Infants of left-handed mothers showed more left hand-use than infants of left-handed fathers or infants of right-handed parents. Indeed, 64% of infants with left-handed mothers had significant left hand-use preference scores whereas none of the infants in the other groups had significant left hand-use preference scores. Possible mechanisms for this maternal effect on infant hand-use preferences are discussed.

Postural and lateral asymmetries in the ontogeny of handedness during infancy.

Ten newborn infants who preferred to lie with their heads turned rightward and ten who preferred to lie with their heads turned leftward had their hand-use preferences for reaching assessed at 8 different ages during the period 12-74 weeks postpartum. Eighteen infants maintained stable hand-use preferences throughout this period and the direction of their neonatal head orientation preference predicted the hand they preferred to use. The neonatal head-turn preference was maintained through the first 2 months and induced lateral asymmetries in visual regard and motor control of the hands. These lateral asymmetries are plausible contributors to mechanisms linking neonatal headturn preference to infant hand-use preference. Thus, the dextral bias in handedness may be derived, in part, from the rightward bias in neonatal head-turn preference.

Hand-use preference for reaching and object manipulation in 6- through 13-month-old infants.

Validity, reliability, and stability of separate assessment of hand-use preferences for reaching, object manipulation, and complementary bimanual action were determined for 6 through 13 month-old male and female infants. Only hand-use preferences for complementary bimanual action varied with age. Females had more distinct hand-use preference than males but only for object manipulation. Comparison of hand-use preference among these different sensorimotor skills can provide a richer evaluation of infant handedness status required for systematic study of the development of neuropsychological functions during infancy.

Caffeine reduces amphetamine-induced activity in asymmetrical interaction.

Caffeine-amphetamine interactions were studied to determine whether attenuation of amphetamine-induced activity by caffeine pretreatment (30 mg/kg) is the result of increased or decreased sensitivity to amphetamine. Caffeine pretreatment attenuated amphetamine activity in the rats without producing a horizontal shift in the dose-response curve. Results support a reduction in sensitivity to amphetamine. A cross-tolerance design revealed an asymmetrical interaction between caffeine and amphetamine. Multiple caffeine treatments (30 mg/kg) produced tolerance and attenuation of subsequent amphetamine activity (1.5 mg/kg). Amphetamine did not produce tolerance or affect subsequent caffeine-induced activity.