A method for release and multiple strand amplification of small quantities of DNA from endospores of the fastidious bacterium Pasteuria penetrans.

AIMS: To establish a reliable protocol to extract DNA from Pasteuria penetrans endospores for use as template in multiple strand amplification, thus providing sufficient material for genetic analyses. To develop a highly sensitive PCR-based diagnostic tool for P. penetrans. METHODS AND RESULTS: An optimized method to decontaminate endospores, release and purify DNA enabled multiple strand amplification. DNA purity was assessed by cloning and sequencing gyrB and 16S rRNA gene fragments obtained from PCR using generic primers. Samples indicated to be 100%P. penetrans by the gyrB assay were estimated at 46% using the 16S rRNA gene. No bias was detected on cloning and sequencing 12 housekeeping and sporulation gene fragments from amplified DNA. The detection limit by PCR with Pasteuria-specific 16S rRNA gene primers following multiple strand amplification of DNA extracted using the method was a single endospore. CONCLUSIONS: Generation of large quantities DNA will facilitate genomic sequencing of P. penetrans. Apparent differences in sample purity are explained by variations in 16S rRNA gene copy number in Eubacteria leading to exaggerated estimations of sample contamination. Detection of single endospores will facilitate investigations of P. penetrans molecular ecology. SIGNIFICANCE AND IMPACT OF THE STUDY: These methods will advance studies on P. penetrans and facilitate research on other obligate and fastidious micro-organisms where it is currently impractical to obtain DNA in sufficient quantity and quality.

Inter- and intra-specific cuticle variation between amphimictic and parthenogenetic species of root-knot nematode (Meloidogyne spp.) as revealed by a bacterial parasite (Pasteuria penetrans).

Specific host-parasite interactions exist between species and strains of plant parasitic root-knot nematodes and the Gram-positive bacterial hyperparasite Pasteuria penetrans. This bacterium produces endospores that adhere to the cuticle of migrating juveniles, germinate and colonise the developing female within roots. Endospore attachment of P. penetrans populations to second-stage juveniles of the root-knot nematode species Meloidogyne incognita and Meloidogyne hapla showed there were interactive differences between bacterial populations and nematode species. Infected females of M. incognita produced a few progeny which were used to establish two nematode lines from single infective juveniles encumbered with either three or 26 endospores. Single juvenile descent lines of each nematode species were produced to test whether cuticle variation was greater within M. hapla lines that reproduce by facultative meiotic parthenogenesis than within lines of M. incognita, which reproduces by obligate parthenogenesis. Assays revealed variability between broods of individual females derived from single second-stage juvenile descent lines of both M. incognita and M. hapla suggesting that progeny derived from a single individual can differ in spore adhesion in both sexual and asexual nematode species. These results suggest that special mechanisms that produced these functional differences in the cuticle surface may have evolved in both sexually and asexually reproducing nematodes as a strategy to circumvent infection by this specialised hyperparasite.

Effect of chronic exposure to welding light on Calabar welders.

It was generally observed that welders in Calabar, Nigeria did not always wear their protective goggles during welding. Since chronic exposure to welding light can impair vision this study was done to assess the effect of exposure to welding light on ocular function of welders in Calabar, Nigeria. There were 195 subjects comprising 110 welders (test) and 85 control subjects. Both groups were all male and had similar age range. The tests employed were clinical examination for ocular disorders, assessment of visual acuity, and opthalmoscopy. Test questionnaire was also used to record information on length of service, precautionary measures at work place, age and past ocular illnesses. The study also compared incidence of ocular disorders between the two groups of welders (arc and carbide welders). The mean ages of the welders and their control were not significantly different (27.53 +/- 10.0 vs 27.78 +/- 8.5 yrs respectively). There was a significantly [P < 0.01] higher incidence of pingueculum, cataract, allergic conjunctivitis, corneal opacity, and keratoconjunctivitis (arc eye) in welders than in their control subjects. However, visual acuity, incidence of pterygium and glaucoma were similar. Between the two groups of welders, the incidence of pterygium, corneal opacity and keratoconjunctivitis was significantly [P < 0.01] higher in arc welders than carbide welders. The incidence of pingueculum and glaucoma were however, similar. In conclusion, chronic exposure to welding light without adequate precaution may cause ocular disorders. Arc welding is more dangerous to ocular function than carbide welding. Length of service and age are predisposing factors to ocular disorders in the welding business.

The interaction between the gelatin-binding domain of fibronectin and the attachment of Pasteuria penetrans endospores to nematode cuticle.

Pasteuria penetrans is a Gram-positive endospore-producing bacterium that is a parasite of root-knot nematodes. Attachment of endospores to the cuticle of the nematode is the first stage in the infection process. Western blot analysis with monoclonal and polyclonal antibodies that recognize the 30 kDa heparin-binding domain (HBD) and the 45 kDa gelatin-binding domain (GBD) fragments of human fibronectin (Fn) revealed a series of polypeptides of approximately 40, 45 and 55 kDa present in crude cuticle extracts of Meloidogyne javanica 2nd-stage juveniles. The results suggest that the structure of the nematode fibronectin is different to the fibronectins so far characterized. Pre-treatment of endospores of Pasteuria with either the HBD or the GBD was found to inhibit binding to the nematode cuticle. The larger GBD fragment was the most effective at blocking adhesion. Pre-treatment of the GBD fragment with gelatin prevented the GBD fragment from inhibiting endospore attachment to the nematode cuticle.

What makes genetically modified organisms so distasteful?

The debate concerning genetically modified organisms goes on unabated and reflects some genuine concerns. I suggest that a significantly large number of educated people believe that moving genes around between species is intuitively wrong and that this is based on an essentialist view of the world. This essentialist view has a long history that dates back to Plato and Aristotle and was eventually overthrown by the population thinking of Charles Darwin. The essentialist, who is antipathetic to population thinking, will naturally find the transfer of a gene from one organism to another distasteful, and this, I argue, is the result of Platonic thinking, which still remains and casts its spell over us today.

Cuticle heterogeneity as exhibited by Pasteuria spore attachment is not linked to the phylogeny of parthenogenetic root-knot nematodes (Meloidogyne spp.).

The cuticle is a major barrier prohibiting the infection of nematodes against micro-organisms. The attachment of bacterial spores of the nematode hyperparasite Pasteuria penetrans (PP1) to field populations of root-knot nematodes (RKN, Meloidogyne spp.) from Burkino Faso, Ecuador, Greece, Malawi, Senegal and Trinidad and Tobago were assayed in standard attachment tests. The attachment of spore population PP1 to different field populations of root-knot nematode showed that the rates of attachment differed between countries. Similar tests were also undertaken on P. penetrans spores from these countries against 2 species of RKN, M. incognita and M. arenaria. The results showed a high degree of variability in spore attachment with no clear distinction between the 2 species of nematode. It has been hypothesized that Pasteuria spore attachment is linked to nematode species designations and this study clearly shows that this is not the case. Further tests showed that variation in spore attachment was not linked to nematode phylogeny. The results therefore beg the question of how do parthenogenetic root-knot nematodes maintain cuticle variability in the face of such an aggressive hyperparasite.

Post-translational hydroxylation at the N-terminus of the prion protein reveals presence of PPII structure in vivo.

The transmissible spongiform encephalopathies are characterized by conversion of a host protein, PrP(C) (cellular prion protein), to a protease-resistant isoform, PrP(Sc) (prion protein scrapie isoform). The importance of the highly flexible, N-terminal region of PrP has recently become more widely appreciated, particularly the biological activities associated with its metal ion-binding domain and its potential to form a poly(L-proline) II (PPII) helix. Circular dichroism spectroscopy of an N-terminal peptide, PrP(37-53), showed that the PPII helix is formed in aqueous buffer; as it also contains an Xaa-Pro-Gly consensus sequence, it may act as a substrate for the collagen-modifying enzyme prolyl 4-hydroxylase. Direct evidence for this modification was obtained by mass spectrometry and Edman sequencing in recombinant mouse PrP secreted from stably transfected Chinese hamster ovary cells. Almost complete conversion of proline to 4-hydroxyproline occurs specifically at residue Pro44 of this murine protein; the same hydroxylated residue was detected, at lower levels, in PrP(Sc) from the brains of scrapie-infected mice. Cation binding and/or post-translational hydroxylation of this region of PrP may regulate its role in the physiology and pathobiology of the cell.

Hippocampal sclerosis in a two-year-old with temporal lobe epilepsy: case report with pathological confirmation.

The case of a 2-year-old girl who underwent anterior temporal lobectomy for intractable epilepsy is described. Hippocampal sclerosis (HS) was present on histopathological examination. We believe this to be one of the youngest reported patients with pathologically confirmed HS. This has implications for the possible etiology of HS.

Intracarotid amobarbital procedure and prediction of postoperative memory in patients with left temporal lobe epilepsy and hippocampal sclerosis.

PURPOSE: Although temporal lobe epilepsy (TLE) patients with dominant hemisphere hippocampal sclerosis generally have good cognitive outcome after anterior temporal lobectomy (ATL), a minority of patients experience at least mild post-ATL decline on one or more standardized measures of episodic and semantic memory. The goal of this investigation was to determine whether memory outcome in this group could be predicted from preoperative intracarotid amobarbital procedure (IAP) recognition memory scores. METHODS: Data from 22 left TLE patients were studied retrospectively. All were left hemisphere language dominant and had IAP scores for each hemisphere, a significant degree of pathology-confirmed left hippocampal sclerosis (HS+), and no positive MRI findings other than atrophy. Cognitive outcome status was represented by the number of pre- to post-ATL declines across three tests, as defined by 90th percentile Reliable Change Index (RCI) criteria. RESULTS: Only 14% of the sample exhibited decline on more than one memory test. Low right IAP (left hemisphere injection) scores and relatively high preoperative cognitive ability and age at surgery predicted a greater risk of post-ATL memory decline. CONCLUSIONS: A minority of left TLE HS+ patients experience at least a mild degree of RCI-defined decline in episodic or semantic memory after ATL. The right hemisphere IAP memory score, which reflects the functional reserve of the contralateral hemisphere, can help predict the risk of postoperative memory decline for TLE patients in whom HS+ is likely based on the presence of hippocampal atrophy on MRI or early age of seizure onset.

Confrontation naming after anterior temporal lobectomy is related to age of acquisition of the object names.

Decline in visual confrontation naming ability may occur as a postacute complication of left anterior temporal lobectomy (ATL) for the treatment of intractable mesial temporal lobe epilepsy. In this study of 26 left ATL patients who demonstrated postsurgery decline on a standardized naming measure, it was hypothesized that naming performance would be significantly associated with specific attributes of the object names. We investigated the relation between performance on the Boston Naming Test (BNT) and the following attributes of the test items: living versus nonliving category (L/NL), word length (WL), written word frequency (WF), and age of acquisition (AoA). Regression analyses revealed that AoA and WF were significant predictors of preoperative group performance. AoA was the only significant predictor of performance after left ATL. For the 17 individuals who demonstrated a statistically meaningful decline on the BNT, as indicated by a Reliable Change Index, individual logistic regressions demonstrated that AoA was the strongest and most consistent predictor of postoperative success/failure for items that had been named correctly preoperatively. Consistent with the literature on naming errors in elderly normals and patients with aphasia or semantic dementia, the results provide evidence that object names learned in late childhood are among the most vulnerable when there is a decline in object naming ability. Investigation of additional attributes and semantic knowledge for the concepts represented by the pictured objects will be necessary to determine whether the naming deficit associated with TLE and ATL reflects an impairment of phonological word-form retrieval, semantics, or both.

Synaptophysin immunoreactivity in temporal lobe epilepsy-associated hippocampal sclerosis.

We have previously devised a semiquantitative grading system for hippocampal sclerosis (HS) in specimens resected for intractable temporal lobe epilepsy. The grades range from zero to four based on the amount and distribution of neuronal loss and gliosis. In the present study hippocampal sections from 25 patients who had temporal lobe epilepsy and had previously been assigned a grade were examined with synaptophysin immunohistochemistry, and the synaptic content in specific hippocampal fields was correlated with the results of the HS grading system. There was evidence of both significant synaptic loss and increased synaptic density in different fields of the hippocampus with increasing HS. A marked decrement of synaptic inmmunostaining was present in fields CA1 and CA4 that were highly correlated with HS grade. Sector CA4 seemed to respond in a more graded or continuous way to the pathological insults occurring in temporal lobe epilepsy than did CA1, which appeared to exhibit an all or nothing response. Also, while the width of the outer part of the molecular layer of the dentate (mid) gyrus decreased with increasing HS grade, the inner part of the mid became wider and showed an increased synaptic density so that the overall width of the mid was increased in the high-grade group. We conclude that quantitative measurement of synaptic loss in CAI and CA4 using synaptophysin immunohistochemistry is a sensitive method for detecting HS and correlates well with the empirically derived HS grading scale, with CA4 exhibiting a more graded response than CA1. In addition, a plasticity response in the inner part of the mld in patients with high-grade HS has been confirmed and quantitated.

Attachment tests of Pasteuria penetrans to the cuticle of plant and animal parasitic nematodes, free living nematodes and srf mutants of Caenorhabditis elegans.

Populations of Pasteuria penetrans isolated from root-knot nematodes (Meloidogyne spp.) and cyst nematodes (Heterodera spp.) were tested for their ability to adhere to a limited selection of sheathed and ex-sheathed animal parasitic nematodes, free living nematodes, including Caenorhabditis elegans wild type and several srf mutants, and plant parasitic nematodes. The attachment of spores of Pasteuria was restricted and no spores were observed adhering to any of the animal parasitic nematodes either with or without their sheath or to any of the free living nematodes including C. elegans and the srf mutants. All spore attachment was restricted to plant parasitic nematodes; however, spores isolated from cyst nematodes showed the ability to adhere to other genera of plant parasitic nematodes which was not the case with spores isolated from root-knot nematodes. The results are discussed in relationship to cuticular heterogeneity.

Predatory behaviour of trapping fungi against srf mutants of Caenorhabditis elegans and different plant and animal parasitic nematodes.

The initial infection process of nematode-trapping fungi is based on an interaction between the trapping structure of the fungus and the surface of the nematode cuticle. A bioassay was designed to investigate the predatory response of several isolates of nematode-trapping fungi against 3 mutants of Caenorhabditis elegans (AT6, AT10 and CL261), which have been reported to differ in the reaction of their cuticle to antibodies and lectins. The bioassay was also applied to infective larvae of animal (Haemonchus contortus, Teladorsagia (Ostertagia) circumcincta and Trichostrongylus axei) and plant (Meloidogyne spp.) parasitic nematodes. Differences in trapping ability were most marked in the first 24 h, and were density dependent. Although the isolate of Arthrobotrys responded very rapidly in the first 24 h, Duddingtonia flagrans was generally the most effective isolate and Monacrosporium responded relatively poorly throughout all experiments. All the fungi tested trapped the srf mutants of C. elegans more efficiently than the wild type, and there were differences between the different srf mutants of C. elegans. Differences in trapping ability were also observed between different isolates of D. flagrans; similarly, differences in trapping behaviour were observed not only amongst the different species of plant-parasitic nematodes, but also between the sheathed and exsheathed larvae of the animal-parasitic nematodes.

Prediction of presence of hippocampal sclerosis from intracarotid amobarbital procedure memory asymmetry scores and epilepsy on set age.

Identification of the pathological status of the hippocampus prior to surgery is important since the absence of hippocampal sclerosis (HS) carries risks to memory function following anterior temporal lobectomy (ATL). We studied 62 patients undergoing ATL (31 L, 31 R) for intractable epilepsy of temporal lobe origin in whom no pathology was identified apart from HS. An intracarotid amobarbital procedure (IAP) was performed as part of the preoperative evaluation. All patients were left hemisphere dominant for language. IAP memory testing was according to the protocol of Loring. We examined IAP memory asymmetry scores at four levels of difference (<2, > or =2, > or =4, > or =6) as a function of the presence (HS+) or absence (HS-) of HS. A logistic regression analysis was performed with HS+ as the dependent variable, and age at onset of epilepsy, age at time of surgery, gender, side of surgery and significant IAP memory asymmetry as independent variables. At each level of memory asymmetry, onset age and memory asymmetry were the only predictors of HS+. Younger age at onset was associated with HS+. Curves were constructed showing probability of HS+ for age at onset for each level of asymmetry. These can be used to predict the likelihood of presence of HS based on age at onset of epilepsy and the IAP memory asymmetry score. It is concluded that IAP memory asymmetry scores reflect the functional and pathological status of the hippocampus, and greater asymmetry increases the probability of finding HS in the resected hippocampus.

Spitting automatism in complex partial seizures: a nondominant temporal localizing sign?

PURPOSE: Spitting as an ictal automatism has been rarely reported. We aimed to establish its potential lateralizing and localizing significance. METHODS: Review of patients undergoing surgery for intractable epilepsy at two comprehensive epilepsy centers. RESULTS: Five patients were found who had spitting as a stereotyped automatism of their complex partial seizures. All had evidence of right temporal ictal onset and underwent resective surgery. Two had tumors; one, a cavernous angioma; one, hippocampal gliosis, and one, hippocampal sclerosis. We found no instances of ictal spitting in patients with left hemisphere onset. CONCLUSIONS: Spitting as an automatism in complex partial seizures, although uncommon, may be a localizing sign to the nondominant temporal lobe.

Meloidogyne incognita Surface Antigen Epitopes in Infected Arabidopsis Roots.

Surface-coat epitopes of Meloidogyne incognita were detected in root tissues of Arabidopsis thaliana during migration and feeding site formation. A whole-mount root technique was used for immunolocalization of surface coat epitopes in A. thaliana, with the aid of a monoclonal antibody raised specifically against the outer surface of infective juveniles of M. incognita. The antibody, which was Meloidogyne-specific, recognized a fucosyl-bearing glycoprotein in the surface coat. During migration in host tissues the surface coat was shed, initially accumulating in the intercellular spaces next to the juvenile and later at cell junctions farther from the nematode. Upon induction of giant cell formation, the antibody bound to proximally located companion cells and sieve elements of the phloem.

Synaptophysin immunohistochemistry densitometry measurement in resected human hippocampus: implication for the etiology of hippocampal sclerosis.

Synaptophysin (SY) is a protein expressed at presynaptic vesicles. SY immunohistochemistry (IHC) was undertaken in surgically resected hippocampal specimens from 25 patients with intractable epilepsy. All were investigated with chronic ictal EEG videotelemetry, which showed a temporal onset in each case, and all had normal magnetic resonance imaging (MRI). The density of reaction product of SY IHC was used to generate optical density (OD) measurements as an estimate of synaptic density in CA1 and CA4 fields (ODCA1 and ODCA4), and inner third and outer two-thirds of the molecular layer of the dentate gyrus (ODIML and ODOML). There was significant correlation between ODCA1 (r=0.619, P=0.001) and ODCA4 (r=0.639, P=0.001) and onset age of epilepsy. There was no correlation between ODCA1 and duration of epilepsy. There was correlation between ODCA4 and duration (r=-0.412, P=0.041), but partial correlations showed no significant correlation between ODCA4 and duration when controlling for onset, although correlation between ODCA4 and onset when controlling for duration remained significant (r=0.536, P < 0.01). Univariate ANOVA revealed onset age to be the only predictor of ODCA1 and ODCA4. Chronological age and duration were not predictors. There was no correlation between ODIML or ODOML and either onset age or duration. ODIML and ODOML were not predicted by onset age, duration or chronological age. These findings support the hypothesis that the major cause of hippocampal sclerosis is an age specific insult rather than the result of repeated seizures.

Prediction of verbal memory loss in individuals after anterior temporal lobectomy.

PURPOSE: Decreased memory function represents the area of greatest neuropsychological morbidity after anterior temporal lobectomy (ATL), particularly for left ATL candidates. We wished to identify easily derived demographic and neuropsychological predictors of risk of pre- to postoperative memory decline using only information available preoperatively. METHODS: We assessed decline in memory as measured by the California Verbal Learning Test (CVLT) by deriving multiple regression equations using the following measures as independent variables: age at onset, chronological age at time of surgery, sex, Full Scale IQ (FSIQ), level of education, and preoperative memory scores. In all, 203 patients (93 males, 110 females), undergoing ATL (107 left, 96 right) with preoperative and 6-month postoperative testing, were examined. RESULTS: The combination of age, FSIQ, sex, side of surgery and preoperative score was highly predictive (p-values < 0.0001) of postoperative memory scores. Higher postoperative scores were associated with higher preoperative score, younger chronological age, higher FSIQ, female sex, and right side of resection. Reliable change index (RCI) values were used to estimate meaningful decline on the total score across five trials. Logistic regression analysis showed preoperative score and age to be predictors of RCI decline for left-sided resections. Sensitivity of decline (> or =90th centile RCI) prediction was 56%, and specificity was 95%. Validation in 30 patients from a separate population of patients undergoing left ATL produced similar figures. CONCLUSIONS: The derived regression equations can accurately predict verbal memory decline on a list-learning task in approximately 50% of individual patients undergoing ATL, and false-positive prediction errors are very rare.