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.

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.

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.

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.

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.

Relationship of hippocampal sclerosis to duration and age of onset of epilepsy, and childhood febrile seizures in temporal lobectomy patients.

Controversy exists as to whether hippocampal sclerosis (HS) is a preexisting cause or a consequence of seizures. We investigated 122 consecutive patients who underwent anterior temporal lobectomy for intractable epilepsy between 1989 and 1992. MRI scans were normal apart from evidence of HS in 5 cases. The degree of HS was graded from 0 to 4. There was a significant inverse correlation between age of seizure onset and grade of HS (P < 0.0001), and a positive correlation between duration of epilepsy and grade of HS (P < 0.001). Using a dichotomous grouping of HS (HPSC - for grades 0 and 1 [no/mild HS], and HPSC + for grades 3 and 4 [moderate/marked HS]), there was a positive correlation between HPSC + and a history of childhood febrile seizures (CFS) (P = 0.003), earlier age of onset of epilepsy (P < 0.001) and longer duration of epilepsy (P < 0.001). There was no correlation with history of particularly prolonged individual seizures. Partial correlations after controlling for age at onset of epilepsy showed that there was no longer a significant relationship between HPSC + and duration of epilepsy. After controlling for duration of epilepsy, the relationship between HPSC + and age of onset remained significant (P < 0.001). The correlation between HPSC + and CFS, controlling for age at onset, was not significant. A series of logistic regression analyses showed age at onset to be the only predictor of HPSC +. It is concluded that this is supportive evidence for preexisting HS being a cause of temporal lobe epilepsy and not a consequence of seizures.

Differentiating central neurocytoma. Case report.

In 1976 a patient underwent partial resection of an intraventricular tumor that showed central neurocytoma. No other tumor pattern was observed. In 1994 this patient underwent a second operation for removal of the tumor, at which time foci of tumor were diagnosed as central neurocytoma and ganglioglioma. This is the first reported case of differentiation of central neurocytoma into ganglioglioma, a sequence of events termed differentiating central neurocytoma.

Hemispherectomy for intractable seizures: long-term results in 17 patients followed for up to 38 years.

Seventeen patients who underwent hemispherectomy for intractable epilepsy between 1950 and 1971 were reviewed to evaluate outcome for seizure control and the development of late complications. Sixteen had complete resection and in one the frontal pole was preserved. The follow-up period was 19 to 38 years (mean 28 years). One patient was lost to follow-up review 10 years after surgery. Three patients had died but none of the deaths were related to the surgery or to epilepsy. Ten patients had no postoperative complications, and three developed late complications: two had elevated intracranial pressure with enlargement of the remaining lateral ventricle after 13 and 16 years, and one had recurrent bleeding into the cerebrospinal fluid after 6 years. All were treated surgically and have since remained well. Eight patients (47%) had no seizures after surgery and eight (47%) were almost seizure-free. It is concluded that classical hemispherectomy is an effective operation for control of some types of epilepsy. The late complications, which occurred in 17% of the cases in this series, can be successfully treated. This series presents the longest follow-up results after hemispherectomy reported to date.

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.

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.

Cortical resections for intractable epilepsy of extratemporal origin: experience with seventeen cases over eleven years.

Seventeen patients underwent surgery between 1981 and 1990 for intractable partial epilepsy arising outside the temporal lobe. Twelve had frontal seizure onset, two parietal, two occipital and one diffusely in the hemisphere. Localization was achieved using extraoperative electrocorticography (ECoG) in five cases and intraoperative ECoG was employed in 12. Fifteen patients underwent cortical resections, but two did not subsequently have a resection. Both of these had porencephalic cysts. Of the 15 who had resections six (40%) were seizure free after a mean of 7.3 years. One (7%) was almost seizure free, six (40%) had worthwhile improvement. Pathological examination revealed oligodendroglioma in three, recurrent meningioma in one, vascular malformations in two, glial hamartoma in one and gliosis in six. One case with gliosis initially was shown to have an underlying malignant astrocytoma 2 years later. All these patients had CT abnormalities prior to surgery. Two patients (13%) had no worthwhile improvement. Pathology in these two was ischaemic neurons and arachnoid thickening. Both had normal CT findings preoperatively. One patient had an increased hemiparesis postoperatively. There were three cases of postoperative infection. It is concluded that extratemporal resection can achieve good results for seizure control and intraoperative ECoG is an effective technique for localizing the epileptogenic area. The presence of a structural lesion carries a particularly favourable prognosis for seizure outcome and surgery should, therefore, be strongly considered in patients with intractable partial epilepsy who have evidence of underlying structural pathology.

Acute spontaneous spinal epidural haematoma with temporary resolution.

A case of acute spontaneous spinal epidural haematoma is presented which underwent resolution of symptoms and signs before relapsing again hours later. Cases of spontaneous remission of this condition have been reported but not subsequent relapse. Conservative management of apparently resolving cases may therefore be inappropriate.

Temporal lobectomy for intractable epilepsy: experience with 58 cases over 21 years.

Sixty patients with intractable complex partial seizures underwent surgery between 1969 and 1990, and 58 of these underwent a temporal resection. Of 39 patients who had chronic subdural electrocorticography (ECoG) 37 subsequently underwent an anterior temporal lobectomy (ATL) and two had the electrodes removed without resection. Two patients had bilateral depth electrodes placed and then had an ATL. Fourteen patients with evidence of temporal structural lesions had temporal resections with intraoperative ECoG and five had resection without ECoG. Mean length of follow-up for all patients was 6 years. Fifty-five per cent of cases were seizure-free postoperatively, 7% almost seizure-free, 21% had worthwhile improvement and 17% no improvement. The outcome for patients with structural lesions was particularly good. Nine patients complained of mild memory impairment postoperatively and one had a severe amnestic problem. One patient with an unsuspected tumour developed a hemiparesis and dysphasia. One other patient had persistent dysphasia. No mortality was attributable to the surgery. It is concluded that ATL is an excellent operation for the treatment of intractable epilepsy arising from the temporal lobe and chronic subdural ECoG is a safe and reliable method for localizing the seizure origin.

Diversity and partial characterization of putative virulence determinants in Pasteuria penetrans, the hyperparasitic bacterium of root-knot nematodes (Meloidogyne spp.).

Antigens recognized by monoclonal antibodies (Mabs) raised to the surface of the obligate nematode hyperparasite Pasteuria penetrans were characterized. Using the attachment of spores of the bacterium to host nematodes to determine the biological variability present on the spore surface greatly underestimated the amount of surface heterogeneity present compared with estimates from immunological techniques. This heterogeneity differed not only between different individual spores from the same population but also between different spore populations. None of the Mabs completely inhibited any spore population from attaching to the nematode cuticle, suggesting that the mechanism of attachment may be more complex than previously supposed. Chemical degradation of one particular epitope recognized by monoclonal antibody PP1/117, and designated ep117, occurred after treatment with NaOH, periodate or Proteinase K, suggesting that an O-linked glycoprotein may be involved. Fibronectin, which had been found to bind to Pasteuria spores through hydrophobic interactions, also prohibited the Mab from recognizing ep117. However, SDS-PAGE of spore extracts followed by immunoblotting showed that none of the Mabs could detect this epitope and so ep117 may be conformational in nature. Thus, the conformation of any particular epitope recognized by a Mab may be important in determining to which nematode a particular spore will attach. The distribution of a particular epitope within a population of spores will in turn therefore determine its virulence on a particular nematode.

Comparison of two sympatric Pasteuria populations isolated from a tropical vertisol soil.

An isolate of Pasteuria (designated PPMJ) recovered from the root-knot nematode Meloidogyne javanica, was characterized using host preference, spore morphometrics, and serology, and compared with another sympatric Pasteuria isolate (designated PPHC) collected from the cyst nematode, Heterodera cajani. PPMJ spores were larger (x 1.5) than the PPHC spores and had a mean diameter of 3.4 µm after fixation for electron microscopy. The central body of PPMJ spores was about twice as big as the central body of PPHC spores. The host preference tests, based on spore attachment to the nematode cuticle, revealed that Meloidogyne incognita, M. javanica, M. hapla, Pratylenchus coffeae, and Pratylenchus sp. were hosts of PPMJ but not of PPHC. It was found that males of Radopholus similis were hosts of PPHC. Western blot analysis of spore extracts probed with a polyclonal antiserum raised against PPHC spores showed an antigenic ladder which had similarities to lipopolysaccharide; another antiserum revealed differences in the molecular weight of antigens of the different spore isolates. Population diversity can therefore be vastly altered by the maintenance and culture of the bacterium on a particular host. The implications of these results are discussed in relation to the use of Pasteuria as a biological control agent.

Modulation of spore adhesion of the hyperparasitic bacterium Pasteuria penetrans to nematode cuticle.

Monoclonal antibodies (mAb) raised to the cuticule surface of second-stage juveniles (J2) of the nematode Heterodera cajani were partially characterized by immunofluorescence and Western blot analysis. Five antigens with relative molecular weights (M(r)) 55, 80, 110, 180 and 210 kDa were identified with six mAb. Pasteuria spores, originating from the same population of H. cajani to which the antibodies were raised, were tested for their ability to attach to J2, which had been pretreated with each of the mAb. Monoclonal antibody HC/129 was found to reduce spore attachment by 42%, whereas HC/145 increased spore attachment by 124%. This is the first record of an antibody binding to the cuticle and increasing spore attachment, and suggests that components of the cuticle involved in inhibiting spore attachment may be masking the Pasteuria receptor present on the cuticle.

Anterior temporal lobectomy, hippocampal sclerosis, and memory: recent neuropsychological findings.

Anterior temporal lobectomy (ATL) is an effective and increasingly utilized treatment for nonlesional, intractable mesial temporal lobe epilepsy. However, this surgery results in domain-specific neuropsychological morbidity for a subset of patients. Within the past decade, multidisciplinary studies have revealed that left ATL patients without significant sclerosis in the resected hippocampus are most at risk for a substantial postacute decline in the ability to encode new verbal information. These patients are also at risk for a significant decrement in confrontation naming and other retrieval-based language abilities. The memory deficit is not attributable to this disruption of language. A relationship between hippocampal sclerosis (HS) status and memory performance has not been identified consistently in right ATL patients, but investigation of new visuospatial measures continues. The influence of variables other than HS on neuropsychological outcome is also discussed.

Results of cortical resection for intractable epilepsy using intra-operative corticography without chronic intracranial recording.

Twenty-four patients with intractable partial epilepsy underwent surgery between 1969 and 1988. Localization was by non-invasive means using scalp EEG and CT. In 12 cases the focus was temporal and in eight frontal. Craniotomy was undertaken with intraoperative electrocorticography (ECoG). Ten cases had a standard temporal lobectomy, seven a topectomy, four topectomy with tumour excision and one tumour excision alone. Two cases did not have a resection. Pathology revealed a mass lesion in 12 cases, hippocampal sclerosis in two and gliosis in six. Mean length of follow-up was 7.4 was. Fourteen patients (64%) were seizure free, two (9%) almost seizure free, four (18%) had worthwhile improvement and two (9%) no improvement. Of the temporal resections, 9 out of 12 (75%) were seizure free and of the extratemporal resections 5 out of 10 (50%) were seizure free. Removal of a mass lesion carried the most favourable prognosis for seizure outcome: 10 our of 12 (83%) of the cases with mass lesions, but 3 out of 6 (50%) of the cases with gliosis were seizure free. In two of the frontal resections removal of tumour resulted in disappearance of contralateral frontal independent spikes. It is concluded that where sufficient information exists to localize seizure activity by non-invasive means, invasive recording is unnecessary. The utility of intraoperative ECoG is discussed. It may be of limited use in cases of standard resection of when a mass lesion is present, but ECoG can prove useful to delineate the epileptogenic area in cases where there is o mass lesion and the results can still be rewarding.