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Biomedical subjects

A J Green

Publications and source records attributed to A J Green.

At least 19 recordsLinked to original sources
PubMed1997

Increased S100beta in the cerebrospinal fluid of patients with frontotemporal dementia.

Levels of S100beta, a calcium-binding protein found in astrocytes, were measured using a sandwich ELISA in the cerebrospinal fluid (CSF) of patients with frontotemporal dementia and Alzheimer's disease and compared with controls. Mean CSF S100beta concentrations were significantly raised in patients with frontotemporal dementia when compared with healthy controls (0.49 +/- 0.28 vs. 0.22 +/- 0.08 ng/ml, P < 0.001). There was no correlation between age at disease onset, disease severity or length of illness. The increased concentration of CSF S100beta seen in frontotemporal dementia may reflect the marked astrocytosis seen in this condition.

Adult↗
PubMed1997

Identification of the tuberous sclerosis gene TSC1 on chromosome 9q34.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by the widespread development of distinctive tumors termed hamartomas. TSC-determining loci have been mapped to chromosomes 9q34 (TSC1) and 16p13 (TSC2). The TSC1 gene was identified from a 900-kilobase region containing at least 30 genes. The 8.6-kilobase TSC1 transcript is widely expressed and encodes a protein of 130 kilodaltons (hamartin) that has homology to a putative yeast protein of unknown function. Thirty-two distinct mutations were identified in TSC1, 30 of which were truncating, and a single mutation (2105delAAAG) was seen in six apparently unrelated patients. In one of these six, a somatic mutation in the wild-type allele was found in a TSC-associated renal carcinoma, which suggests that hamartin acts as a tumor suppressor.

Amino Acid Sequence↗
PubMed1997

A specific and sensitive ELISA for measuring S-100b in cerebrospinal fluid.

A sensitive, simple and specific sandwich ELISA for S-100b is described. This method involves the binding of a monoclonal anti-S-100b antibody to the wall of a microtitre plate. This capture antibody is subsequently incubated with S-100b standard, control or patient sample in the form of cerebrospinal fluid (CSF). After incubation, the microtitre plate is washed and horseradish peroxidase-labelled polyclonal anti-S-100b is added (detector antibody). The amount of detector antibody bound to the microtitre plate is proportional to the amount of S-100b in the sample. The assay has a lower limit of detection of 0.04 ng/ml and shows < 0.006% reactivity with the closely related polypeptide S-100a. The assay has a mean within-batch precision of 9.3 and 5.6% at S-100b concentrations of 0.38 and 0.8 ng/ml, respectively. The between batch precision is 8.9 and 8.1% at S-100b concentrations of 0.12 and 0.34 ng/ml, respectively. The recovery of S-100b from CSF spiked with 0.5 ng/ml was 94% with a CV of 8.5%. The assay may be completed in less than 5 h using precoated microtitre plates, thus lending itself to routine use in clinical laboratories. Using this ELISA, 154 CSF samples were analysed and 19% of samples were found to have elevated levels. The highest levels were found in patients with cerebral haemorrhage or central nervous system malignancy. S-100b concentrations from individuals without evidence of neurological disease were found to be less than 0.4 ng/ml. Only 5% of patients with multiple sclerosis were found to have elevated CSF S-100b concentrations. Serial CSF samples taken from a patient with an infected in-dwelling shunt showed a dramatic decline, suggesting that S-100b is rapidly cleared.

Enzyme-Linked Immunosorbent Assay↗
PubMed1997

A novel bacterial vector system for monitoring protein-protein interactions in the cAMP-dependent protein kinase complex.

A bacterial expression vector is described for investigation of protein-protein interactions. Important features of the vector include partition of the cI repressor of bacteriophage lambda into two functional domains separated by a multicloning site, and low level auto-regulated expression of human genes as C-terminal fusions to the DNA-binding domain of cI. Two different reporter systems have been employed; expression of either a suppressor tRNA or the alkaline phosphatase gene is dependent in both cases on the extent of repression of the major leftward promoter of lambda (lambdaP(L)). The cAMP-dependent protein kinase (PKA) has been used as a model protein complex because both homodimer and heterodimer interactions are known to occur and because cAMP acts as a modulator of these interactions. It has been shown that the product of the repressor gene with newly incorporated expressed polylinker restriction sites still functions as a repressor. Substitution of the dimerisation domain of the cI repressor with the regulatory subunit of PKA does not diminish the ability of a cI fusion protein to repress expression of the reporter gene from lambdaP(L), indicating that the regulatory subunit of PKA dimerises the fusion protein in the Escherichia coli cytoplasm. Substitution instead with the catalytic subunit of PKA destroys the repression ability of cI, which is partially restored by separate expression of the regulatory subunit within the same cell. Complete restoration is achieved using a host E. coli strain which has lost its ability to synthesise cAMP and again this can be reversed by the addition of exogenous cAMP to these cells. Human PKA has been reconstituted in the E. coli cytoplasm, where all subunit interactions appear functional and respond as expected to the allosteric modulator cAMP.

Alkaline Phosphatase↗
PubMed1997

Tuberous sclerosis-like lesions in epileptogenic human neocortex lack allelic loss at the TSC1 and TSC2 regions.

Glioneuronal malformations with a striking histological resemblance to cortical tubers of tuberous sclerosis, but no extracerebral stigmata of this phacomatosis, are frequently encountered in patients with chronic pharmacoresistant epilepsies. It is controversial as to whether these lesion represent a forme fruste of tuberous sclerosis or a distinct entity. The recently reported loss of heterozygosity (LOH) at the regions of the TSC1 or TSC2 locus in hamartomas obtained from different organs of patients with established tuberous sclerosis, including cortical tubers, stimulated us to examine epilepsy-associated tuberous sclerosis-like glioneuronal malformations with respect to LOH at the TSC1 and TSC2 loci of chromosomes 9q34 and 16p 13.3, respectively. The analysis was carried out on DNA derived from paraffin-embedded brain tissues of 11 patients. For 5 patients, peripheral blood leukocytes were also available for DNA extraction. We performed microsatellite analysis with five markers on chromosome 9 and four markers on chromosome 16. In addition, polymerase chain reaction-restriction fragment length polymorphism (RFLP) analysis was performed using a polymorphic EcoRV restriction site in exon 40 of the TSC2 gene. No LOH was identified in any of the cases. These findings do not support a relationship between the epilepsy-associated glioneuronal lesions and tuberous sclerosis. However, tuberous sclerosis is genetically heterogeneous and microsatellite and RFLP analysis cannot exclude small deletions or point mutations. Thus, given the histopathological similarity of glioneuronal malformations in epilepsy patients to cortical tubers, further molecular genetic studies will be needed as our understanding of the molecular basis of tuberous sclerosis increases to completely clarify the relationship of these lesions to tuberous sclerosis.

Adolescent↗
PubMed1997

Parenting stress and anger expression as predictors of child abuse potential.

To explore one potential pathway to physical child abuse, the present investigation used hierarchical regression analysis using measures of parenting stress and anger expression to jointly predict child abuse potential. The Parenting Stress Index, State-Trait Anger Expression Inventory, and the Child Abuse Potential Inventory were administered to two different samples of New Zealand parents. As expected, both studies revealed parenting stress and anger expression and were individually positively correlated with child abuse potential: the major finding involved the strong point contribution of parenting stress and anger expression in predicting Child Abuse Potential Inventory scores. Application of findings for intervention and prevention are discussed.

Adult↗
PubMed1997

Using a phenomenological research technique to examine student nurses' understandings of experiential teaching and learning: a critical review of methodological issues.

This paper provides a report of the usage of a phenomenological research methodology to investigate the influence on clinical practice of pre- and post-registration nurse education which makes explicit use of experiential teaching and learning approaches. The primary aim of the research was to explore the use of a phenomenological research methodology to examine the students' understanding of experiential teaching and learning. The claims made for the use of experiential teaching and learning approaches in both pre- and post-registration nurse courses and how clinical practice is influenced by the experiential learning elements of pre- and post-registration nurse education were also examined. The first stage of the enquiry involved focused non-directive interviews with members of BSc Nursing Studies and MSc Mental Health Branch programmes. Both programmes claim to make use of experiential teaching and learning. The data were analysed using a technique developed by Giorgi. Previous experiences of experiential teaching and learning were probed, student interpretations differentiated, and the relationship between course-based learning using experiential approaches and the implications for it's influence on practice were examined. The second stage of the enquiry has followed up the initial findings, exploring the students' experience of experiential approaches on their courses both in the classroom and in work-based learning situations. The findings are presented and discussed in the context of other studies from both nurse and higher education. Throughout the paper methodological concerns arising are discussed. The paper concludes with the identification of methodological problems arising from the research strategy: the implications of the power nexus created when teachers research students, and issues relating to the use of a phenomenological methodology in a longitudinal study.

Clinical Competence↗
PubMed1997

The X-linked gene G4.5 is responsible for different infantile dilated cardiomyopathies.

Barth syndrome (BTHS) is an X-linked disorder characterized clinically by the associated features of cardiac and skeletal myopathy, short stature, and neutropenia. The clinical manifestations of the disease are, in general, quite variable, but cardiac failure as a consequence of cardiac dilatation and hypertrophy is a constant finding and is the most common cause of death in the first months of life. X-linked cardiomyopathies with clinical manifestations similar to BTHS have been reported, and it has been proposed that they may be allelic. We have recently identified the gene responsible for BTHS, in one of the Xq28 genes, G4.5. In this paper we report the sequence analysis of 11 additional familial cases: 8 were diagnosed as possibly affected with BTHS, and 3 were affected with X-linked dilated cardiomyopathies. Mutations in the G4.5 gene were found in nine of the patients analyzed. The molecular studies have linked together what were formerly considered different conditions and have shown that the G4.5 gene is responsible for BTHS (OMIM 302060), X-linked endocardial fibroelastosis (OMIM 305300), and severe X-linked cardiomyopathy (OMIM 300069). Our results also suggest that very severe phenotypes may be associated with null mutations in the gene, whereas mutations in alternative portions or missense mutations may give a "less severe" phenotype.

Abnormalities, Multiple↗
PubMed1997

Female germline mosaicism in tuberous sclerosis confirmed by molecular genetic analysis.

We have investigated a family in which three siblings with the autosomal dominant disorder tuberous sclerosis had unaffected parents. The family were typed for polymorphic markers spanning the two genes known to cause tuberous sclerosis located at 9q34 (TSC1) and 16p13.3 (TSC2). TSC1 markers showed different maternal and paternal haplotypes in affected children, excluding a mutation in TSC1 as the cause of the disease. For the TSC2 markers all the affected children had the same maternal and paternal haplotypes, as did three of their unaffected siblings. Mutation screening by RT-PCR and direct sequencing of the TSC2 gene identified a 4 bp insertion TACT following nucleotide 2077 in exon 18 which was present in the three affected children but not in five unaffected siblings or the parents. This mutation would cause a frameshift and premature termination at codon 703. Absence of the mutation in lymphocyte DNA from the parents was consistent with germline mosaicism and this was confirmed by our finding of identical chromosome 16 haplotypes in affected and unaffected siblings, providing unequivocal evidence of two different cell lines in the gametes. Molecular analysis of the TSC2 alleles present in the affected subjects showed that the mutation had been inherited from the mother. This is the first case of germline mosaicism in tuberous sclerosis proven by molecular genetic analysis and also the first example of female germline mosaicism for a characterized autosomal dominant gene mutation apparently not associated with somatic mosaicism.

Adult↗
PubMed1997

Mutations in the TSC2 gene: analysis of the complete coding sequence using the protein truncation test (PTT).

Mutations in the TSC2 gene on chromosome 16p13.3 are responsible for approximately 50% of familial tuberous sclerosis (TSC). The gene has 41 small exons spanning 45 kb of genomic DNA and encoding a 5.5 kb mRNA. Large germline deletions of TSC2 occur in <5% of cases, and a number of small intragenic mutations have been described. We analysed mRNA from 18 unrelated cases of TSC for TSC2 mutations using the protein truncation test (PTT). Three cases were predicted to be TSC2 mutations on the basis of linkage analysis or because a hamartoma from the patient showed loss of heterozygosity for 16p13.3 markers. Three overlapping PCR products, covering the complete coding sequence of mRNA, were generated from lymphoblastoid cell lines, translated into 35S-methionine labelled protein, and analysed by SDS-PAGE. PCR products showing PTT shifts were directly sequenced, and mutations confirmed by restriction enzyme digestion where possible. Six PTT shifts were identified. Five of these were caused by mutations predicted to produce a truncated protein: (i) a sporadic case showed a 32 bp deletion in exon 11, and a mutant mRNA without exon 11 was produced; the normal exon 10 was also spliced out; (ii) a sporadic case had a 1 bp deletion in exon 12 (1634delT); (iii) a TSC2-linked mother and daughter pair had a G-->T transversion in exon 23 (G2715T) introducing a cryptic splice site causing a 29 bp truncation of mRNA from exon 23; (iv) a sporadic case showed a 2 bp deletion in exon 36; (v) a sporadic case showed a 1 bp insertion disrupting the donor splice site of exon 37 (5007+2insA), resulting in the use of an upstream exonic cryptic splice site to cause a 29 bp truncation of mRNA from exon 37. In one case, the PTT shift was explained by in-frame splicing out of exon 10, in the presence of a normal exon 10 genomic sequence. Alternative splicing of exon 10 of the TSC2 gene may be a normal variant. Three 3rd base substitution polymorphisms were also detected during direct sequencing of PCR products. Confirmed mutations were identified in 28% of the families studied and on the assumption that half of the sporadic cases should have TSC2 mutations, a crude estimate of the detection rate would be 60%. This compares favourably with other screening methods used for TSC2, notably SSCP, and since PTT involves much less work it may be the method of choice.

Alternative Splicing↗
PubMed1997

Spectrum of clinical features associated with interstitial chromosome 22q11 deletions: a European collaborative study.

We present clinical data on 558 patients with deletions within the DiGeorge syndrome critical region of chromosome 22q11. Twenty-eight percent of the cases where parents had been tested had inherited deletions, with a marked excess of maternally inherited deletions (maternal 61, paternal 18). Eight percent of the patients had died, over half of these within a month of birth and the majority within 6 months. All but one of the deaths were the result of congenital heart disease. Clinically significant immunological problems were very uncommon. Nine percent of patients had cleft palate and 32% had velopharyngeal insufficiency, 60% of patients were hypocalcaemic, 75% of patients had cardiac problems, and 36% of patients who had abdominal ultrasound had a renal abnormality. Sixty-two percent of surviving patients were developmentally normal or had only mild learning problems. The majority of patients were constitutionally small, with 36% of patients below the 3rd centile for either height or weight parameters.

Adolescent↗
PubMed1996

Clonality of tuberous sclerosis harmatomas shown by non-random X-chromosome inactivation.

Tuberous sclerosis (TSC) is an autosomal dominant condition characterised by tumour-like malformations (hamartomas) in the brain and other organs. A proportion of hamartomas from patients with TSC show loss of heterozygosity (LOH) for DNA markers in the region of either the TSC1 gene on chromosome 9q34 or the TSC2 gene on 16p13.3. This implies that these lesions are clonal. We have studied X-chromosome inactivation, as a marker of clonality, in 13 hamartomas from females with TSC. The hamartomas comprised five renal angiomyolipomas, three fibromas and seven other lesions. In previous studies, four of the lesions showed LOH. A polymerase chain reaction assay was used to analyse differential methylation of an HpaII restriction site adjacent to the androgen-receptor triplet-repeat polymorphism on Xq11-12. In 12 of the lesions, there was a skewed inactivation pattern with one X chromosome being fully methylated and the other unmethylated. Normal tissue showed a random pattern of inactivation. These data confirm that most TSC hamartomas are clonal in origin. This is an intriguing finding, since these lesions are composed of more than one cell type.

Angiomyolipoma↗
PubMed1996

Clinical evidence of efficient tumor targeting based on single-chain Fv antibody selected from a combinatorial library.

We present a system for cancer targeting based on single-chain Fv (scFv) antibodies selected from combinatorial libraries, produced in bacteria and purified by using an engineered tag. Combinatorial libraries of scFv genes contain great diversity, and scFv antibodies with characteristics optimized for a particular task can be selected from them using filamentous bacteriophage. We illustrate the benefits of this system by imaging patients with carcinoembryonic antigen (CEA)-producing cancers using an iodine-123 labeled scFv anti-CEA selected for high affinity. All known tumor deposits were located, and advantages over current imaging technology are illustrated. ScFvs are produced in a cloned form and can be readily engineered to have localizing and therapeutic functions that will be applicable in cancer and other diseases.

Adult↗
PubMed1996

Loss of heterozygosity in tuberous sclerosis hamartomas.

We have previously described in tuberous sclerosis (TSC) hamartomas the phenomenon of loss of heterozygosity (LOH) for DNA markers in the region of both the TSC2 gene on chromosome 16p13.3 and the TSC1 gene on 9q34. We now describe the spectrum of LOH in 51 TSC hamartomas from 34 cases of TSC. DNA was extracted from leucocytes or normal paraffin embedded tissue, and from frozen paraffin embedded hamartoma tissue from the same patient. The samples were analysed for 11 markers spanning the TSC1 locus and nine markers spanning the TSC2 locus. Twenty-one of 51 hamartomas showed LOH (41%). There was significantly more LOH on 16p13.3, with 16 hamartomas showing LOH around TSC2, and five in the vicinity of TSC1. No hamartoma showed LOH for markers around both loci. All the areas of LOH on chromosome 9 were large, but the smallest region of overlap lay between the markers D9S149 and D9S114, providing independent evidence for the localisation of the TSC1 gene. These data show that LOH is a common finding in a wide range of hamartomas, affecting the same TSC locus in different lesions from the same patient but not affecting both loci. These data support the hypothesis that both the TSC genes act as tumour suppressors and that the manifestations of TSC in patients with germline TSC mutations rise from "second hit" somatic mutations inactivating the remaining normal copy of the TSC gene.

Angiomyolipoma↗
PubMed1996

Down syndrome in association with features of the androgen insensitivity syndrome.

Three cases of Down syndrome (DS) are reported in association with features of the androgen insensitivity syndrome (AIS). All were 47, XY, +21 and reared as females. One case had a normal female phenotype, and two cases showed minimal clitoromegaly and labial fusion. Minor genital underdevelopment has been reported as common in males with DS; however, AIS has not previously been associated with DS. Androgen binding studies in genital skin fibroblasts were normal in two cases and in the 46,XY brother of the third who has perineal hypospadias. Mutation screening of the androgen receptor (AR) gene by PCR-SSCP was normal in all cases. Normal androgen binding and the absence of an identified mutation in the coding region of the AR gene is very unusual in AIS, particularly in the complete form. This finding suggests the operation of hitherto unrecognised genes on chromosome 21 with a role in androgen response and sex differentiation.

Adolescent↗
PubMed1996

An autosomal dominant syndrome of renal and anogenital malformations with syndactyly.

We describe a family with autosomal dominant inheritance of anal anomalies, renal tract abnormalities, genital malformations, and syndactyly. These clinical manifestations do not clearly fall into any previously described syndrome. A mother and daughter had almost identical congenital malformations, short stature, and unusual facies. The proband was born with anal stenosis, a rectovaginal fistula, clitoral hypertrophy, a pelvic right kidney, and syndactyly of both feet. Her daughter had the same anal, clitoral, and foot anomalies, a solitary pelvic kidney, and no fistula. This family is likely to represent autosomal dominant inheritance of a new combination of malformations, which may overlap with the Townes-Brocks syndrome, but does not fall into a current diagnostic category.

Anal Canal↗
PubMed1995

Experiential learning and teaching--a critical evaluation of an enquiry which used phenomenological method.

Experiential teaching methods have been recommended as the most effective medium for students to acquire the interpersonal skills and self-awareness required for the mental health nurse. This enquiry developed from the premise that there is potential for students to learn effectively in experiential teaching sessions. The authors experience of teaching in mental health gave focus to the question as to whether nurse teachers consider, and then use, a particular experiential learning framework when facilitating experiential teaching methods. The enquiry was an individual case study of one nurse teacher, and the data were collected through non-participant observation and focussed, non-directive interviewing. The philosophical perspective and methodology was phenomenological with the data analysed using phenomenological guidelines. This was used as it concentrated on the nurse teachers understanding of experiential learning as a phenomenon. The results concluded that the nurse teacher had a clear understanding of experiential learning and aspects of her interpretation manifested themselves within the classroom as part of experiential teaching. When compared with the literature there were commonalities with the meaning of experiential learning held by the nurse teacher studied. The focus of this paper is to report briefly on the enquiry, and then offer a detailed critical description and evaluation of the methodological approach used.

Faculty, Nursing↗