Mutation scanning methods for the analysis of parasite genes

Molecular variation is widespread in parasite populations, and its analysis has important implications for studying aspects relating to the function and organisation of genes, and the taxonomy, phylogeny and population genetics of parasites. This article reviews some PCR-based mutation scanning techniques that have advantages over currently used DNA methods for the analysis of genetic variation in parasites. The review is technical and describes briefly the principles of relevant techniques, examines some of their advantages and disadvantages and gives several examples for possible applications.     

Endoscopic orbital decompression for dysthyroid eye disease

Orbital decompression for dysthyroid eye disease is traditionally performed either through an external approach or transantrally. The introduction of endoscopic sinus surgery over the last decade has led to a better understanding of the intranasal surgical landmarks of orbital walls and the development of endoscopic orbital decompression. We have adopted the endoscopic technique as the treatment of choice for orbital decompression in dysthyroid eye disease for the last two years. The surgical technique is described and illustrated, and the results of our first series of 15 patients (30 orbits) are presented.     

Allergic eye disease mechanisms

The histologic distinction between epithelial peritoneal mesothelioma and papillary serous carcinoma diffusely involving the peritoneum may be difficult. Although some investigators have indicated that immunohistochemistry can facilitate this differential diagnosis. only a few studies using a limited number of markers have been published. In this study, the immunoreactivity of keratin 5/6, vimentin, epithelial membrane antigen, thrombomodulin, calretinin, MOC-31, Ber-EP4, carcinoembryonic antigen, TAG-72 (B72.3), CD15 (Leu-M1), placental alkaline phosphatase, CA19-9, CA-125, HBME-1, 44-3A6, and S-100 protein was investigated in 35 epithelial peritoneal mesotheliomas, and 45 papillary serous carcinomas [30 ovarian (10 primary and 20 metastatic to the peritoneum) and 15 papillary serous carcinomas of the peritoneum]. After analyzing the results, it is concluded that calretinin, thrombomodulin, and keratin 5/6 are the best positive markers for differentiating epithelial malignant mesotheliomas from papillary serous carcinomas diffusely involving the peritoneum. The best diagnostic discriminators among the antibodies considered to be negative markers for mesothelioma are MOC-31, B72.3, Ber-EP4, CA19-9, and Leu-M1. Immunostaining for carcinoembryonic antigen, placental alkaline phosphatase, epithelial membrane antigen, vimentin, HBME-1, 44-3A6, CA-125, or S-100 have little or no diagnostic utility in establishing the differential diagnosis between these conditions. The results of this study also confirm previous observations indicating that both papillary serous carcinomas of the peritoneum and serous carcinomas of the ovary have a similar phenotype and, therefore, immunohistochemical studies are not useful in separating these entities.     

Mutation screening of interferon-gamma (IFNgamma) as a candidate gene for asthma

BACKGROUND: Reduced levels of interferon gamma (IFNgamma) mRNA and protein have been detected in the bronchoalveolar lavage fluid of atopic asthmatics. IFNgamma is secreted by TH1 cells while IL-4 and IL-5 are secreted by TH2 cells and an imbalance in the TH1/TH2 response may be responsible for atopic asthma. The gene for IFNgamma is located on chromosome 12; a region of the genome which has been shown in linkage studies to be associated with asthma. OBJECTIVE: To determine if there are any mutations present in the coding exons and 5' flanking region of the IFNgamma gene in atopic asthmatic subjects compared with controls to explain the lower levels of this cytokine as an inherited, rather than acquired, factor in the asthmatic subjects. METHODS: The four exons and 5' flanking region of the IFNgamma gene were amplified by polymerase chain reaction (PCR) from genomic DNA of 265 individuals from a Western Australian and a Venezuelan population. The PCR products were examined by single strand conformational polymorphism and heteroduplex analyses to see if there were any changes in the DNA migration patterns which would suggest the presence of a sequence variation. RESULTS: The four exons and the 5' flanking region of the IFNgamma gene were amplified from 265 individuals from two populations. Single strand conformational polymorphism and heteroduplex analyses did not reveal any mutations in the regions examined. CONCLUSION: The gene for IFNgamma appears to be highly conserved as no sequence variations were detected in 265 individuals. These results suggest that mutations of the IFNgamma gene are unlikely to be a significant cause of an inherited asthma diathesis.     

Mutation analysis of S182 (presenilin-I) in patients with familial Alzheimer''s disease and its biological function

OBJECTIVE: We studied the long-latency response of the orbicularis oris muscle elicited with transcranial magnetic stimulation in patients with hemifacial spasm (HFS) and evaluated the excitability of the facial nucleus. METHODS: We compared the thresholds on both sides in 8 normal volunteers and 7 patients with hemifacial spasm. The thresholds were determined as the lowest intensity required to produce motor evoked potentials with an amplitude of at least 50 microV in the orbicularis oris muscle. Average values were given as means +/- standard deviation. Wilcoxon's rank sum test was used for comparisons between the sides of normal subjects and of patients with HFS with respect to the threshold stimulus. RESULTS: There was no significant difference between the thresholds on the two sides of the normal subjects (mean 1.88+/-5.30%, P > 0.05). In patients with HFS, there was a significant difference between the thresholds on the spasm side and the normal side (mean 20.7+/-13.0%, P < 0.05) In one patient studied after MVD, the difference between both sides disappeared. CONCLUSION: The difference between the thresholds in patients with HFS and the normalization in threshold after MVD suggested that the mechanism of HFS was hyperexcitability of the facial nucleus.     

Systematic screening for diabetic eye disease in insulin dependent diabetes

It has been demonstrated that alveolar macrophages (AM) are permissive for human immunodeficiency virus (HIV-1) after in vitro infection. However, data concerning in vivo infection of AM by HIV-1 still conflict. Therefore, we investigated AM collected by bronchoalveolar lavage from 15 HIV-1-infected patients. HIV-1 p24 and Gp120 antigens and viral RNA were not detected by immunocytochemistry and in situ hybridization, respectively, using 35S-labeled 3 kb Pol-Env, 0.350 kb Gag, and 0.150 kb U5 LTR cRNA probes. In contrast, when using polymerase chain reaction on DNA extracted from purified AM, HIV-1 DNA was detected in the seven patients tested. After short-term culture of alveolar cells from three HIV-1-infected patients and in vitro stimulation with granulocyte/macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor-alpha (TNF-alpha), HIV-1 replication was observed in most of the AM. These results demonstrate that AM are latently infected by HIV-1 in vivo but are not a site for viral replication. In contrast, HIV-1 replication occurs when AM are withdrawn from their local environment, enhanced by GM-CSF and TNF-alpha stimulation. This suggests either a negative control or an inadequate stimulation of HIV-1 replication in the alveolar environment.     

Detection of sight-threatening diabetic eye disease

Blindness due to diabetes mellitus is potentially preventable in the majority of patient. Early detection of sight-threatening changes is associated with a better outcome, indicating the need to screen for retinopathy. At least 50% of diabetic patients do not attend a hospital, so that diabetologists and ophthalmologists are unable to screen the diabetic population comprehensively. Although in theory all patients has access to general practitioners, these may lack training or confidence to screen for retinopathy. Hospital based or community optometrists using direct ophthalmoscopy or slit lamps and technicians performing fundus photography are alternatives which may be more effective. Further studies are required to examine the effectiveness of optometry screening. Initial studies using fundus photography raised concerns about the sensitivity of the technique, but these have been partially addressed by improvements in methodology and technology. As well as technicological effectiveness, factors affecting patient uptake of screening services still need to be addressed.     

Natural history of thyroid eye disease

During antibody screening of a Taenia saginata oncosphere cDNA library a clone (R-Tso2) sharing a high degree of homology at both the DNA and amino acid levels with the small heat-shock protein (shsp) family was identified. The R-Tso2 clone was a full-length sequence (1162 bp) with an open reading frame of 945 bp and 314 amino acids, corresponding to a deduced molecular mass of 35.6 kDa and isoelectric point of 5.6. R-Tso2 had the highest degree of homology with the Schistosoma mansoni major egg antigens, showing the characteristic shsp 100 amino-acid sequence motif duplicated. The R-Tso2 expression product was not immuno-precipitated by any serum from a panel of serum samples obtained from bovine, porcine and human hosts suffering from either T. saginata or T. solium cysticercosis.     

Psychopharmacological drugs and the eye (author's transl)

Early and late side-effects of psychopharmacological drugs on the eyes are discussed. The action of antidepressants, neuroleptics, tranquilizers and other groups on the eye are discussed separatedly. The importance of early glaucoma detection and treatment as well as that of pigment and metabolites deposits in the eye tissues is emphasized.     

Propanediol utilization genes (pdu) of Salmonella typhimurium: three genes for the propanediol dehydratase

The propanediol utilization (pdu) operon of Salmonella typhimurium encodes proteins required for the catabolism of propanediol, including a coenzyme B12-dependent propanediol dehydratase. A clone that expresses propanediol dehydratase activity was isolated from a Salmonella genomic library. DNA sequence analysis showed that the clone included part of the pduF gene, the pduABCDE genes, and a long partial open reading frame (ORF1). The clone included 3.9 kbp of pdu DNA which had not been previously sequenced. Complementation and expression studies with subclones constructed via PCR showed that three genes (pduCDE) are necessary and sufficient for propanediol dehydratase activity. The function of ORF1 was not determined. Analyses showed that the S. typhimurium propanediol dehydratase was related to coenzyme B12-dependent glycerol dehydratases from Citrobacter freundii and Klebsiella pneumoniae. Unexpectedly, the S. typhimurium propanediol dehydratase was found to be 98% identical in amino acid sequence to the Klebsiella oxytoca propanediol dehydratase; this is a much higher identity than expected, given the relationship between these organisms. DNA sequence analyses also supported previous studies indicating that the pdu operon was inherited along with the adjacent cobalamin biosynthesis operon by a single horizontal gene transfer.     

Phenol sulfotransferases: candidate genes for Batten disease

Batten disease (juvenile-onset neuronal ceroid lipofuscinosis; JNCL) is an autosomal recessive neurodegenerative disorder, characterized by the cytosomal accumulation of autofluorescent proteolipopigments in neurons and other cell types. The Batten disease gene (CLN3) has not yet been identified, but has been mapped to a small region of human chromosome area 16p12.1-p11.2. We recently reported the fortuitous discovery that the cytosolic phenol sulfotransferase gene (STP) is located within this same interval of chromosome 16p. Since phenol sulfotransferase is expressed in neurons, can sulfate lipophilic phenolic compounds, and is mapped near CLN3, STP is considered as a candidate gene for Batten disease. YAC and cosmid cloning results have further substantiated the close proximity of STP and a highly related sulfotransferase (STM), encoding the catecholamine-preferring enzyme, to the CLN3 region of chromosome 16p. In this report, we summarize some of the recent progress in the identification of two phenol sulfotransferase genes (STP and STM) as positional candidate genes for Batten disease.     

Mutation analysis using the restriction site mutation (RSM) assay

Although two decades of research suggests that the hippocampus plays a special role in place learning, the present paper describes a series of studies using swimming pool spatial tasks that show that hippocampal rats have considerable place learning ability, which includes the abilities of finding, remembering, and searching for places. The same studies also show that when environmental cues are uninformative, as is the case early in original learning and again in reversal learning, hippocampal rats are impaired. Since control rats quickly resolve spatial ambiguity in these situations, it is argued that they must have a system with which they can calibrate spatial cues. The discussion considers the possibility that they use dead reckoning with path integration, a spatial strategy that provides guidance based on cues generated by a point of reference and subsequent self-movement and not the cues in the environment through which they are moving. With path integration an animal can monitor its location and at the same time attach spatial meaning to cues that it encounters. An ability to recalibrate external cues may provide the tuning that allows control rats to quickly acquire place responses while hippocampal rats are constrained by the processes of associative learning.     

Rehabilitation after serious perforating eye injuries (author''s transl)

In a recent study of perforating eye injuries the authors found that in 21% there was no binocular function. To prevent complications arising from this, early fitting of hard contact lenses seems necessary. The problems of fitting contact lenses to posttraumatically aphakic eyes are discussed, with special regard to aniseikonia.     

The distirubtion of active genes (globin) and inactive genes (keratin) in fractionated chicken erythroid chromatin

Repeatable fractionation of sheared chromatin from purified populations of chicken erythroid cells has been achieved, based on the Bio-Gel procedure of Janowski et al. ((1972) Karolinska Symp. 5, 112). For reticulocytes, 3-5% of chromatin DNA is excluded from Bio-Gel A-50 m (peak I) and over 90% elutes in the included volume of the column(peak II). Peak I material has a higher protein/DNA ratio than peak II chromatin and the two fractions have melting profiles characteristic of "active" and "inactive" chromatin, respectively. In cells prelabeled with [3H]uridine or [3H]leucine there was very pronounced preferential association of radioactivity with the "active" peak I chromatin. The distribution of "active" (globin) and "inactive" (keratin) gene sequences in the DNA of fractions from peak I and peak II chromatin was determined with complimentary DNA (cDNA) probes to chicken globin mRNA and chicken feather keratin mRNA. While slight enrichment for globin gene sequences was found in peak I (relative to DNA in these fractions), some 80% of the total globin hybrid formed was found in peak II fractions. Experiments with the keratin cDNA probe showed that these genes were equally distributed in both chromatin fractions rather than being confined to the "inactive" peak II material. The hybridization data in particular question the validity of claims for fractionation of chromatin into "active" and "inactive" material.