Verification of the folkloric and anecdotal antidiabetic effects of Hypoxis hemerocallidea (Fisch., C.A. Mey. & Avé-Lall) and isolated, ß-sitosterol using early-stage type II spontaneous diabetic mutant BKS-Leprdb mice.

BACKGROUND: Previous studies in our laboratory in ex vivo assays have demonstrated H. hemerocallidea extract as potential antidiabetic agent through increased insulin release from pancreatic beta cells. Thus, for this study the early stage type II spontaneous diabetic mutant mice model was used to evaluate and determine the degree of the antidiabetic efficacy of H. hemerocallidea. METHODS: Eight-weeks-old type II spontaneous pre-diabetic mutant BKS-Leprdb mice were fed with feed supplemented with either H. hemerocallidea extract, isolated compound (ß-sitosterol) or chlorpropamide (positive control) for 4 weeks. The haematological parameters, clinical chemistry, glucose tolerance, feed intake, faecal output and body weights were measured. RESULTS: The blood glucose concentrations of all the animals treated with plant extract, ß-sitosterol compound and non-treated pre-diabetic animals did not return to baseline levels. Only the ß-sitosterol treatment and positive control groups resulted in a respective small decrease of 5.8 and 5.2% in the mouse weights over the study period, with no significant changes (p > 0.05) in food intake. However, there was a general trend for decrease in faecal output for all the groups. Albumin, triglycerides, and total cholesterol levels in ß-sitosterol and chlorpropamide-treated animals were lower, relative to untreated-animals. Animals fed with plant extract showed large amounts of internal fat. There were no significant changes (p > 0.05) in total serum protein, globulin, alanine aminotransferase, alkaline phosphatase, urea nitrogen and creatinine attributed to administration of treatments. In all groups, some animals showed lesions associated with cardiac puncture. Few animals except animals treated with plant extract, showed presence of a left-ventricular hypertrophic cardiomyopathy. The liver and kidneys for all groups appeared macroscopically normal and the thymuses were small (±2 mg). There were pathological signs in some of the animals particularly in myocardial fibres, renal tubular, glomerular, hepatocyte granularity and pancreas islets. However, there was no significance trend between the groups. CONCLUSION: Based on the results, none of the treatments could be considered highly effective for the management of type II pre-diabetes as sole therapeutic intervention.

Morphology of Emoleptalea Nwanedi n. Sp. from Schilbe Intermedius from Nwanedi-Luphephe Dam, Limpopo Province, South Africa.

A new species, Emoleptalea nwanedi n. sp. is described from the intestine of Schilbe intermedius, the silver catfish or butter barbel, from the Nwanedi-Luphephe Dam in the Limpopo Province of South Africa. Fish were collected using gill nets where after they were euthanised and dissected. The parasites were sampled, fixed in 70 % EtOH and stained with Van Cleave's haematoxylin. This species represents an addition to the African cluster of Emoleptalea species previously described and differs from the known species due to its unique size, equal size of oral and ventral suckers, position of ovary and seminal receptacle, number of vitelline follicles and their size, as well as the unique ciliated receptors on the wall of the acetabulum. This is the first record of this parasite from the silver catfish and from southern Africa.

Cercariae developing in Lymnaea natalensis Krauss, 1848 collected in the vicinity of Pretoria, Gauteng Province, South Africa.

Freshwater snails are known to serve as first intermediate hosts for various parasitic diseases such as schistosomosis and fasciolosis. Snails were collected on several occasions in the proximity of Pretoria, South Africa and their cercarial sheddings were studied. This article describes three different types of cercariae shed by the freshwater snail, Lymnaea natalensis, viz. a fork-tailed cercaria of a Trichobilharzia sp., an avian parasite belonging to the family Schistosomatidae, an echinostomatid cercaria of the family Echinostomatidae, also avian parasites and a xiphidiocercaria of the family Plagiorchiidae which parasitise avians and amphibians. The morphology of these cercariae was studied by light and scanning electron microscopy.

Altered expression of the mRNA stability factor HuR promotes cyclooxygenase-2 expression in colon cancer cells.

Cyclooxygenase-2 (COX-2) expression is normally tightly regulated. However, constitutive overexpression plays a key role in colon carcinogenesis. To understand the molecular nature of enhanced COX-2 expression detected in colon cancer, we examined the ability of the AU-rich element-containing (ARE-containing) 3' untranslated region (3'UTR) of COX-2 mRNA to regulate rapid mRNA decay in human colon cancer cells. In tumor cells displaying enhanced growth and tumorigenicity that is correlated with elevated COX-2, vascular endothelial growth factor (VEGF), and IL-8 protein levels, the corresponding mRNAs were transcribed constitutively and turned over slowly. The observed mRNA stabilization is owing to defective recognition of class II-type AREs present within the COX-2, VEGF, and IL-8 3'UTRs; c-myc mRNA, containing a class I ARE decayed rapidly in the same cells. Correlating with cellular defects in mRNA stability, the RNA-binding of trans-acting cellular factors was altered. In particular, we found that the RNA-stability factor HuR binds to the COX-2 ARE, and overexpression of HuR, as detected in tumors, results in elevated expression of COX-2, VEGF, and IL-8. These findings demonstrate the functional significance rapid mRNA decay plays in controlling gene expression and show that dysregulation of these trans-acting factors can lead to overexpression of COX-2 and other angiogenic proteins, as detected in neoplasia.

ATP reorients the neck linker of kinesin in two sequential steps.

Recent models of the kinesin mechanochemical cycle provide some conflicting information on how the neck linker contributes to movement. Some spectroscopic approaches suggest a nucleotide-induced order-to-disorder transition in the neck linker. However, cryoelectron microscopic imaging suggests instead that nucleotide alters the orientation of the neck linker when docked on the microtubule surface. Furthermore, since these studies utilized transition state or non-hydrolyzable nucleotide analogs, it is not clear at what point in the ATPase cycle this reorientation of the neck linker occurs. We have addressed this issue by developing a strategy to examine the effect of nucleotide on the orientation of the neck linker based on the technique of fluorescence resonance energy transfer. Transient kinetic studies utilizing this approach support a model in which ATP binding leads to two sequential isomerizations, the second of which reorients the neck linker in relation to the microtubule surface.

HuR, a RNA stability factor, is expressed in malignant brain tumors and binds to adenine- and uridine-rich elements within the 3' untranslated regions of cytokine and angiogenic factor mRNAs.

Tumors of the central nervous system (CNS) often have sustained expression of labile genes, including angiogenic growth factors and immunosuppressive cytokines, which promote tumor progression. Stabilization of the RNA transcripts for these genes, such as vascular endothelial growth factor (VEGF), is an important molecular pathway for this up-regulation. HuR, a member of the Elav family of RNA-binding proteins, has been implicated in this pathway through its binding to adenine and uridine (AU)-rich stability elements (ARE) located in the 3' untranslated regions (3'-UTRs) of the mRNA. Whereas three of the Elav family members (Hel-N1, HuC, and HuD) are restricted to young and mature neurons, HuR is more broadly expressed, including proliferating cells of the developing CNS. Because RNA stabilization of labile genes may promote tumor growth, we analyzed and compared the expression pattern of HuR in 35 freshly resected and cultured CNS tumors to determine whether there was any correlation with tumor grade or histological type. We found that HuR mRNA was consistently expressed in all of the tumors, regardless of cell origin or degree of malignancy. Using a novel HuR-specific polyclonal antibody, we found that strong HuR protein expression was limited to high-grade malignancies (glioblastoma multiforme and medulloblastoma). Within the glioblastoma multiforme, prominent HuR expression was also detected in perinecrotic areas in which angiogenic growth factors are up-regulated. To further define its role as a potential RNA stabilizer, we analyzed whether HuR could bind to the stability motifs within the 3'-UTRs of cytokines and growth factors linked to brain tumor progression. We used a novel ELISA-based RNA binding assay and focused on the 3'-UTRs of angiogenic factors VEGF, COX-2, and (interleukin) IL-8 as well as the immunomodulating factors IL-6, transforming growth factor (TGF)-beta and tumor necrosis factor (TNF)-alpha as potential RNA ligands. Our results indicated overall a very high binding affinity to these RNA targets. A comparison of these ligands revealed a hierarchy of binding affinities with the angiogenic factors, and TGF-beta showing the highest (Kd of 1.8-3.4 nM), and TNF-alpha the lowest (Kd of 18.3 nM). The expression pattern of HuR, coupled with the RNA binding data, strongly suggests a role for this protein in the posttranscriptional regulation of these genes in CNS tumors.

Morphology and life history of Petasiger variospinosus (trematoda: echinostomatidae) in the Free State, South Africa.

Specimens of the freshwater snail Bulinus tropicus (Krauss, 1848) collected in the Free State, South Africa shed cercariae with an oral collar bearing 27 spines. Tadpoles of the African clawed toad Xenopus laevis laevis Daudin, 1802 collected from the same waters harbored metacercariae with a similar collar of spines. Adults were obtained after feeding infected tadpoles to laboratory-reared reed cormorants, Phalacrocorax africanus (Gmelin, 1789). The parasite was identified as Petasiger variospinosus (Odhner, 1910), the life cycle was experimentally completed, and stages described by the use of light and scanning electron microscopy.

Analysis of the 5' end of the mouse Elavl1 (mHuA) gene reveals a transcriptional regulatory element and evidence for conserved genomic organization.

mHuA (Elavl1) belongs to a highly conserved family of genes encoding RNA-binding proteins and has been linked to cell growth and proliferation through its regulation of mRNA stability. Here, we use an RNase protection assay to demonstrate that the mHuA transcript is relatively abundant in a range of mouse tissues, with the highest levels being found in lung and embryonic stem cells. We then cloned and mapped an 18 kb DNA fragment which encompasses the 5' end of the mHuA gene. The genomic organization in this region is similar to the neural-restricted family members, Hel-N1 (ELAVL2) and mHuD (Elavl4). The first exon is lengthy and untranslated, and the second exon, which includes the methionine start site, ends between the ribonucleoprotein motifs of the first RNA binding domain. Mapping of the mHuA transcript by primer extension demonstrated three potential transcription-initiation sites which were detected consistently among different tissues and cell lines. Analysis of the sequence flanking these sites revealed the presence of transcriptional elements including TATA, CREB, c-ets, and AP1 sites. Transfection analysis of this promoter region using a luciferase-reporter-gene assay indicated strong transcriptional activity both in HeLa and in mouse macrophage (RAW) cells which is consistent with the ubiquitous expression pattern of mHuA. Thus, while the genomic organization of mHuA is similar to the neural-restricted members of the Elav family, the promoter element differs substantially both by sequence analysis and transcriptional activity in non-neural cell types.

RNA-binding analyses of HuC and HuD with the VEGF and c-myc 3'-untranslated regions using a novel ELISA-based assay.

Human members of the ELAV family, referred to as ELAV-like proteins (ELPs), include HuC, HuD, Hel-N1 and HuR. These proteins bind to AU-rich elements in the 3'-untranslated regions (3'-UTRs) of many growth-related mRNAs, including c-myc and VEGF, and may participate in regulating the stability of these transcripts. Here, I have developed an enzyme-linked immunosorbent assay (ELISA) which can rapidly assess the RNA-protein-binding properties of ELPs. With this assay, I demonstrate that HuC and HuD bind to the VEGF 3'-UTR regulatory segment (VRS) and to the c- myc 3'-UTR in a specific and concentration-dependent pattern, with both proteins showing a greater affinity for the VRS. Further analysis of the VRS indicated that the binding affinity was greater for the 3'-end where the majority of AU motifs reside. Binding to the VRS could be competed by both proteins as well as a poly(U) ribohomopolymer. The binding could not be competed by other ribohomopolymers or serum from patients with high titer anti-HuD antibodies. In summary, this assay provides a rapid analysis of ELP-RNA binding which can be utilized for further characterization of RNA-binding properties and for identification of competitor molecules for in vivo functional analysis of ELPs.

Representation and classification of breath sounds recorded in an intensive care setting using neural networks.

OBJECTIVE: Develop and test methods for representing and classifying breath sounds in an intensive care setting. METHODS: Breath sounds were recorded over the bronchial regions of the chest. The breath sounds were represented by their averaged power spectral density, summed into feature vectors across the frequency spectrum from 0 to 800 Hertz. The sounds were segmented by individual breath and each breath was divided into inspiratory and expiratory segments. Sounds were classified as normal or abnormal. Different back-propagation neural network configurations were evaluated. The number of input features, hidden units, and hidden layers were varied. RESULTS: 2127 individual breath sounds from the ICU patients and 321 breaths from training tapes were obtained. Best overall classification rate for the ICU breath sounds was 73% with 62% sensitivity and 85% specificity. Best overall classification rate for the training tapes was 91% with 87% sensitivity and 95% specificity. CONCLUSIONS: Long term monitoring of lung sounds is not feasible unless several barriers can be overcome. Several choices in signal representation and neural network design greatly improved the classification rates of breath sounds. The analysis of transmitted sounds from the trachea to the lung is suggested as an area for future study.

A proposed method for the measurement of anesthetist care variability.

OBJECTIVE: Some critical events in anesthesiology occur as seemingly preventable misadventures, their exact origins indeterminable. Inexperienced anesthetists, anesthesia machine malfunctions, lack of vigilance and human error inevitably initiate some incidents. Anesthesia training improves recognition and decision-making. Avoiding crisis initiation and amelioration of those that do occur is one role of the consultant anesthesiologist. Safe patient care requires medical and procedural knowledge, technical expertise, and control of resources in a complex milieu. Anesthesia simulators are clinical laboratories where anesthetists can sharpen both cognitive and manual skills. Dynamic scenarios allow opportunities for anesthetists to explore and experience crises as they develop and apply their knowledge while attempting to manage these events. Simulator-based scenarios are reproducible and large amounts of useful data can be collected and saved. The authors hypothesize these data can be utilized to compare performance of anesthetists and to measure improvement of individual anesthetists over time. METHODS: We have designed "Stable Anesthesia," a prototypic scenario to test anesthetists' capabilities under the stress of performance guidelines. Three subjects performed anesthesia using the simulator and this protocol. Data from the simulator were archived by the system and analyzed by the authors. RESULTS: A simple mathematical analysis gave good separation of data from three subjects of different training level. CONCLUSIONS: It is suggested that the use of the techniques mentioned here may be of value in the development of a standardized testing protocol for anesthetists.

Hu antigen specificities of ANNA-I autoantibodies in paraneoplastic neurological disease.

Despite a broad clinical spectrum, paraneoplastic enecephalomyelitis/sensory neuronopathy (PEM/SSN) is characterized by the presence of a common autoantibody, referred to as anti-Hu or type I anti-neuronal nuclear antibody (ANNA-1). The target of these antibodies is a family of four Hu antigens: three (Hel-N1, HuC, HuD) are neural-specific, while the fourth (HuR) is ubiquitous. Here, we have analysed by enzyme-linked immunosorbent assay (ELISA) the immunoreactivity of all four Hu antigens in serum from 75 patients with ANNA-1 autoantibodies and looked for clinical correlations. IgG in all the patients' sera bound to each of the four antigens, and the titers correlated with those of the ANNA-I immunofluorescence assay. Median titers for the neural-specific antigens (range: 56, 892-90,051) were significantly higher than for HuR (36,799). Patients with gastrointestinal dysmotility or subacute sensory neuronopathy had the highest median titers to all four antigens, while patients with sensorineural deafness had the lowest titers. The results indicate a heterogeneous immune response to individual Hu antigens in patients with PEM/SSN, and that the titers to these antigens as a group, rather than individually, correlate with clinical profile. Furthermore, these results suggest that ELISA analysis of a single neural-specific Hu antigen is sufficient for serological screening in PEM/SSN.

Charcot-Marie-Tooth phenotype produced by a duplicated PMP22 gene as part of a 17p trisomy-translocation to the X chromosome.

The Charcot-Marie-Tooth disease type 1A (CMT1A) phenotype is most often associated with a 1.5 megabase (mb), tandem duplication of chromosome 17 band p12 (17p12). The prevailing hypothesis is that the demyelinating neuropathy results from a dosage effect of the peripheral myelin protein gene PMP22 which is included within this duplication. We present a patient with clinical and electrophysiological features of CMT1A in whom an extra PMP22 gene resulted from a rare unbalanced translocation of 17p to the X chromosome. This finding further supports the hypothesis of gene dosage as the basis for CMT1A. Moreover, this case highlights the importance of fluorescence in situ hybridization (FISH) as an alternative molecular technique in the diagnosis of CMT1A.

Localization of HuC (ELAVL3) to chromosome 19p13.2 by fluorescence in situ hybridization utilizing a novel tyramide labeling technique.

HuC is a neural-specific member of the Elav family of RNA-binding proteins. This highly conserved gene family plays a crucial role in neurogenesis, and HuC (HGMW-approved symbol ELAVL3) is expressed at an early stage of neural development. Using a novel tyramide fluorescence in situ hybridization (T-FISH) technique, we localized HuC to chromosome 19p13.2. This localization was confirmed by radiation hybrid mapping and coincides with that of HuR (HGMW-approved symbol ELAVL1), another elav family member. Dual T-FISH analysis with HuC and HuR probes, however, indicated distinct loci, with HuC being centromeric to HuR. This study demonstrates the utility of T-FISH in colocalizing two genes on the same chromosomal preparation using only biotinylated probes.

HuR, a novel target of anti-Hu antibodies, is expressed in non-neural tissues.

Paraneoplastic encephalomyelitis (PEM) is characterized by a diverse set of clinical signs that are limited to the nervous system. The serologic hallmark of PEM is the presence of circulating autoantibodies, collectively referred to as 'anti-Hu,' which immunoreact specifically with members of the Elav protein family. Until recently, the ELAV antigens were only detected in neurons, thus strongly supporting a role for anti-Hu antibodies in the selective neural tissue injury in PEM. The identification of HuR, however, a new member with a broad, non-neural pattern of RNA expression, raises several fundamental questions regarding PEM. First, why are non-neural tissues spared in PEM? Second, why is PEM predominantly associated with neuroendocrine tumors? To begin addressing these questions, we sought to determine whether the antibody response to HuR differs from the neural-specific counterparts in patients with PEM, and to characterize the protein expression pattern of this novel antigen in peripheral tissues and tumors. Using sera from 11 patients with Hu-positive PEM, we found that the majority of samples (73%) were weakly or non-reactive for recombinant HuR on Western blot, in contrast to consistently strong immunoreactivity with the neural-specific members HuD and Hel-N1. We also demonstrate that HuR is expressed at the protein level in both non-neural tissues and non-neuroendocrine tumors. These findings suggest that immunoreactive differences among Elav family members may contribute to the neural-restrictive pattern of tissue injury in patients with PEM.

Differential expression of the neuroendocrine genes Hel-N1 and HuD in small-cell lung carcinoma: evidence for down-regulation of HuD in the variant phenotype.

Hel-NI and HuD belong to the elav gene family and have gained recent attention as potential neuroendocrine markers for small-cell lung carcinoma (SCLC). Members of this conserved family normally appear at different stages of neuronal maturation, raising the possibility that their expression patterns in SCLC reflect the degree of neuroendocrine differentiation. I have utilized a ribonuclease protection assay to analyze Hel-NI and HuD expression in cultured SCLC cells with high (classic phenotype) and low (variant phenotype) levels of neuroendocrine differentiation. Hel-NI was detected in both classic and variant SCLC. Although HuD was detected consistently in classic SCLC, it was low to absent in variant SCLC, indicating a significant down-regulation in that phenotype. The expression patterns of Hel-NI and HuD also were analyzed in 9 primary SCLC and 10 non-SCLC lung-tumor samples. In the majority of SCLC samples, either Hel-NI or HuD was detected exclusively or predominantly, indicating a pattern of variable gene expression similar to cultured SC LC. Neither transcript could be detected in the non-SCLC samples. These data indicate that (i) HuD mRNA expression is associated with a higher level of neuroendocrine differentiation in SCLC, (ii) Hel-NI and HuD expressions are variable in both primary and cultured SCLC and (iii) HuD and Hel-NI, in combination, are neurogenetic markers for SCLC.

Description of the adult and larval stages of Tylodelphys xenopi (Trematoda:Diplostomidae) from southern Africa.

The strigeoid metacercaria Diplostomulum xenopi is commonly found in the pericardial cavity of Xenopus laevis laevis. This paper provides the first description of the adult obtained from the intestine of an experimental host, the darter, Anhinga melanogaster. Natural cercarial infections were found in specimens of the freshwater snail Bulinus tropicus collected from dams in the Free State. South Africa. The life cycle was experimentally completed and all stages were described by light and scanning electron microscopy.

An expert-guided decision tree construction strategy: an application in knowledge discovery with medical databases.

With the steady growth in electronic patient records and clinical medical informatics systems, the data collected for routine clinical use have been accumulating at a dramatic rate. Inter-disciplinary research provides a new generation of computation tools in knowledge discovery and data management is in great demand. In this study, an expert-guided decision tree construction strategy is proposed to offer an user-oriented knowledge discovery environment. The strategy allows experts, based on their expertise and/or preference, to override inductive decision tree construction process. Moreover, by reviewing decision paths, experts could focus on subsets of data that may be clues to new findings, or simply contaminated cases.

Neuron-specific hel-N1 and HuD as novel molecular markers of neuroblastoma: a correlation of HuD messenger RNA levels with favorable prognostic features.

Hel-N1 and HuD belong to the elav gene family and encode neuron-specific RNA-binding proteins that are temporally regulated in neural development. Recently, these genes have been detected in small cell lung carcinoma, a neuroendocrine tumor, with HuD down-regulated in poorly differentiated, variant subsets. We, therefore, sought to determine: (a) the extent to which Hel-N1 and HuD are expressed in neuroblastoma (NB); and (b) whether the individual patterns of expression are associated with clinical features of the tumor. We used a sensitive and quantitative RNase protection assay that reliably distinguishes between these homologous genes, and with it we show that Hel-N1 and HuD transcripts were detected in 100% of cultured cells (11 of 11) and 97% of primary tumor samples (35 of 36). Densitometric quantification of transcripts indicated that the levels of HuD and Hel-N1 varied in all samples. In primary NB tissue, samples that expressed the highest Hel-N1 or HuD levels were N-myc unamplified. With HuD, the level in unamplified primary tumors was significantly higher than that of amplified tumors (0.80 +/- 0.12 versus 0.33 +/- 0.12, P < 0.02). HuD expression in prognostically favorable tumor stages was also significantly higher than unfavorable stages (0.98 +/- 0.19 versus 0.47 +/- 0.08, P < 0.03). In summary, the ubiquitous detection of HuD and Hel-N1 in NB indicates that they are molecular neuronal markers of this tumor. Furthermore, high HuD mRNA levels may predict a clinically favorable outcome.

Localization of human elav-like neuronal protein 1 (Hel-N1) on chromosome 9p21 by chromosome microdissection polymerase chain reaction and fluorescence in situ hybridization.

Hel-N1 is a member of the highly conserved elav family of neuronal genes. It shares considerable sequence homology with HuD, another human member, and both genes are expressed in brain. HuD was recently mapped to chromosome 1p34. Here, we have utilized chromosome microdissection polymerase chain reaction and fluorescence in situ hybridization to map Hel-N1 to chromosome 9p21. The different chromosomal locations of these homologous genes underscore their distinct identities.