Short communication: Epidemiological assessment of Strongyloides stercoralis in Fijian children.

As a part of the lymphatic filariasis (LF) transmission assessment survey (TAS)/soil-transmitted helminths (STH) prevalence survey in Western Division of Fiji, a pilot screen for Strongyloides stercoralis (SS) in school children was undertaken using a combination of the Baermann concentration (BC) method and real-time PCR assays. Using BC, faecal samples collected from 111 children of 7 schools were examined. A single child was positive for larvae of SS and underwent a clinical examination finding an asymptomatic infection. Other members of this child's household were screened with BC, finding none infected. Aliquots of 173 faecal samples preserved in ethanol originating from all schools were examined by real-time PCR, and the prevalence of SS infection was 3.5%. Our study confirms the existence of SS infection on Fiji and showed that assessing SS prevalence alongside TAS/STH survey is a convenient access platform, allowing introduction of other surveillance techniques such as BC and real-time PCR.

Manipulation of a quasi-natural cell block for high-efficiency transplantation of adherent somatic cells

Recent advances have raised hope that transplantation of adherent somatic cells could provide dramatic new therapies for various diseases. However, current methods for transplanting adherent somatic cells are not efficient enough for therapeutic applications. Here, we report the development of a novel method to generate quasi-natural cell blocks for high-efficiency transplantation of adherent somatic cells. The blocks were created by providing a unique environment in which cultured cells generated their own extracellular matrix. Initially, stromal cells isolated from mice were expanded in vitro in liquid cell culture medium followed by transferring the cells into a hydrogel shell. After incubation for 1 day with mechanical agitation, the encapsulated cell mass was perforated with a thin needle and then incubated for an additional 6 days to form a quasi-natural cell block. Allograft transplantation of the cell block into C57BL/6 mice resulted in perfect adaptation of the allograft and complete integration into the tissue of the recipient. This method could be widely applied for repairing damaged cells or tissues, stem cell transplantation, ex vivo gene therapy, or plastic surgery.

Manipulation of a quasi-natural cell block for high-efficiency transplantation of adherent somatic cells.

Recent advances have raised hope that transplantation of adherent somatic cells could provide dramatic new therapies for various diseases. However, current methods for transplanting adherent somatic cells are not efficient enough for therapeutic applications. Here, we report the development of a novel method to generate quasi-natural cell blocks for high-efficiency transplantation of adherent somatic cells. The blocks were created by providing a unique environment in which cultured cells generated their own extracellular matrix. Initially, stromal cells isolated from mice were expanded in vitro in liquid cell culture medium followed by transferring the cells into a hydrogel shell. After incubation for 1 day with mechanical agitation, the encapsulated cell mass was perforated with a thin needle and then incubated for an additional 6 days to form a quasi-natural cell block. Allograft transplantation of the cell block into C57BL/6 mice resulted in perfect adaptation of the allograft and complete integration into the tissue of the recipient. This method could be widely applied for repairing damaged cells or tissues, stem cell transplantation, ex vivo gene therapy, or plastic surgery.

Crumbs interacts with Xpd for nuclear division control in Drosophila.

Crumbs (Crb) family proteins are crucial for cell polarity. Recent studies indicate that they are also involved in growth regulation and cancer. However, it is not well-understood how Crb participates in mitotic processes. Here, we report that Drosophila Crb is critically involved in nuclear division by interacting with Xeroderma pigmentosum D (XPD). A novel gene named galla-1 was identified from a genetic screen for crb modifiers. Galla-1 protein shows homology to MIP18, a subunit of the mitotic spindle-associated MMS19-XPD complex. Loss-of-function galla-1 mutants show abnormal chromosome segregation, defective centrosome positions and branched spindles during nuclear division in early embryos. Embryos with loss-of-function or overexpression of crb show similar mitotic defects and genetic interaction with galla-1. Both Galla-1 and Crb proteins show overlapping localization with spindle microtubules during nuclear division. Galla-1 physically interacts with the intracellular domain of Crb. Interestingly, Galla-1 shows little binding to the Drosophila homolog of XPD, but a related protein Galla-2 binds both Crb and Xpd. Loss-of-function galla-2 mutants show similar mitotic defects as galla-1 and strong genetic interaction with crb. Xpd can form a physical complex with Crb. In imaginal disc, Crb overexpression causes tissue overgrowth as well as DNA damages marked by H2Av phosphorylation. These phenotypes are suppressed by reduction of Xpd. Taken together, this study identifies a novel Crb-Galla-Xpd complex and its function for proper chromosome segregation during nuclear division, implicating a potential link between Crb and Xpd-related genome instability.

Minimum-intensity projection for in-depth morphology study of mesiobuccal root.

OBJECTIVE: This micro-computed tomography (MCT) study investigated the utility of thin-slab minimum-intensity projection (TS-MinIP) technique as an adjunct to 3-dimensional (3D) modeling for in-depth morphology study. STUDY DESIGN: One hundred one extracted maxillary first molars were scanned for microtomographic analysis (SkyScan). Two-dimensional TS-MinIP and 3D images of mesiobuccal (MB) roots were produced and analyzed to record the number and configurations of the canals, the incidence and location of accessory canals, loop, and intercanal connections, and number of foramina. RESULTS: Multiple-canal MB roots were present in 76.2%, and all of the roots had intercanal communications. Weine type III configuration was the most common in the multiple-canal roots. Accessory canals were found in 78.2% of the roots. Configurations that were nonclassifiable were found in 10.9% of the MB roots. CONCLUSIONS: MB root canal anatomy was complex, and MinIP may serve as an adjunct to 3D modeling for in-depth morphology study.

Clonorchis sinensis infection is positively associated with atopy in endemic area.

BACKGROUND: Epidemiologic studies have suggested that helminth infections play a protective role against allergy; this inverse association, however, has not been consistent. Clonorchis sinensis, the liver fluke of human, is prevalent in the Far East. The association between C. sinensis infection and allergy has not yet been reported. OBJECTIVE: We evaluated the association between clonorchiasis and atopy or allergic diseases in adults in endemic areas of clonorchiasis. METHODS: A total of 1116 subjects (males 419, females 697; age range, 30-86; mean age=61 years) were recruited from two endemic areas of C. sinensis in Korea. Clonorchiasis was confirmed by stool examination. Allergic symptoms were evaluated with a modified ISAAC questionnaire, and atopy was defined by skin prick test for common inhalant allergens. Total serum IgE and C. sinensis-specific IgE level was measured by ELISA and methacholine bronchial provocation test was performed to evaluate airway hyperresponsiveness (AHR). RESULTS: Clonorchiasis was positively associated with atopy [odds ratio (OR), 1.856; 95% confidence interval (CI), 1.199-2.873] and high levels of total serum IgE (OR, 1.455; 95% CI, 1.050-2.016). Higher association with clonorchiasis was shown in subjects who showed both atopy and high total serum IgE levels (OR, 2.540; 95% CI, 1.448-4.455). Clonorchiasis had no association with wheezing, AHR, asthma or allergic rhinitis. CONCLUSION AND CLINICAL RELEVANCE: Clonorchiasis was positively associated with atopy in adults in endemic area.

Detection of Clonorchis sinensis in stool samples using real-time PCR.

Human clonorchiasis, caused by infection with the trematode Clonorchis sinensis, is a common health problem in East Asia. In an attempt to develop a new, sensitive method for the diagnosis of the disease, the use of a real-time PCR (targeting the internal-transcribed-spacer-2 sequence of the parasite) to detect C. sinensis-specific DNA in faecal samples has recently been evaluated. The PCR-based assay, which included an internal control to detect any inhibition of the amplification by faecal constituents in the sample, was performed on stool samples and on DNA controls representing a wide range of intestinal microorganisms. The assay appeared very specific, only showing positivity with C. sinensis and Opisthorchis felineus. The sensitivity of the assay was explored by testing 170 preselected samples of human faeces, from an endemic area of South Korea, which had known (microscopically-determined) densities of C. sinensis eggs. The sensitivity of the assay was 100% for the 74 samples that each had > 100 eggs/g and 91.4% for the other 70 samples found egg-positive by microcopy (i.e. those that had

Specific hippocampal choline decrease in an animal model of depression.

A decreased level of the hippocampal choline signal was found in patients with depression in previous proton magnetic resonance spectroscopy ((1)H-MRS) studies. The objective of this study is to compare choline levels before and after the forced swimming test (FST), an animal model of depression typically used for assessing antidepressant activity. (1)H-MRS spectra were obtained from both the left and right hippocampus. After the FST, rats showed a significant decrease of the choline/creatine (Cho/Cr, p = 0.037) and choline/N-acetylaspartate (Cho/NAA, p = 0.048) ratios in the left hippocampus, but not in the right hippocampus. This finding was analogous to results from patients with depression. It suggests that decreased Cho/Cr and Cho/NAA ratios in the left hippocampal regions might be considered to be biomarkers in rats with depression.

Oral administration of pravastatin reduces egg cholesterol but not plasma cholesterol in laying hens.

The effect of different 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGR)-inhibitors (statins) on plasma and egg cholesterol in laying hens was investigated. Forty-four ISA brown layers were fed lovastatin, simvastatin, or pravastatin at 0.03 or 0.06% for 4 wk. Cholesterol levels in plasma, liver, and egg yolk as well as hen laying performance were studied. Egg weight was significantly lowered with all statin treatments, although egg production was relatively unaffected. Total plasma cholesterol was significantly reduced by 0.06% lovastatin, 0.03% simvastatin, and 0.06% simvastatin, in agreement with previous reports, whereas pravastatin had no effect. In contrast, liver cholesterol concentrations showed a significant decrease in response to 0.03 and 0.06% pravastatin, implying selective regulation of liver cholesterol synthesis. Furthermore, oral administration with 0.06% pravastatin reduced egg cholesterol levels by almost 20% compared with the control diet. This suggests that pravastatin, unlike other classes of statin, may be a good candidate for commercial production of low cholesterol eggs with limited impact on hen physiology and egg production.

Clonorchis sinensis: molecular cloning and characterization of 28-kDa glutathione S-transferase.

A 28-kDa glutathione S-transferase (Cs28GST) was purified from a Clonorchis sinensis cytosolic fraction through anion-exchange and glutathione-affinity column chromatographies. A monoclonal antibody raised against Cs28GST reacted specifically to the C. sinensis antigen among trematode proteins. A putative peptide of 212 amino residues deduced from a cDNA clone appeared homologous with 28-kDa GST of trematodes, and its secondary structural elements predicted a GSH-binding site. Recombinant Cs28GST showed GST enzyme activity with CDNB substrate and was sensitive to the model inhibitors. The recombinant Cs28GST was antigenically indistinguishable from the native form and was recognized specifically by C. sinensis-infected human sera. The Cs28GST was localized in the tegument and underlying mesenchymal tissues. It is suggested that Cs28GST may play significant physiological roles against bioreactive molecules and be a useful reagent for serodiagnosis of clonorchiasis.

Molecular cloning of the anther-preferential nonspecific lipid transfer protein cDNA in hot pepper by mRNA differential display.

To better understand the molecular control of anther development, an anther-preferential mRNA was isolated from hot pepper (Capsicum annuum) using mRNA differentially display. Using the displayed fragment as a probe, a full-length cDNA named CaLTP was isolated. A nucleotide sequence analysis of CaLTP revealed that the clone contains an open reading frame of 123 amino acids, which exhibits a 60-23% identity with nonspecific lipid transfer proteins (nsLTP). Northern and RT-PCR analysis of the clone confirmed that CaLTP mRNA was predominant to anther tissues. The basal expression level in the leaves was slightly induced only by abscisic acid (ABA) treatment. Southern analysis reveals that CaLTP is present as a single-copy gene in hot pepper genome. We hypothesize that CaLTP might have an important role in protecting the reproductive tissues from environmental stresses.

Localization of cytoskeletal proteins in Pneumocystis carinii by immuno-electron microscopy.

Pneumocystis carinii causes serious pulmonary infection in immunosuppressed patients. This study was undertaken to observe the cytoskeletal proteins of P. carinii by immuno-electron microscopy. P. carinii infection was experimentally induced by immunosuppression of Sprague-Dawley rats for seven weeks, and their lungs were used for the observations of this study. The gold particles localized actin, tropomyosin, and tubulin. The actin was irregularly scattered in the cytoplasm of the trophic forms but was much more concentrated in the inner space of the cell wall of the cystic forms called the inner electron-lucent layer. No significant amount of tropomyosin was observed in either trophic forms or cystic forms. The tubulin was distributed along the peripheral cytoplasm and filopodia of both the trophic and cystic forms rather than in the inner side of the cytoplasm. Particularly, in the cystic forms, the amount of tubulin was increased and located mainly in the inner electron-lucent layer of the cell wall where the actin was concentrated as well. The results of this study showed that the cell wall of P. carinii cystic forms is a structure whose inner side is rich in actin and tubulin. The location of the actin and tubulin in P. carinii suggests that the main role of these proteins is an involvement in the protection of cystic forms from the outside environment by maintaining rigidity of the cystic forms.

Control of clonorchiasis by repeated treatments with praziquantel.

The present study aimed to evaluate control efficacy of clonorchiasis by two schemes of repeated treatment with praziquantel at two endemic villages in China. Residents of one village at Guangxi Autonomous Region were treated and examined 6-monthly and of another at Liaoning Province 12-monthly. In residents that took 25 mg/kg x 3 (total 75 mg/kg) of praziquantel every 6 months for one year the egg positive rate showed a significant drop from 69.0% to 17.1%. In contrast, a group of same praziquantel medication once showed a slight marginal decrease in the egg rate from 18.9% to 12.2% after one year. Of 39 subjects examined 3 times, 56.4% were cured, 7.7% persistently positive, one (2.6%) reinfected after cure or newly infected, but 25.6% were persistently negative. The present finding suggests that 6-monthly medication with 75 mg/kg of praziquantel should effectively lower the prevalence but incomplete for control of clonorchiasis in heavy endemic areas.

Purification of a 68-kDa cysteine proteinase from crude extract of Pneumocystis carinii.

The present study intended to verify activities of cysteine proteinase of Pneumocystis carinii from rats and to purify the enzyme. In order to exclude the contamination of host-derived enzymes, concentrates of P. carinii was primarily treated with a mixture of proteinase inhibitors before lysis of P. carinii. A 68-kDa cysteine proteinase was finally purified from the crude extract of P. carinii by 4 sequential chromatographic methods. The enzyme showed an optimal activity at pH 5.5 in 0.1 M sodium acetate, and its activity was specifically inhibited by L-trans-epoxy-succinylleucylamido (4-guanidino) butane (E-64) and iodoacetic acid, suggesting that the enzyme is a cysteine proteinase. The 68-kDa proteinase weakly digested macromolecules such as collagen, hemoglobin and fibronectin. The present study demonstrated the activity of cysteine proteinase at the 68-kDa band of P. carinii, and purified and characterized the molecule.

Genetic heterogeneity of Pneumocystis carinii from rats of several regions and strains.

Pneumocystis carinii is a major opportunistic pathogen which has been found in the lungs of a wide variety of mammalian host species, and the fact suggests the possibility of intraspecific variation. Until now, P. carinii from different mammalian species are differentiated as subspecies, and the rats are known to be infected by two subspecies. The present study investigated genetic heterogeneity of P. carinii isolates from two strains of rats in Korea and China by molecular karyotyping, RFLP and sequencing analysis. Karyotypes of P. carinii were grouped into three, two from two strains of rats in Korea and one from rats in China. However RFLP of PCR product of ribosomal and MSG gene of the P. carinii isolates showed same pattern. The sequence homology rates of alpha-tubulin DNA of the P. carinii isolates were 96% in Seoul Wistar rats, 93% in Seoul Sprague-Dawley rats, and 85% in Chinese Sprague-Dawley rats. The present finding confirmed that P. carinii from rats in Korea are grouped into two karyotype strains which are different from that of P. carinii from rats in China. The Chinese isolate shows a little different sequences of alpha-tubulin DNA.

Partial characterization of a 17 kDa protein of Clonorchis sinensis.

A 17 kDa protein from Clonorchis sinensis adults was purified by a procedure including Sephacryl S-200 HR gel filtration and Q-Sepharose anion exchange chromatography. The protein was proved to be a cysteine protease as it showed hydrolytic activity toward Cbz-Phe-Arg-AMC in the presence of dithiothreitol and was inhibited by specific inhibitors such as iodoacetic acid or trans epoxy-succinly-L-leucyl-amido(4-guanidino) butane. The polyclonal antibody raised against the protein reacted to 17 kDa proteins of trematodes such as Paragonimus westermani, Fasciola hepatica, Opisthorchis viverrini, Gymnophalloides seoi, and Metagonimus yokogawai. The antibody recognized the 17 kDa and 16 kDa cysteine proteases purified from C. sinensis, P. westermani, and G. seoi as well. These results suggest that the 17 kDa protein may be a cysteine protease commonly present in trematodes.

La48Br81Os8: isolated clusters in an unusual superstructure with significantly greater intercluster bonding.

Exploration of reactions in the La-Br-Z system for Z = Fe, Ru, and Os in welded Nb containers at 900-950 degrees C resulted in only the title phase. The La48Br81Os8 stoichiometry is very close to that of known triclinic Pr6Br10Os but with an approximately 32-times larger cell, 138 independent atoms, and completely different intercluster connectivities in a complex monoclinic superstructure (a = 33.076(5) A, b = 23.466(3) A, c = 23.537(2) A, beta = 110.701(4) degrees, P2(1)/c (No. 14), Z = 4, 23 degrees C). Tetragonally compressed, approximately 16 e- lanthanum octahedra centered by Os are heavily interbridged by Br, including Br(f-a) (f = face) and Br(i-a-a) functions, to increase coordination numbers about some Br (to 4) and La (to 6) and to give an average of 19.63 bonded Br/La6Os vs the usual 18. These result in a cell volume 10% less than for an equivalent (hypothetical) La6Br10Os and Br-Br contacts as short as 3.30 A. Increased polar La-Br interactions presumably drive these changes. Optimal atom sizes for this structure have been found so far only in this novel compound.

Is Pneumocystis carinii vertically transmitted to neonatal rats?

Pneumocystis carinii is a pulmonary pathogen of immunocompromised humans or other mammals. Its infection results from activation of organisms involved in latent infection or from new infection through the air. Almost all children are known to be infected within 2 to 4 years of birth, though prenatal transplacental transmission has not yet been demonstrated. In this study we observed experimental P. carinii infection in neonatal rats, thus investigating the possibility of transplacental vertical transmission by Diff-Quik staining of the lung impression smears and in-situ hybridization for lung sections. The positive rate of P. carinii infection in immunosuppressed maternal rats was 100%, but that in normal maternal rats was 0%. Cystic forms of P. carinii were observed in three of six 1-week old neonatal rats born of heavily infected mothers, but none of them was positive by in-situ hybridization. Five weeks after birth, cystic forms were detected in four neonatal rats. In the lobes of the lungs, no predilection site of P. carinii was recognized. Counts of cystic forms on smears and the reactivity of in-situ hybridization in the lungs of neonatal rats were significantly lower than in maternal rats. The present findings suggest that P. carinii is rarely transmitted through the placenta and proliferates less successfully in the lungs of neonatal rats than in mothers.