Comparative study of PCR-based Cryptosporidium discriminating techniques with a review of the literature

We identified the species or genotypes of the six Cryptosporidium isolates from patients and C. parvum strain HNJ-1 using the seven previously described species-differentiation and genotyping PCR protocols for detection of Cryptosporidium parasites. In addition, we also discussed about the usefulness of these PCR-based protocols on the basis of the reports previously published. Cryptosporidium diagnostic fragment was amplified by PCR with each primer pair, targeting the 18S ribosomal RNA (18SrRNA), Cryptosporidium oocyst wall protein (COWP). Heat shock protein 70 (HSP70), Polythreonine (Poly-T), Thrombospondin related adhesive protein of Cryptosporidium-1 (TRAP-C1), and unknown gene locus, in all isolates from patients and the strain HNJ-1. The RFLP profiles of 18SrRNA, COWP, HSP70, Poly-T, and TRAP-C1 PCR products in all isolates from patients were found to be the same among isolates, and were correspondent to those of C. parvum human genotype. While the RFLP profiles of HNJ-1 were strictly different from those of isolates from patients, and were correspondent to C. parvum cattle genotype. In addition, nucleotide sequences in 18 SrRNA gene of all isolates from patients and HNJ-1 were found to be identical to that of C. parvum, human or cattle genotype, respectively. Therefore, the isolates from patients and HNJ-1 were identified as C. parvum human and cattle genotype, respectively. According to the reports related to the PCR-based protocols applied in the present study, RFLP profiles targeting the HSP70, Poly-T, TRAP-C1 genes had been revealed in only a few species or genotypes, but those of 18SrRNA and COWP genes were in all species and genotypes. However, we supposed that it was difficult to distinguish between human or cattle genotype and other species or genotypes by RFLP profiles of 18SrRNA or COWP because the RFLP profiles of human or cattle genotype were identical or similar to those of other species or genotypes. On the other hand, it has been known that the nucleotide sequences in 18SrRNA or COWP gene are different among Cryptosporidium species and/or genotypes. Therefore, the direct sequencing method targeting the variable regions which can be used to distinguish among Cryptosporidium species, as well as the genotypes within C. parvum in either 18SrRNA or COWP gene is the most useful tool for accurate identification of Cryptosporidium isolates.     

Microvascular changes in large flame burn wound in sheep

Advances in local wound management with early excisional therapy have decreased morbidity and mortality of massive third-degree burn patients. Although blood redistribution within burned tissue is of clinical interest, few studies have longitudinally determined the regional blood flow of various layers of the burn wound. We used a conscious ovine model in which animals were subjected to 40% third degree burn. Burned tissue was divided into the four layers (i.e. skin, panniculus carnosus, adipose tissue, and skeletal muscle), and regional blood flow was determined separately, with fluorescent microspheres, while measuring systemic hemodynamics and total burned tissue microvascular fluid flux. The subburn adipose tissue exhibited a remarkable biphasic alteration in regional blood flow, whereas the skin layer showed only decreased blood flow during the whole experimental period. The increase in blood flow to the adipose tissue seems to be related to a sustained fluid filtrate in the postresuscitation period, resulting in edema formation mainly located in the adipose tissue at the endpoint.     

ABCA4 gene mutations in Japanese patients with Stargardt disease and retinitis pigmentosa

PURPOSE: To evaluate photoreceptor cell-specific adenosine triphosphate (ATP)-binding cassette transporter (ABCA4) gene mutations in Japanese patients with Stargardt disease (STGD) and the correlation of these mutations to clinical phenotypes. METHODS: Serum was obtained from 10 unrelated Japanese patients with STGD and 96 unrelated Japanese patients with autosomal recessive retinitis pigmentosa (arRP). All 50 ABCA4 gene exons of the patients with STGD were screened for mutations by a combination of single-strand conformation polymorphism analysis and polymerase chain reaction (PCR) direct-sequencing techniques. By restriction enzyme digestion, primer extension analysis, and PCR direct sequencing techniques, the patients with arRP were screened for three segregated, presumably null ABCA4 gene mutations observed in Japanese patients with STGD. RESULTS: Three novel, presumably null mutations of the ABCA4 gene, IVS7-45_952delinsTCTGACC, IVS12+2T-->G, and 1894delA, were identified. The Arg2149stop mutation that had been found in a white patient with STGD in a prior study was also found in a Japanese patient. Two arRP-affected siblings and two unrelated patients with STGD were found to be homozygous for the same IVS12+2T-->G mutation, and three other arRP-affected siblings were carriers of the IVS12+2T-->G mutation and/or the IVS7-45_952delinsTCTGACC mutation. These three siblings with arRP showed only atrophic degeneration in the macula early after the onset of the disease, and STGD had been diagnosed. CONCLUSIONS: Three novel ABCA4 gene mutations were identified in Japanese patients with STGD and arRP. Mutations in the ABCA4 gene can cause panretinal degeneration that changes its clinical appearance from STGD to arRP over time.     

Pharmacokinetic and clinical studies on teicoplanin for sepsis by methicillin-cephem resistant Staphylococcus aureus in the pediatric and neonate field

Pharmacokinetics, clinical efficacy and safety of teicoplanin (TEIC) were evaluated in pediatric and neonate patients with MRSA sepsis in the dosages approved in overseas. The administrated dose for pediatrics patients was 10 mg/kg once at hour 0, 12 and 24, followed by every 24 hours intervals. In neonates patients, first dose was 16 mg/kg, then 8 mg/kg every 24 hours intervals. 1. Pharmacokinetic results. All 17 patients (9 neonates and 8 pediatrics) who received TEIC were evaluated for pharmacokinetics. Trough concentrations were analyzed in 16 patients (9 neonates and 7 pediatrics) excluding one patient for lack of measurement of drug concentration at day 7. No patient with a concentration exceeding 60 micrograms/mL in peak or trough concentrations were reported. Mean concentrations in trough at day 3, 4 and 7 in neonates were 15.2, 14.7 and 17.8 micrograms/mL, and in pediatrics were 12.5, 12.2 and 13.1 micrograms/mL, respectively. These results were similar to those reported in foreign pediatrics and neonates patients. 2. Efficacy and safety results. Since no patient was excluded, all patients were evaluated for efficacy and safety. Microbiological efficacy as well as clinical cure were secondarily evaluated in 2 patients for whom MRSA was isolated from blood. Clinical efficacy rate was 76.5% (13/17) and number of cases in judgments of excellent, good, fairly improved and no change were 12, 1, 3 and 1 cases respectively. The patients for whom MRSA was isolated from blood were judged as MRSA eradicated case and cured without any additional anti-MRSA drugs. Adverse events were reported in 2 neonates and 3 pediatric patients. Possibly related adverse events to study drug (adverse drug reactions) were: 1 case of respiratory disorder, thrombocythemia, gamma-GTP increased, GOT increased and GPT increased in 3 pediatrics. These results suggest that an application of overseas dose regimen of TEIC for neonate and pediatrics is appropriate in Japan.     

QSAR evaluation of the Ch'an Su and related bufadienolides against the colchicine-resistant primary liver carcinoma cell line PLC/PRF/5(1)

QSAR analysis has been used to identify the essential structural requirements for increasing the inhibitory activities of selected bufadienolides from the Chinese drug Ch'an Su (and other sources) against the primary liver carcinoma cell line PLC/PRF/5 (PLC) and the derived colchicine-resistant line (COL). The variable substituent domain of the proposed pharmacophore of the bufadienolides was investigated using a Comparative Molecular Field Analysis (CoMFA) approach. A model with considerable predictive ability was obtained. In addition, the CoMFA results agreed well with the pharmacophore bufadienolide model for the parent PLC line proposed earlier.     

Mutational analysis of conserved hydrophobic amino acid residues in the N-terminal region of DnaA protein

DnaA is the initiator of chromosomal DNA replication in E. coli. We previously reported that conserved hydrophobic amino acid residues in the N-terminal region of DnaA (I26 and L40) are essential for DNA replication in vivo and in vitro using mutant DnaA proteins (DnaAI26S and DnaAL40S). In this study, we introduced further random mutations to find intragenic suppressors for dnaAI26S or dnaAL40S. By direct DNA sequence, a mutation which causes substitution of the Ser (Ile, in the wild-type DnaA) with Phe (DnaAI26F or DnaAL40F) was found in all of the suppressors. Site-directed mutational analysis showed that DnaAI26L, and DnaAL40I, but not DnaAI26S or DnaAL40S, were active for oriC DNA replication in cells. Furthermore, purified DnaAI26F but not DnaAI26S was active for oriC DNA replication in a crude extract. These results strongly suggest that hydrophobic amino acid residues in these positions of DnaA (I26 and L40) are important for the function of this protein as an initiator of DNA replication both in vivo and in vitro.     

Discrimination between composite resin and teeth using fluorescence properties

The differentiation of composite resin from teeth using fluorescence emission was investigated as basic research for the visual detection of resin filled teeth in mass dental health examinations. Fluorescence spectra were taken from extracted human maxillary central incisors and 12 types of light-cured composite resins with a maximum of 15 shades via excitation using light with wavelengths of 400-500 nm. The fluorescence intensity ratio of resin to tooth was lowest around 500 nm for all the resins. The fluorescent images were taken based on spectroscopic results, which confirmed discrimination between the resin part and the tooth in the resin filled tooth.     

A mixture of ethylene vinyl alcohol copolymer and ethanol yielding a nonadhesive liquid embolic agent to treat cerebral arteriovenous malformations: initial clinical experience

OBJECT: The authors report their clinical experience with their new nonadhesive liquid embolic agent, an ethylene vinyl alcohol copolymer (EVAL)/ethanol mixture, to treat arteriovenous malformations (AVMs). METHODS: Between June 1995 and April 2001, 57 patients with confirmed AVMs underwent embolization of their lesions with the EVAL/ethanol mixture. In 87 procedures consisting of one to three stages, the authors embolized 185 feeding arteries to occlude as much of the AVM as possible. Repeated injections under fluoroscopic control could be performed smoothly without encountering cementing of the catheter to the vessel wall. Among the 87 embolizations undertaken in 57 patients, seven procedures (8%) in six patients produced new postembolization symptoms. Resolution of these symptoms occurred within hours or days after four of the seven procedures; permanent neurological deficits remained after the other three procedures (3.4%). Of the 57 patients, three underwent postembolization radiosurgery, and 54 underwent radical treatment with microsurgical extirpation. Histopathological examination of the 54 specimens disclosed mild inflammation within the embolized lumen without inflammatory reactions in the media or adventitia. Follow-up angiograms obtained 3 years after radiosurgery was administered showed that in all three patients treated in this fashion the nidus had completely disappeared. CONCLUSIONS: The EVAL/ethanol mixture is handled easily and appears to be an effective and safe agent for preoperative embolization of AVMs.     

A nonadhesive liquid embolic agent composed of ethylene vinyl alcohol copolymer and ethanol mixture for the treatment of cerebral arteriovenous malformations: experimental study

OBJECT: The authors have developed a mixture of ethylene vinyl alcohol copolymer (EVAL) and iopamidol, which is dissolved in ethanol, as an alternative solvent to provide a safe means of embolizing arteriovenous malformations (AVMs). METHODS: A two-stage delivery technique is required to prevent premature precipitation in the catheter when using this material: the catheter is first infused with 30% ethanol and this is followed by the delivery of the EVAL-ethanol mixture. Acute angiographic changes were analyzed after superselective delivery of dimethyl sulfoxide (DMSO) and 30% ethanol into the renal artery of rabbits. Histological changes following the embolization of the renal artery achieved using the EVAL-ethanol mixture were recorded at 1 hour and at 2 and 16 weeks after the procedure. Although DMSO always produced severe, rapidly progressive vasospasm in the renal artery during a 1- to 60-minute postinfusion, 30% ethanol did not. Microscopically, the lumens of embolized vessels examined 1 hour after embolization with EVAL-ethanol appeared to be filled with EVAL sponges, leaving almost no open spaces. The space between the EVAL sponges and the inner surface of the vessels was filled with fresh thrombus. In the vessel walls of specimens examined 2 weeks after embolization there was no or a slight inflammatory reaction. Scattered in the EVAL sponges were almost equal numbers of neutrophilic granulocytes and mononuclear cells, indicative of a mild inflammatory response. In specimens examined 16 weeks postembolization, the changes noted at 2 weeks were intensified. There was no definite histopathological evidence of mural hemorrhage, perivascular extravasation of the mixture, or perivascular hemorrhage in any specimen that was examined. CONCLUSIONS: Although the degree of permanence of this embolization material is yet unknown, the mixture was easy to handle, and appeared safe and effective for AVM embolization. Its nonadhesive characteristic and its ability to be infused by repeated injections make it an attractive alternative to currently available materials. The good results obtained in this study led us to undertake a clinical trial, the results of which are contained in a companion article in this issue of the Journal of Neurosurgery.     

Carotenoids in cancer chemoprevention

Various natural carotenoids, besides -carotene, were proven to have anticarcinogenic activity, and some of them showed more potent activity than -carotene. Thus, these carotenoids (-carotene, lutein, zeaxanthin, lycopene, rbeta -cryptoxanthin, fucoxanthin, astaxanthin, capsanthin, crocetin and phytoene), as well as -carotene, may be useful for cancer prevention. In the case of phytoene, the concept of 'bio-chemoprevention', which means biotechnology-assisted method for cancer chemoprevention, may be applicable. In fact, establishment of mammalian cells producing phytoene was succeeded by the introduction of crtB gene, which encodes phytoene synthase, and these cells were proven to acquire the resistance against carcinogenesis. Antioxidative phytoene-containing animal foods may be classified as a novel type of functional food, which has the preventive activity against carcinogenesis, as well as the ability to reduce the accumulation of oxidative damages, which are hazardous for human health.