Molecular identification and phylogenetic analysis of important medicinal plant species in genus Paeonia based on rDNA-ITS, matK, and rbcL DNA barcode sequences.

This study was performed to identify and analyze the phylogenetic relationship among four herbaceous species of the genus Paeonia, P. lactiflora, P. japonica, P. veitchii, and P. suffruticosa, using DNA barcodes. These four species, which are commonly used in traditional medicine as Paeoniae Radix and Moutan Radicis Cortex, are pharmaceutically defined in different ways in the national pharmacopoeias in Korea, Japan, and China. To authenticate the different species used in these medicines, we evaluated rDNA-internal transcribed spacers (ITS), matK and rbcL regions, which provide information capable of effectively distinguishing each species from one another. Seventeen samples were collected from different geographic regions in Korea and China, and DNA barcode regions were amplified using universal primers. Comparative analyses of these DNA barcode sequences revealed species-specific nucleotide sequences capable of discriminating the four Paeonia species. Among the entire sequences of three barcodes, marker nucleotides were identified at three positions in P. lactiflora, eleven in P. japonica, five in P. veitchii, and 25 in P. suffruticosa. Phylogenetic analyses also revealed four distinct clusters showing homogeneous clades with high resolution at the species level. The results demonstrate that the analysis of these three DNA barcode sequences is a reliable method for identifying the four Paeonia species and can be used to authenticate Paeoniae Radix and Moutan Radicis Cortex at the species level. Furthermore, based on the assessment of amplicon sizes, inter/intra-specific distances, marker nucleotides, and phylogenetic analysis, rDNA-ITS was the most suitable DNA barcode for identification of these species.

Molecular identification of the traditional herbal medicines, Arisaematis Rhizoma and Pinelliae Tuber, and common adulterants via universal DNA barcode sequences.

Methods to identify Pinelliae Tuber and Arisaematis Rhizoma are required because of frequent reciprocal substitution between these two herbal medicines and the existence of several closely related plant materials. As a result of the morphological similarity of dried tubers, correct discrimination of authentic herbal medicines is difficult by conventional methods. Therefore, we analyzed DNA barcode sequences to identify each herbal medicine and the common adulterants at a species level. To verify the identity of these herbal medicines, we collected five authentic species (Pinellia ternata for Pinelliae Tuber, and Arisaema amurense, A. amurense var. serratum, A. erubescens, and A. heterophyllum for Arisaematis Rhizoma) and six common adulterant plant species. Maturase K (matK) and ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) genes were then amplified using universal primers. In comparative analyses of two DNA barcode sequences, we obtained 45 species-specific nucleotides sufficient to identify each species (except A. erubescens with matK) and 28 marker nucleotides for each species (except P. pedatisecta with rbcL). Sequence differences at corresponding positions of the two combined DNA barcodes provided genetic marker nucleotides that could be used to identify specimens of the correct species among the analyzed medicinal plants. Furthermore, we generated a phylogenetic tree showing nine distinct groups depending on the species. These results can be used to authenticate Pinelliae Tuber and Arisaematis Rhizoma from their adulterants and to identify each species. Thus, comparative analyses of plant DNA barcode sequences identified useful genetic markers for the authentication of Pinelliae Tuber and Arisaematis Rhizoma from several adulterant herbal materials.

Effect of distal graft anastomosis site on retrograde perfusion and flow patterns of native coronary vasculature.

BACKGROUND: To select the site of a target vessel for distal anastomosis surgeons use different approaches. Some try to place the graft as close to the stenosis as possible, whereas others routinely anastomose the graft onto the distal portion. In this latter case the proximal portion and its tributaries are perfused from the graft in a retrograde rather than an antegrade fashion. The aim of this study was to investigate the effect of local hemodynamics associated with the different location of distal anastomoses on flow patterns in the proximal native artery and its branches. METHODS: Computational fluid dynamic and in vitro model studies were carried out in a control model composed of a straight tube (host) with a 45E side branch and models in which the proximal end of the host had various degrees of stenosis; a 45E end-to-side "graft" anastomosis was introduced either proximal (upstream) or distal (downstream) to the branch. RESULTS: Placing the graft proximal to the branch largely preserved the flow patterns that were seen in the control model. Placing the graft distal to the branch, however, introduced an extensive region of relatively stagnant flow in the native vessel near the branch. Such regions are known to promote thrombus formation that could ultimately lead to occlusion of the retrograde portion of the host vessel. CONCLUSIONS: This study suggests that, although often less convenient surgically, long-term outcome of coronary artery bypass grafting may be improved by placing grafts in the most proximal portion of the native vessel, as close to the occlusion or stenosis as possible for better preservation of a proximal artery and its branches.

Identification and characterization of a new member of the placental prolactin-like protein-C (PLP-C) subfamily, PLP-Cbeta.

We have isolated a complementary DNA (cDNA) clone that encodes a new member of the PRL-like protein-C (PLP-C) subfamily of the PRL gene family. The clone was amplified from a 13.5-day-old mouse conceptus cDNA library by PCR using primers based on conserved regions of PLP-C sequences. The full-length cDNA encodes a predicted protein of 241 residues, which contains a putative signal sequence and 2 putative N-linked glycosylation sites. The predicted protein shares 55-66% amino acid identity with mouse PLP-Calpha and rat PLP-D, PLP-H, PLP-Cv, and PLP-C and also contains 6 homologously positioned cysteine residues. Thus, we named this protein PLP-Cbeta for consistency. We have also isolated rat PLP-Cbeta from rat placenta cDNA library. Surprisingly, two messenger RNA (mRNA) isoforms of rat PLP-Cbeta were isolated: one mRNA (rPLP-Cbeta) encodes a 241-amino acid product, but another mRNA (rPLP-Cbetadelta39) lacks 39 bases that encode for a region rich in aromatic amino acids. The 39-bp region corresponds to exon 3 of other PLP-C subfamily members, such as PLP-Calpha, PLP-Cv, and d/tPRP. It suggests that the two isoforms are probably generated by an alternative splicing from a single gene. RT-PCR analysis revealed that the rPLP-Cbeta form was dominantly expressed in placenta, although both isoforms are coexpressed during placentation. The mouse PLP-Cbeta mRNA expression, which was specific to the placenta, was first detected by Northern analysis on embryonic day 11.5 (E 11.5) and persisted until birth. However, in situ hybridization analysis revealed mPLP-Cbeta expression on E 10.5 in specific trophoblast subsets, such as giant cells and spongiotrophoblast cells. mPLP-Cbeta mRNA was detected in the labyrinthine zone on E 18.5, suggesting that spongiotrophoblast cells had penetrated the labyrinthotrophoblast zone. Consistent with the observed expression in trophoblast giant cells, PLP-Cbeta expression was also detected in in vitro differentiated Rcho-1 cells, which express the trophoblast giant cell phenotype. In summary, overall high amino acid identity (79%), the locations of cysteine residues, and consensus sites for N-linked glycosylation between mouse and rat PLP-Cbeta clearly indicate that PLP-Cbeta is a bona fide member of the PLP-C subfamily. The conservation between mouse and rat, the presence of alternative isoforms, and the pattern of expression during gestation suggest the biological significance of PLP-Cbeta during pregnancy.

Mediastinal irradiation: A risk factor for atherosclerosis of the internal thoracic arteries.

Previous radiotherapy to the thorax is a risk factor for coronary artery disease. Patients with radiation-induced atherosclerosis tend to be young and frequently have lesions involving the coronary ostia and left anterior descending artery. Bypass is often the most suitable method of revascularization, and given the young age of the patient, arterial conduits would be considered superior to vein grafts. However, the internal thoracic arteries can lie within the radiation field and may not be free of atherosclerosis. A 40-year-old man who required coronary artery bypass grafting for multivessel coronary artery disease 11 years following radiotherapy for Hodgkin's lymphoma is reported. Preoperative angiography showed that the right internal thoracic artery had significant atherosclerosis and was unsuitable as a conduit.

The molecular basis of the obese mutation in ob2J mice.

The recessive ob2J mutation in mice results in an obese phenotype that is identical to that of the original ob allele. Initial studies indicated that ob2J mice fail to synthesize ob RNA in adipose tissue. Here we report the genomic organization of the mouse obese gene and establish the molecular genetic basis of the ob2J mutation. The ob2J mutation is the result of the insertion of a retroviral-like tranposon in the first intron of the ob gene. The insertion is a member of the ETn family of transposons and contains several splice acceptor and polyadenylation sites. This leads to the production of chimeric RNAs in which the ob first exon is spliced to sequences in the ETn insertion. As a consequence mature ob RNA is not synthesized, and leptin, the encoded protein, is not produced.

Expression of ob gene in adipose cells. Regulation by insulin.

The product of the recently cloned mouse obese (ob) gene is likely to play an important role in a loop regulating the size of the adipose tissue mass. The hormonal regulation of the ob gene could affect adiposity. To investigate this point, the effect of insulin on ob gene expression was examined in cells of the 3T3-F442A preadipocyte clonal line. ob mRNA is absent from exponentially growing, undifferentiated cells as well as from confluent preadipose cells. Terminal differentiation of preadipose to adipose cells leads to the expression of ob mRNA detected by a sensitive and quantitative ribonuclease protection assay. In adipose cells, the level of ob mRNA is sensitive to insulin in the nanomolar range of concentrations with an increase from an average of 1 copy to 5-10 copies/cell. The effect of insulin was fully reversible and takes place primarily at a transcriptional level. The ob mRNA shows a rapid turnover, with a half-life of approximately 2 h in the absence or presence of insulin. The level of secreted Ob protein is also regulated by insulin. These results indicate that the ob gene is expressed in mature fat cells only and support the possibility that insulin is an important regulator of ob gene expression.

Neodymium:yttrium-aluminum-garnet laser vaporization for palliation of obstructing esophageal carcinoma.

Palliative therapy for obstructing esophageal carcinoma is more often necessary than curative surgery. The neodymium:yttrium-aluminum-garnet laser was used for vaporization of obstructing esophageal carcinoma in 18 patients requiring 24 treatments. Three women and 15 men (age range 42 to 87 years) had esophageal carcinoma (seven squamous cell and nine adenocarcinoma). Twelve tumors were at the esophagogastric junction, four at the midesophagus, and two in the cervical esophagus. Lengths varied from 3 to 7 cm. Inoperability was due to diffuse metastases in eight patients, local invasion in five, poor operative risk in one patient, and patient refusal for operative treatment in four patients. Energy use was 1000 to 22,600 J per session (mean 6120 J). Good results were achieved in 16 patients (88.9%): Seven returned to full diet, five to soft diet, and four to full fluids without dysphagia. Four patients required retreatment 1 to 3 months later because of recurrent dysphagia. One patient was not benefited by the treatment and died of carcinomatosis 1 week later. No intraoperative complications occurred. Postoperatively, one patient had laryngeal edema and another had a bronchoesophageal fistula 3 weeks later. The mean survival time is 3 1/2 months. Neodymium:yttrium-aluminum-garnet laser vaporization for obstructing esophageal carcinoma is effective palliation regardless of histologic tumor type. It can be performed under direct vision with a low frequency of postoperative complications.

PMN superoxide radical production following a metabolic-endocrine simulation of trauma.

Serious infections following major trauma remain inexplicably high. Metabolic and endocrine changes after injury have been suggested as being responsible for many of the documented defects in the polymorphonucleocyte (PMN). The in vitro bactericidal activity of normal human PMNs has been examined in this laboratory by assaying the activity of the PMN membrane bound enzyme NADPH oxidase and hence O2- production of the PMN in a metabolic/endocrine milieu designed to simulate moderately severe trauma. This was accomplished by incubating the PMN with physiological and trauma serum concentrations of insulin, glucose, cortisol, epinephrine, and glucagon. The results indicate that the O2- production of the PMN is significantly enhanced in this environment. It would appear that exogenous glucose alone was responsible for this enhanced O2- production.

Effect of antibiotics and sedatives on normal neutrophil nicotinamide-adenine dinucleotide phosphate-reduced oxidase activity.

The effects of antibiotics and other commonly used medications on the human polymorphonuclear neutrophil leukocytes' (PMNs) nicotinamide-adenine dinucleotide phosphate-reduced (NADPH) oxidase activity have been investigated in vitro. Five antibiotics (penicillin G sodium, cefamandole nafate, metronidazole hydrochloride, clindamycin phosphate, and tobramycin sulfate, and a triple combination of penicillin G sodium-metronidazole hydrochloride-tobramycin sulfate) and two sedatives (morphine sulfate and diazepam) were incubated with normal human PMNs at therapeutic, infratherapeutic, and supratherapeutic drug levels. The superoxide dismutase-inhibitable, NADPH-dependent reduction of cytochrome C in the PMNs was studied after stimulation with formyl-methionyl-leucine-phenylalanine. Tobramycin sulfate and the triple combination of penicillin G sodium-metronidazole hydrochloride-tobramycin sulfate significantly reduced the NADPH oxidase activity at all dosages studied. Clindamycin phosphate, morphine sulfate, and diazepam also showed significant reduction at therapeutic and supratherapeutic concentrations. Penicillin G sodium, cefamandole nafate, and metronidazole hydrochloride did not cause a decrease in enzyme activity at any levels tested. We conclude that NADPH oxidase activity can be adversely affected by the circulating levels of common antibiotics and sedatives.