Oral Rehabilitation of Patients Sustaining Orofacial Injuries: The UPenn Initiative.

Tissue injuries in the oral and maxillofacial structures secondary to trauma, warfare, ablative cancer, and benign tumor surgery result in significant losses of speech, masticatory and swallowing functions, aesthetic deformities, and overall psychological stressors and compromise. Optimal oral rehabilitation remains a formidable challenge and an unmet clinical need due to the influence of multiple factors related to the physiologic limitations of tissue repair, the lack of site and function-specific donor tissues and constructs, and an integrated team of multidisciplinary professionals. The advancements in stem cell biology, biomaterial science, and tissue engineering technologies, particularly the 3-dimensional bioprinting technology, together with digital imaging and computer-aided design and manufacturing technologies, have paved the path for personalized/precision regenerative medicine. At the University of Pennsylvania, we have launched the initiative to integrate multidisciplinary health professionals and translational/clinical scientists in medicine, dentistry, stem cell biology, tissue engineering, and regenerative medicine to develop a comprehensive, patient-centered approach for precision and personalized reconstruction, as well as oral rehabilitation of patients sustaining orofacial tissue injuries and defects, especially oral cancer patients.

Two in vitro methods for screening potential parasiticides against Ichthyophthirius multifiliis using Tetrahymena thermophila.

Ichthyophthirius multifiliis (Ich) is a ciliate parasite that infects many species of freshwater fishes worldwide and causes heavy economic losses in aquaculture. Currently, parasiticides for controlling this parasite are limited, and few pond-practical chemical therapies exist. Hence, the search for new parasiticides is urgently needed. One challenge confronting the screening of potential parasiticides is the difficulty in raising enough parasite for efficacy testing as Ich is an obligate parasite. This study used species of Tetrahymena, Ich-related and cultivable ciliate protozoa, to evaluate two in vitro methods to test parasiticides. Plate counting and MTS assays (CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay) were used to compare lethal concentrations or median lethal concentrations (LC50) of copper sulphate, formalin and malachite green between T. thermophila and Ich theronts or between T. thermophila and Ich tomonts. The parasiticides that killed T. thermophila have been demonstrated to kill theronts or tomonts. These in vitro methods using T. thermophila can be used to screen novel parasiticides against Ich.

Visible red and infrared light alters gene expression in human marrow stromal fibroblast cells.

OBJECTIVES: This study tested whether or not gene expression in human marrow stromal fibroblast (MSF) cells depends on light wavelength and energy density. MATERIALS AND METHODS: Primary cultures of isolated human bone marrow stem cells (hBMSC) were exposed to visible red (VR, 633 nm) and infrared (IR, 830 nm) radiation wavelengths from a light emitting diode (LED) over a range of energy densities (0.5, 1.0, 1.5, and 2.0 Joules/cm2) Cultured cells were assayed for cell proliferation, osteogenic potential, adipogenesis, mRNA and protein content. mRNA was analyzed by microarray and compared among different wavelengths and energy densities. Mesenchymal and epithelial cell responses were compared to determine whether responses were cell type specific. Protein array analysis was used to further analyze key pathways identified by microarrays. RESULT: Different wavelengths and energy densities produced unique sets of genes identified by microarray analysis. Pathway analysis pointed to TGF-beta 1 in the visible red and Akt 1 in the infrared wavelengths as key pathways to study. TGF-beta protein arrays suggested switching from canonical to non-canonical TGF-beta pathways with increases to longer IR wavelengths. Microarrays suggest RANKL and MMP 10 followed IR energy density dose-response curves. Epithelial and mesenchymal cells respond differently to stimulation by light suggesting cell type-specific response is possible. CONCLUSIONS: These studies demonstrate differential gene expression with different wavelengths, energy densities and cell types. These differences in gene expression have the potential to be exploited for therapeutic purposes and can help explain contradictory results in the literature when wavelengths, energy densities and cell types differ.

Molecular cloning and expression analysis of jasmonic acid dependent but salicylic acid independent LeWRKY1.

Various plant genes can be activated or inhibited by phytohormones under conditions of biotic and abiotic stress, especially in response to jasmonic acid (JA) and salicylic acid (SA). Interactions between JA and SA may be synergistic or antagonistic, depending on the stress condition. In this study, we cloned a full-length cDNA (LeWRKY1, GenBank accession No. FJ654265) from Lycopersicon esculentum by rapid amplification of cDNA ends. Sequence analysis showed that this gene is a group II WRKY transcription factor. Analysis of LeWRKY1 mRNA expression in various tissues by qRT-PCR showed that the highest and lowest expression occurred in the leaves and stems, respectively. In addition, LeWRKY1 expression was induced by JA and Botrytis cinerea Pers., but not by SA.

Identification of miRNAs and their targets in wheat (Triticum aestivum L.) by EST analysis.

MicroRNAs (miRNAs) are a newly discovered class of noncoding small RNAs that regulate gene expression by directing target mRNA cleavage or translational inhibition. A large number of miRNAs have been identified in plants. Increasing evidence has shown that miRNAs play multiple roles in plant biological processes. So far, identification of miRNAs has been limited to a few model plant species, whose genomes have been sequenced. Wheat (Triticum aestivum L.) is one of the most important cereal crops worldwide. To date, only a few conserved miRNAs have been predicted in wheat. Here, we showed the conserved miRNAs identified in wheat by expressed sequence tag (EST) analysis. All previously known miRNAs from Arabidopsis, rice, and other plant species were used in a BLAST search against the wheat EST database to identify novel wheat miRNAs by a series of filtering criteria. By this strategy, we identified 62 conserved miRNAs, belonging to 30 miRNA families, 48 of which were newly discovered in wheat. These newly identified wheat miRNAs may regulate 287 potential targets, which are involved in development, signal transduction, metabolic pathways, disease resistance, ion transportation, and environmental stress response.

DPSC-Derived Extracellular Vesicles Promote Rat Jawbone Regeneration.

Repair and functional reconstruction of large jawbone defects remain one of the challenges in the field of head and neck surgery. The recent progress in tissue engineering technologies and stem cell biology has significantly promoted the development of regenerative reconstruction of jawbone defects. The multiple trophic activities of extracellular vesicles (EVs) produced by mesenchymal stem cells (MSCs) may play a critical role in their therapeutic effects. Accumulating evidence has shown the promise of dental pulp stem cells (DPSCs) in bone regeneration, but less is known about the regenerative effects of DPSC-EVs on jawbone defects. The purpose of this study is to explore the osteogenic effects of DPSC-EVs on jawbone marrow-derived MSCs (JB-MSCs) in vitro and their osteoinductive effects in a mandibular bone defect model in rats. Our results showed that JB-MSCs could efficiently uptake DPSC-EVs, which in turn significantly promoted the expression of osteogenic genes, such as runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), and osteocalcin (OCN), as well as the osteogenic differentiation capability of JB-MSCs. Meanwhile, we found that the pro-osteogenic effect in vitro induced by DPSC-EVs was comparable to that induced by BMP-2 (bone morphogenetic protein 2), currently the only Food and Drug Administration-approved osteoinductive growth factor. In vivo, animals that were locally treated with DPSC-EVs laden with a commercially available collagen membrane exhibited a relatively fast wound closure and increased new bone density at the mandible defects. Our results provide evidence for the osteogenic and osteoinductive effects of DPSC-EVs on jawbone regeneration. Due to the accessibility, rapid proliferation, and osteogenic propensity of DPSCs, DPSC-EVs may represent a safe cell-free therapeutic approach for craniofacial bone regeneration.

The effects of food on the pharmacokinetics of mitiglinide tablets in healthy volunteers and a novel mass-spectrometric (UPLC-MS/MS) method for such studies.

WHAT IS KNOWN AND OBJECTIVE: Mitiglinide (MGN) is a new insulinotropic agent of the glinide class with rapid onset. The effects of food intake on the pharmacokinetic (PK) profile of mitiglinide tablets after single oral administration have not yet been reported in healthy adults. We aimed to assess the effects of food intake on the PK properties of mitiglinide (MGN) tablets, using a novel analytical method, after single escalating oral doses in healthy Chinese volunteers. METHODS: In this open-label, randomized, single-dose (three distinct doses), two-way crossover PK study, three doses of MGN 5, 10 or 20 mg were administered to healthy adult volunteers after an overnight fast (fasted condition) or low-fat breakfast (fed condition) (period 1). After 7 days, the participants received the same dose under the opposite fed/fasted condition (period 2). Serial blood samples were obtained before and through 8 h after study drug administration. Concentrations of MGN in plasma were determined using UPLC-MS/MS. Adverse events (AEs) were monitored and recorded on each in-clinic day. RESULTS AND DISCUSSION: Twenty-four Chinese volunteers (eight [four men, four women] volunteers per group) were enrolled in the study. The extent of absorption of MGN was similar in both fed and fasted conditions at single doses in the range 5-20 mg. Food intake was associated with decreases in C(max) by 60·4% to 65·2% in the three dose groups and greatly delayed T(max) [0·36(Standard deviation 0·16) vs. 1·75(0·92) hours with 5 mg, 0·29(0·19) vs. 1·97(0·81) hours with 10 mg and 0·30(0·10) vs. 1·18(0·68) hours with 20 mg; all, P < 0·05]. t(1/2) , CL/F and V/F (P > 0·05) were unaffected. MRT(0-8) at the 5 and 10-mg doses, but not at the 20-mg dose, were markedly lower in fasted volunteers than fed volunteers (P < 0·05). WHAT IS NEW AND CONCLUSIONS: Using a novel UPLC-MS/MS method, we showed that food intake affected the rate but not the extent of absorption of MGN within the 5- to 20-mg dose range. Gender did not appear to affect the PK properties of MGN in either fasted or fed states. MGN should be preferably taken before food.

Molecular cloning and expression analysis of heat-shock protein 70 in orange-spotted grouper Epinephelus coioides following heat shock and Vibrio alginolyticus challenge.

In this study, the complementary (c)DNA encoding heat-shock protein 70 (Hsp70) of orange-spotted grouper Epinephelus coioides (OsgHsp70) was cloned. OsgHsp70 was 2206 bp and encoded 652 amino acids with predicted molecular mass of 70·89 kDa and theoretical isoelectric point of 5·48. Three Hsp70 family signatures, bipartite nuclear localization signal sequence (NLS) and cytoplasmic characteristic motif (EEVD) were observed in the OsgHsp70, which shared high similarity in amino-acid sequences with the Hsp70 gene of other vertebrates. The results indicated that the OsgHsp70 is a member of the heat-shock protein 70 family. The Hsp70 messenger (m)RNA expressions were quantified by real-time PCR following heat shock, bacterial infection and immunization with formalin-killed Vibrio alginolyticus, a kind of bacterial pathogen that causes septicaemia. Hsp70 mRNA expression in gill, kidney, spleen, thymus gland, muscle and total-blood samples increased at first and then decreased gradually following heat shock. A similar time-dependent pattern was observed following V. alginolyticus pathogen challenge, in which Hsp70 mRNA expression peaked at 24 h after live bacterial infection and 3 days after dead bacterial vaccination. The results indicated that the Hsp70 gene was inducible and involved in the fish immune response.

Extracellular Vesicles of GMSCs Alleviate Aging-Related Cell Senescence.

Healthy aging is a complex biological process with progressive accumulation of senescent cells characterized by stable cell cycle arrest, resulting in impaired homeostasis, regenerative potential, and gradual functional decline in multiple tissues and organs, whereby the aberrant activation of mammalian target of rapamycin (mTOR) signaling networks plays a central role. Herein, we explored the effects of extracellular vesicles (EVs) released by gingiva-derived mesenchymal stem cells (GMSC-EVs) on oxidative stress-induced cellular senescence in human endothelial cells and skin fibroblasts and their antiaging potentials. Our results showed that GMSC-EVs robustly abrogated oxidative stress-induced upregulation in the expression of cellular senescence-related genes, such as ß-galactosidase, p21, p53, and γH2AX, and mTOR/pS6 signaling pathway, in human umbilical vein endothelial cells (HUVECs) and skin fibroblasts. Meanwhile, GMSC-EVs restored oxidative stress-induced impairment in proliferation and tube formation by HUVECs. Systemic administration of GMSC-EVs attenuated aging-associated elevation in the expression levels of p21, mTOR/pS6, interleukin 6, and tumor necrosis factor α in skin and heart tissues of aged mice. These findings suggest that GMSC-EVs could be a potential alternative source of cell-free product for attenuation of aging-related skin and vascular dysfunctions due to their potent inhibitory effects on oxidative stress-induced cellular senescence in endothelial cells and skin fibroblasts.

Effect of lncRNA MALAT1 on rats with myocardial infarction through regulating ERK/MAPK signaling pathway.

OBJECTIVE: To explore the effect of the long non-coding ribonucleic acid (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) on rats with myocardial infarction (MI) by regulating the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) signaling pathway. MATERIALS AND METHODS: The Sprague- Dawley (SD) rat model of MI was established, and lncRNA MALAT1 was overexpressed using pcDNA-MALAT1 plasmids (MALAT1 group, n=10) and silenced using RNA interference technique (siMALAT1 group, n=10). The Sham group (n=10) was also set up. The transfection efficiency of lncRNA MALAT1 in rats was detected via Reverse Transcription-Polymerase Chain Reaction (RT-PCR). 2 weeks after the successful modeling, the cardiac function indexes were measured through magnetic resonance imaging (MRI) and echocardiography (ECG). The myocardial tissue injury was observed via hematoxylin-eosin (HE) staining, and the apoptosis of myocardial tissues was detected via terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Moreover, the levels of the serum inflammatory factors were detected via enzyme-linked immunosorbent assay (ELISA), the messenger RNA (mRNA) expressions of Collagen I and III, the apoptosis, the and pathway genes were detected via RT-PCR. The expressions of ERK/MAPK pathway-related proteins in myocardial tissues were detected via Western blotting. RESULTS: The expression of lncRNA MALAT1 was remarkably increased in the MALAT1 group but evidently declined in the siMALAT1 group (p<0.05), indicating the successful transfection. The fractional shortening (FS, %) and ejection fraction (EF, %) were significantly restored in siMALAT1 group (p<0.05), suggesting that the silence of MALAT1 can improve the cardiac function after acute MI. The results of the HE staining and TUNEL assay manifested that siMALAT1 group had milder myocardial injury and decreased apoptosis compared with MALAT1 group. In the MALAT1 group, the mRNA expressions of Collagen I and III, Caspase3, ERK2, and MAPK were remarkably increased (p<0.05), while the mRNA expression of Bcl-2 was remarkably decreased (p<0.05). The above expressions had the opposite trends in siMALAT1 group. Besides, the protein expressions of ERK2 and MAPK in MALAT1 group were significantly increased (p<0.05). CONCLUSIONS: The downregulation of lncRNA MALAT1 can significantly improve the cardiac function after MI in SD rats mainly by inhibiting the ERK/MAPK pathway.

Induction of Salivary Gland-Like Cells from Dental Follicle Epithelial Cells.

The dental follicle (DF), most often associated with unerupted teeth, is a condensation of ectomesenchymal cells that surrounds the tooth germ in early stages of tooth development. In the present study, we aim to isolate epithelial stem-like cells from the human DF and explore their potential differentiation into salivary gland (SG) cells. We demonstrated the expression of stem cell-related genes in the epithelial components of human DF tissues, and these epithelial progenitor cells could be isolated and ex vivo expanded in a reproducible manner. The human DF-derived epithelial cells possessed clonogenic and sphere-forming capabilities, as well as expressed a panel of epithelial stem cell-related genes, thus conferring stem cell properties (hDF-EpiSCs). When cultured under in vitro 3-dimensional induction conditions, hDF-EpiSCs were capable to differentiate into SG acinar and duct cells. Furthermore, transplantation of hDF-EpiSC-loaded native de-cellularized rat parotid gland scaffolds into the renal capsule of nude mice led to the differentiation of transplanted hDF-EpiSCs into salivary gland-like cells. These findings suggest that hDF-EpiSCs might be a promising source of epithelial stem cells for the development of stem cell-based therapy or bioengineering SG tissues to repair/regenerate SG dysfunction.

Loss of Notch3 Signaling Enhances Osteogenesis of Mesenchymal Stem Cells from Mandibular Torus.

Mandibular torus (MT) is a common intraoral osseous outgrowth located on the lingual surface of the mandible. Histologic features include hyperplastic bone consisting of mature cortical and trabecular bone. Some theories on the etiology of MT have been postulated, such as genetic factors, masticatory hyperfunction, trauma, and continued growth, but the underlying mechanism remains largely unknown. In this study, we investigated the potential role of mesenchymal stem cells (MSCs) derived from human MT in the pathogenesis of bone outgrowth. We demonstrated that MT harbored a distinct subpopulation of MSCs, with enhanced osteogenic and decreased adipogenic differentiation capacities, as compared with their counterparts from normal jaw bone. The increased osteogenic differentiation of mandibular torus MSCs was associated with the suppression of Notch3 signaling and its downstream target genes, Jag1 and Hey1, and a reciprocal increase in the transcriptional activation of ATF4 and NFATc1 genes. Targeted knockdown of Notch3 expression by transient siRNA transfection promoted the expression of osteogenic transcription factors in normal jaw bone MSCs. Our data suggest that the loss of Notch3 signaling may contribute partly to bone outgrowth in MT, as mediated by enhanced MSC-driven osteogenic differentiation in the jaw bone.

Dystroglycan induced muscular dystrophies - a review.

Dystroglycanopathies are muscular dystrophies caused by mutations in genes involved the in O-linked glycosylation of α-dystroglycan. Severe forms of these conditions result in abnormalities in exhibit brain and ocular developmental too, in addition to muscular dystrophy. The full spectrum of developmental pathology is caused mainly by loss of dystroglycan from Bergmann glia. Moreover, cognitive deficits are constant features of severe forms of dystroglycanopathies. However, the precise molecular mechanism leading to neuronal dysfunction in these diseases is not fully known yet. The present review article will discuss the importance of dystroglycan in cerebellar development and associated pathological states.

PHACTR4 regulates proliferation, migration and invasion of human hepatocellular carcinoma by inhibiting IL-6/Stat3 pathway.

OBJECTIVE: Phosphatase and actin regulator 4 (PHACTR4) is one member of the largely uncharacterized PHACTR family of protein phosphatase 1 (PP1)-and actin-binding proteins. PHACTR4 is significantly deleted or mutant in many tumor subtypes, such as breast, colorectal, lung, neural, ovarian, and renal cancers. However, the role of PHACTR4 in human hepatocellular carcinoma (HCC) is completely unknown. MATERIALS AND METHODS: Ten paired HCC tissues and adjacent non-cancerous tissues were used to detect the expression PHACTR4. Real-time PCR was used to detect the mRNA level of PHACTR4 in clinic samples. The protein level of PHACTR4 was determined by Western blot. Retrovirus-based gene transduction was used to generate Flag-tagged PHACTR4 HepG2 stable cell line. BrdU assay was used to determine the cell growth of HepG2 cells. The cell cycle distribution was detected by flow cytometry assay. In vitro scratch wounding and Matrigel invasion assays were used to test the migration and invasion ability of HepG2 cells. RESULTS: The expression of PHACTR4 was noticeably decreased in clinical HCC tissues, compared to the non-tumoral tissues. Overexpression of PHACTR4 inhibited HCC cells proliferation, colony formation, migration and invasion, and resulted in significant cycle arrest. PHACTR4 attenuated both constitutive and IL-6-induced phosphorylation of signal transducer and activator of transcription 3 (Stat3), and inhibited Stat3 downstream genes expression. CONCLUSIONS: Overall, our results suggest that PHACTR4 is a tumor suppressor in HCC by inhibiting IL-6/ Stat3 pathway.

DPSCs from Inflamed Pulp Modulate Macrophage Function via the TNF-α/IDO Axis.

Human dental pulp stem cells (DPSCs) can be isolated from inflamed pulp derived from carious teeth with symptomatic irreversible pulpitis (I-DPSCs), which possess stemness and multidifferentiation potentials similar to DPSCs from healthy pulp. Since macrophages-essential cell players of the pulpal innate immunity-can regulate pulpal inflammation and repair, the authors investigated the immunomodulatory effects of DPSCs/I-DPSCs on macrophage functions and their underlying mechanisms. Similar to DPSCs, I-DPSCs were capable of colony-forming efficiency and adipogenic and osteo/dentinogenic differentiation under in vitro induction conditions. I-DPSCs also expressed a similar phenotypic profile of mesenchymal stem cell markers, except a relatively higher level of CD146 as compared with DPSCs. Coculture of DPSCs or I-DPSCs with differentiated THP-1 cells, the human monocyte cell line, markedly suppressed tumor necrosis factor α (TNF-α) secretion in response to stimulation with lipopolysaccharides (LPS) and/or nigericin. However, unlike TNF-α, the secreted level of interleukin 1ß was not affected by coculture with DPSCs or I-DPSCs. Furthermore, DPSC/I-DPSC-mediated inhibition of TNF-α secretion by macrophages was abolished by pretreatment with 1-methyl-D-tryptophan, a specific inhibitor of indoleamine-pyrrole 2,3-dioxygenase (IDO), but not by NSC-398, a specific inhibitor of COX-2, suggesting IDO as a mediator. Interestingly, IDO expression was significantly augmented in macrophages and mesenchymal stromal cells in inflamed human pulp tissues. Collectively, these findings show that I-DPSCs, similar to DPSCs, possess stem cell properties and suppress macrophage functions via the TNF-α/IDO axis, thereby providing a physiologically relevant context for their innate immunomodulatory activity in the dental pulp and their capability for pulp repair.

Quantification of concanavalin A binding to rat brain microsomal membranes detected by fluorescence polarization technique.

A high sensitive method for detecting the change of microsomal membrane surface oligosaccharides was developed to study the regulatory role of lipid- or peptide-linked mannoside of endoplasmic reticulum in synaptic functions. The binding of concanavalin A to the microsomal membrane surface was measured quantitatively using a microgram-order of rat brain microsomal proteins. The fluorescence polarization of concanavalin A (Con A)-fluorescein isothiocyanate (FITC) conjugate bound to the membrane was analyzed to quantitate the change of binding constant and the number of binding sites. As a control, the non-specific binding of bovine serum albumin-FITC conjugate was measured by the same technique. We measured the change of fluorescence intensity of membrane-bound FITC conjugates by the flow cytometry and found that the intensity of FITC conjugate bound to the membrane increased more than that of free form of the probe. We observed that the alpha-mannosidase-treatment of rat brain microsomes resulted in the increase of binding constant of Con A to the microsomal surface without significant loss of binding sites.

Changes of oligosaccharides and fatty acids in monkey hippocampus by synaptic potentiation.

We measured the release of free fatty acids and structural changes of glycoprotein glycans induced by tetraethylammonium (TEA) salt in hippocampal slices of cynomolgus monkey brain. The release of free fatty acids in the hippocampal slices occurred after synaptic potentiation by TEA in a different manner from rat hippocampus. Arachidonic acid release in monkey hippocampus occurred much faster than that in rat. Several types of glycans of monkey hippocampal glycoproteins were determined depending on the duration time after TEA treatment. 5-Mannose was increased within 2 min, while polysialoglycans were increased after 5 min or later. Comparative study of glycans of monkey and rat hippocampal slices revealed the presence of relatively larger amount of sialo- and multi-anntenary glycans in rat than in monkey. These results indicate that the depolarizing stimulation of monkey hippocampal slices induced the change of glycoprotein glycan structures and release of free fatty acids in a different manner from rat hippocampus.

Significance of the straight-leg-raising test in the diagnosis and clinical evaluation of lower lumbar intervertebral-disc protrusion.

The cases of 113 patients who had protrusion of a lumbar intervertebral disc were analyzed to determine the relationship between the findings at operation and the location of the pain that resulted from the straight-leg-raising test. The study showed a close relationship between the location of the pain and the position of the protrusion of the disc. The degree of limitation of straight-leg raising was also found to have a direct relationship to the size and position of the protrusion and to its relationship to the spinal nerve. The protrusions were classified into three types according to position in relation to the dura mater and to the pattern of pain that was induced by passive straight-leg raising. On straight-leg raising, central protrusions tended to cause pain in the back, lateral protrusions caused pain in the lower extremity, and intermediate protrusions caused both. On this basis, the distribution of pain on straight-leg raising allowed an accurate prediction of the location of the lesion in 100 (88.5 per cent) of the 113 patients.

Effects of cyproheptadine on plasma superoxide dismutase activity and malondialdehyde content in rabbits with hemorrhagic shock.

Profound hemorrhagic shock was produced in thirty rabbits by exsanguination via the carotid artery until blood pressure (BP) reached 5.3 kPa (40 mmHg) and was sustained for a period of 90 minutes. The rabbits were equally divided into cyproheptadine (Cyp) treated group and control group. Blood samples 30 minutes after liquid and blood infusion and administration of Cyp (10 mg/kg) were collected from the carotid artery, and the plasma superoxide dismutase (SOD) activity and malondialdehyde (MDA) content measured. The results showed that Cyp remarkably enhanced the plasma SOD activity (2462 +/- 338 vs 1955 +/- 596, P < 0.01) and reduced MDA content (2.68 +/- 0.24 vs 3.20 +/- 0.49, P < 0.01). We believe that the increase of O2 production plays an important role in the development of shock, the single blood and liquid infusion can not significantly improve the shock conditions. Scavenging oxygen free radicals and alleviating cellular damage and multiple organ failure are the possible mechanisms of cyproheptadine anti-shock effect.

[Genetic diagnosis of phenylketonuria. III. Mutations of phenylalanine hydroxylase gene in Orientals].

Phenylketonuria (PKU) is an autosomal recessive disorder caused by lesions in the phenylalanine hydroxylase (PAH) gene. The recent studies on PAH mutations show the genetic drift of PKU alleles among some Oriental populations. Therefore, we searched for PKU mutations among Japanese, Chinese and Taiwanese. Direct sequencing was conducted on DNA fragments amplified by the polymerase chain reaction, using solid-phase technology involving the biotin-streptavidin system. Two new mutations (R241C and G247V) and two of the known mutant alleles (Y204C and R243Q) were found in two Taiwanese and two Chinese PKU patients, and three known mutations (R111X, Y204C and R413P) were recognized in three Japanese; two new mutations were identified in exon 7 of the PAH gene at codon 241 and codon 247, where the single base changes from C to T and from G to T substituted cysteine for arginine and valine for glycine, respectively. Further all the PAH mutations detected are common in Oriental populations as they have been thus far unreported among Caucasians. From these data as well as the clinical phenotype of the patients, we suggest that the R241C and G247V substitutions may interfere with proper enzyme function, although we have not yet performed functional studies. More detailed studies would be needed to clarify the regional distribution of mutant chromosomes in Oriental populations and other unidentified mutations.