Assessment of heavy metals in benthic macroinvertebrates, water and sediments in River Isiukhu, Kenya.

A major ecological challenge facing freshwater resources such as rivers is the influx of chemical contaminants from anthropogenic sources. A report on the levels of heavy metals, namely Zn, Cu, Cr, Cd and Pb in benthic macroinvertebrates, sediments and water along River Isiukhu, within Lake Victoria catchment area in Kenya is presented. Families of benthic macroinvertebrates that were investigated included Gerridae, Baetidae and Unionidae. Samples were digested using the appropriate acid combination and analysed using Spectra AAS Varian 200. Physicochemical parameters were measured on-site using mobile Hydrolab Quanta. Pearson's correlation matrix and post hoc Tukey's test were used to show the relationship between metal concentrations in the various matrices at significant differences accepted at p ≤ 0.05. Positive correlations were obtained for heavy metal concentrations in sediments and benthic macroinvertebrates with significant correlations observed for Zn (r = 0.655, p = 0.029) and Cu (r = 0.641, p = 0.034). Sediments presented the highest range of heavy metal concentrations compared to water and benthic macroinvertebrates and presented the only matrix with quantifiable Pb levels. The range in heavy metal concentration in benthic macroinvertebrates samples were as follows: Zn, 30.73-46.64, 21.93-38.17, and 26.85-41.69 mg/kg, Cu, 1.17-6.54, 1.11-3.87 and 1.15-5.79 mg/kg, Cr, 0.47-1.61, 0.22-0.74 and 0.25-0.92 mg/kg, for families Unionidae, Baetidae and Gerridae respectively. Heavy metal concentration profile along the river indicated an influx of pollutants from anthropogenic sources due to rapid urbanization along the river.

Decay and persistence of empty bivalve shells in a temperate riverine system.

Bivalve shells can persist over a geological time, acting as important physical resources to the associated fauna. However, few studies have investigated their relevance as persistent long-term ecological attributes to the ecosystem. As such, it is relevant to investigate the shell decays in riverine systems subjected to different environmental conditions. Towards this end, shells of four bivalve species (Anodonta anatina, Corbicula fluminea, Potomida littoralis and Unio delphinus) were made available individually and in clusters of different sizes. The effects of river flow and seasonality were assessed by recording the decay rates of shells in lentic and lotic habitats throughout the year. Our results evidenced that the decays varied among species and depend on shell size, water flow and season. Thin shelled species (A. anatina and U. delphinus) showed the highest mean percentage of decay per month, 3.17% (lotic) and 2.77% (lotic), respectively, and thick shelled species (C. fluminea and P. littoralis) the lowest, 2.02% (lotic) and 1.83% (lotic), respectively. Size was a relevant variable explaining decays, with the smallest shells presenting the highest values, 1.2-2.0 times higher compared to the other size classes. Also, robustness showed to be the most relevant feature explaining the decays in thick shelled species. River flow was also a relevant descriptor of the decays, with higher decays observed in the lotic compared to the lentic habitats. Furthermore, lower decays were observed mainly during summer (lentic site), and autumn (lotic site) associated to the burial effect of leaves. In summary, shells of the native species A. anatina and U. delphinus are expected to persist and contribute less as habitat engineering species, than shells of the native P. littoralis and invasive C. fluminea species. This is especially valid to lotic habitats where the decays were up to 2.13 times higher than in lentic habitats.

Utilization of freshwater mussel (Lamellidens marginalis) for the isolation of proteins through pH shift processing: characterization of isolates.

Study was conducted to use underutilized freshwater mussel (Lamellidens marginalis) for the recovery of proteins using pH shift method and to study the functionality and characteristics of the recovered isolates. From the pH range tested (pH 2.0-13.0), maximum protein yields were obtained during solubilization at pH 2.0 and pH 13.0 (p < 0.05). During the protein recovery process, pH 13.0 was found to have minimal effect on proteins resulting in higher protein yields compared to pH 2.0. Isolates obtained by both acidic and alkaline solubilization processes had low stability and poor gel network. Total lipid content, total myoglobin, and pigment contents were reduced significantly (p < 0.05) during pH shift processing, resulting in whiter protein isolates and protein gels. All the essential amino acids were present in the isolates recovered by acid and alkaline solubilization, indicating the complete recovery of amino acids. No microbial counts were observed in any of the isolates prepared using acid and alkaline-aided processing. Acid and alkaline solubilization (pH shift) process was found to be promising for the recovery of proteins from underutilized freshwater mussel thus by reducing the supply demand gap.

Evaluation of diffusive gradients in thin films for prediction of copper bioaccumulation by yellow lampmussel (Lampsilis cariosa) and fathead minnow (Pimephales promelas).

Using a coupled method of diffusive gradients in thin films (DGT) exposure with aquatic organism bioassays, we assessed the use of DGT as a tool for estimating copper (Cu) bioavailability in contaminated waters. The DGT-accumulated Cu fraction could possibly be used as a surrogate for other assessments of metal bioavailability. The Cu concentrations in fathead minnow (Pimephales promelas) and yellow lampmussel (Lampsilis cariosa) soft tissue were compared with DGT-accumulated Cu after 2, 4, and 6 d of exposure to a Cu concentration series in static, water-only assays. The DGT-accumulated Cu was found to include free Cu ions, labile inorganic Cu complexes, and labile dissolved organic matter Cu complexes, compared with Cu speciation output from the biotic ligand model. Regressions of Cu concentrations between DGT and fathead minnow at 4 and 6 d of exposure demonstrated linear relationships. The Cu bioaccumulated in yellow lampmussel was overpredicted by DGT at Cu concentrations greater than 10 µg L-1 , which may be caused by internal regulation of Cu. The speciation component of the biotic ligand model predicted relationships between inorganic Cu and animal-accumulated Cu that were similar to predicted relationships between DGT-indicated Cu and animal-accumulated Cu at all deployment durations. Environ Toxicol Chem 2018;37:1535-1544. © 2018 SETAC.

Freshwater mussel shells (Unionidae) describe anthropogenic changes to trace element cycling within a North American river.

Bivalve shells provide an unparalleled opportunity for understanding the history of bioavailable trace elements in aquatic systems. The present study analyzed the elements Al, As, Cd, Co, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Se, U, V and Zn in freshwater mussel shells collected from a large floodplain river. Shells were collected fresh, sampled from a historic archive, and retrieved from pre-Columbian archeological sites. The elements As, Co, Cu and Ni varied with time over the course of the 20th century. When compared to the pre-Columbian shells, 20th century shell concentrations for these elements were either consistently higher (Co, Cu and Ni) or lower (As). The 20th century shells also had consistently lower concentrations of Mn and Zn when compared to the pre-Columbian period, however diagenesis is the most likely cause of this difference in Mn. The elements Cd and Fe had little spatial or temporal variation in this data set. Several elements (Al, Cr, Hg, Pb, Se, U, and V) were below method detection limits in most shells. This study demonstrated that mussel shells can be used as archives of environmental history in river systems.

Deep conservation of bivalve nacre proteins highlighted by shell matrix proteomics of the Unionoida Elliptio complanata and Villosa lienosa.

The formation of the molluscan shell nacre is regulated to a large extent by a matrix of extracellular macromolecules that are secreted by the shell-forming tissue, the mantle. This so-called 'calcifying matrix' is a complex mixture of proteins, glycoproteins and polysaccharides that is assembled and occluded within the mineral phase during the calcification process. Better molecular-level characterization of the substances that regulate nacre formation is still required. Notable advances in expressed tag sequencing of freshwater mussels, such as Elliptio complanata and Villosa lienosa, provide a pre-requisite to further characterize bivalve nacre proteins by a proteomic approach. In this study, we have identified a total of 48 different proteins from the insoluble matrices of the nacre, 31 of which are common to both E. complanata and V. lienosa A few of these proteins, such as PIF, MSI60, CA, shematrin-like, Kunitz-like, LamG, chitin-binding-containing proteins, together with A-, D-, G-, M- and Q-rich proteins, appear to be analogues, if not true homologues, of proteins previously described from the pearl oyster or the edible mussel nacre matrices, thus forming a remarkable list of deeply conserved nacre proteins. This work constitutes a comprehensive nacre proteomic study of non-pteriomorphid bivalves that has enabled us to describe the molecular basis of a deeply conserved biomineralization toolkit among nacreous shell-bearing bivalves, with regard to proteins associated with other shell microstructures, with those of other mollusc classes (gastropods, cephalopods) and, finally, with other lophotrochozoans (brachiopods).

Anti-inflammatory activity of cyclo-oxygenase2 inhibitory anionic protein fraction from Lamellidens marginalis (Lamarck).

Aqueous extract of freshwater mussel, Lamellidens marginalis is known to possess potent antioxidant and anti-inflammatory activity. Here, we have made an attempt to purify anti-inflammatory protein from Lamellidens marginalis extract (LME). Aqueous LME was prepared, and total protein was precipitated by 60% ammonium sulfate followed by purification through ion exchange chromatography. Isolated fractions were studied for anti-inflammatory activity in in vitro and in vivo experimental models. Active fractions were characterized by SDS PAGE and HPLC. Protein recovered from ammonium sulfate precipitation showed four distinct peaks in diethyl-aminoethyl cellulose ion exchange chromatography when eluted with stepwise salt gradient. Protein fraction eluted in 0.5 M sodium chloride solution showed maximum specific activity and anti-inflammatory activity in acute model and adjuvant induced chronic inflammation model. This fraction also showed cyclo-oxygenase 2 (COX2) enzyme inhibitory activity in in-vitro system. In SDS-PAGE 0.5 M NaCl fraction showed multiple bands after Coomassie brilliant blue staining and three distinct peaks in HPLC. In this study, we identified an anti-inflammatory protein fraction with high anionic property which could be attributed to inhibition of COX2 enzyme activity.

Effects of Hyriopsis cumingii polysaccharides on angiogenesis, macrophage chemotaxis, proliferation and phagocytosis.

Anti-angiogenic activities of crude Hyriopsis cumingii polysaccharides (HCPS) and its purified fractions (HCPS-1, HCPS-2 and HCPS-3) were evaluated in vivo using the chicken embryo chorioallantoic membrane (CAM) assay. The promoting effects of crude HCPS and its purified fractions on the chemotaxis, proliferation and phagocytosis of peritoneal macrophage were tested by cell model in vitro and cyclophosphamide-induced immuno-suppression animal model in vivo. The results showed that HCPS could significantly suppress the neovascularization of chicken embryo CAM and promote peritoneal macrophage migrating to monocyte chemotactic protein-1 (MCP-1), propagating and devouring sheep red blood cell (SRBC) in a dose-dependent manner. In addition, HCPS-3 showed stronger immunostimulatory activities in vitro than crude HCPS, HCPS-1 and HCPS-2. The beneficial effects of HCPS on the immune system might be, at least in part, attributed to the improvement of chemotaxis, proliferation and phagocytosis of peritoneal macrophage. All these results suggest that HCPS is a potential immunoenhancing and anti-tumor agent.

Bioaccumulation of selected metals in bivalves (Unionidae) and Phragmites australis inhabiting a municipal water reservoir.

Urbanization can considerably affect water reservoirs by, inter alia, input, and accumulation of contaminants including metals. Located in the course of River Cybina, Maltanski Reservoir (Western Poland) is an artificial shallow water body built for recreation and sport purposes which undergoes restoration treatment (drainage) every 4 years. In the present study, we demonstrate an accumulation of nine metals (Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, Zn) in water, sediment, three bivalve species (Anodonta anatina, Anodonta cygnea, Unio tumidus), and macrophyte Phragmites australis collected before complete drainage in November 2012. The mean concentrations of metals in the sediment, bivalves, and P. australis (roots and leaves) decreased in the following order: Fe > Mn > Zn > Cu > Cr > Ni > Pb > Co > Cd. A considerably higher bioconcentration of metals was observed in samples collected from the western and southern sites which undergo a higher degree of human impact. Sediments were found to be a better indicator of metal contamination than water samples. Interspecific differences in levels of metal accumulation were found between investigated unionids. U. tumidus accumulated higher levels of Cr, positively correlated with ambient concentrations, predisposing this species as a potential bioindicator of this metal in aquatic environments. On the other hand, species of Anodonta genus demonstrated higher accumulation of Cu and Cd. Positive correlations were found between Pb content in the sediments and tissues of all three bivalve species. In P. australis, metals were largely retained in roots except for Cd and Pb for which higher concentrations were found in leaves suggesting additional absorption of these metals from aerial sources. P. australis and bivalve from the Maltanski Reservoir may be a potential source of toxic metals for animals feeding upon them and contribute to further contamination in the food chain.

Preliminary characterization of proteoglycan from Hyriopsis cumingii.

OBJECTIVE: To study the preliminary characterizations of Hyriopsis cumingii proteoglycan (HCPG). METHODS: The content of carbohydrate and protein were measured by spectrophotometry. FTIR spectrum was used to analyze the functional groups. Relative molecular mass and amino acid composition were detected by HPLC. GC was utilized to determine the monosaccharide composition. The glycopeptide linkage-bond was detected by using the method of beta-elimination reaction. RESULTS: In HCPG,the content of carbohydrate and protein was 80.06% and 9.42%, respectively. FTIR spectrum showed the characteristic absorptions of polysaccharides and protein. Relative molecular mass of HCPG, determined by size-exclusive HPLC, was 503.1 kDa. GC spectra demonstrated that polysaccharide of HCPG was composed of rhamnose, fucose, mannose, glucose and galactose with a molar ratio of 13.80: 4.51: 7.70: 64.92 : 9.07. Fourteen amino acids (13 known and one unknown) have been detected by pre-column derivation HPLC. From beta-elimination reaction, peptide chain was attached to the carbohydrate chain by O-glycosidic bond. CONCLUSION: Basic characterizations of HCPG have been determined preliminarily.

Polybrominated diphenyl ethers in sediment and caged mussels (Elliptio complanata) deployed in the Niagara River.

Polybrominated diphenyl ethers (PBDEs) were detected in caged mussels (Elliptio complanata), deployed in the Niagara River for 3weeks in 2003, 2004 and 2006 and in sediment collected from the river and tributaries. The highest concentrations (measured as the sum of 17 BDE congeners) were in mussels deployed within tributaries to the American side of the River (Cayuga Creek: 11.5ngg(-1) wet wt.; Two Mile Creek 9.5ngg(-1); and Gill Creek: 5.8ngg(-1)). In addition to contributions from the atmosphere, each urban tributary has multiple potential sources of PBDEs which include storm sewers, sewage treatment plants and industries located upstream of the sampling sites. Total PBDE concentrations in sediment ranged from 0.25 to 188ngg(-1). In general, mean mussel tissue concentrations were positively correlated with sediment BDE concentrations. Total PBDE concentrations were higher in mussels deployed at most sites in the Tonawanda Channel which is highly industrialised, compared with mussels deployed on the Canadian side of the upper Niagara River where the surrounding area is generally rural. The sum of BDE-47, -99, -100, -153, -154 and -209 contributed between 92% and 96% of the total BDE concentrations in all of the mussel samples. BDE-209 represented between 39% and 100% of the total BDE in sediment samples. BSAFs in mussels ranged from 1 to 18 depending on the sampling station. Ratios of BDE-47/99 and BDE-100/99 and congener patterns in mussels and sediment were suggestive of the penta formulation as the historical source.

The first Toll receptor from the triangle-shell pearl mussel Hyriopsis cumingii.

Toll receptor was first discovered in Drosophila and has an important function in the innate immunity of invertebrates. In this study, the Toll receptor HcToll1 from Hyriopsis cumingii with a full length of 3810 bp consisting of a 3687 bp ORF that encodes a total of 1228 amino acids protein was selected for further study. The HcToll1 protein consisted of a signal peptide, 17 LRR domains, 2 LRRCT domains, 1 LRRNT domain, 1 TM domain, and 1 TIR domain. Phylogenetic analysis results showed that HcToll1 was clustered in one group together with other mollusca tolls. RT-PCR analysis results showed that HcToll1 was expressed in all tested tissues such as hemocytes, hepatopancreas, gills, and mantle. qRT-PCR analysis results showed that HcToll1 expression was increased by the presence of Escherichia coli, Vibrio anguillarum, Staphyloccocus aureus, and White Spot Syndrome Virus (WSSV). Over-expression of HcTIR could up-regulate expression of drosomycin gene in Drosophila S2 cells. The results of our study indicated that HcToll1 is a functional Toll and it has an important function in the generation of innate immune responses of H. cumingii against microbial challenge.

Gene identification and recombinant protein of a lysozyme from freshwater mussel Cristaria plicata.

Lysozymes are important proteins to bivalve in the innate immune responses against bacterial infections, and provide nutrition as digestion enzymes. A new LYZ1 from the freshwater mussel Cristaria plicata was cloned by rapid amplification of cDNA ends (RACE) and nested PCR method. The full-length cDNA sequence of CpLYZ1 was 763 bp. The cDNA contained a 5'-terminal untranslated region (UTR) of 21 bp, a 3'- terminal UTR of 259 bp with a 29 bp poly(A) tail, a tailing signal (AATAAA) and the open reading frame of 483 bp. The CpLYZ1 cDNA encoded a polypeptide of 160 amino acids with a predicted molecular mass of 17.8 kDa, and a theoretical isoelectric point of 6.07. The comparison of the deduced amino acid sequences with LYZs from other species showed that the enzyme belonged to i-type lysozyme. The mRNA transcript of CpLYZ1 could be detected in all the examined tissues with the highest expression level in hepatopancreas. The expression levels of CpLYZ1 in hemocytes, hepatopancreas and gill significantly increased after Aeromonas hydrophila challenge. The expression level of CpLYZ1 in hemocytes sharply decreased from 6 h to 24 h and significantly increased at 48 h, and was the highest level in hepatopancreas at 24 h, and was the maximum level in gill at 48 h. Furthermore, the recombinant CpLYZ1 was induced to be expressed as an inclusion body form by IPTG at 37 °C for 4 h, and then was purified by using the Ni(2+) affinity chromatography. The relative enzyme activity of the recombinant CpLYZ1 was influenced on pH and temperature. The optimal pH and temperature was 5.5 and 50 °C, respectively. Against Escherichia coli, A. hydrophila, Staphyloccocus aureus, Bacillus subtilis, Streptococcus sp. and Staphylococcus epidermidis, the recombinant CpLYZ1 had bacteriolytic activity.

Four invertebrate-type lysozyme genes from triangle-shell pearl mussel (Hyriopsis cumingii).

Lysozymes in animals have three types, namely chicken-type, goose-type, and invertebrate-type (i-type) lysozymes and all these 3 types have been found in bivalve mollusks. The i-type lysozymes in mollusks are involved in digestion and innate immunity. In this study, four different lysozyme genes that belong to i-type were identified from Hyriopsis cumingii. The HcLyso1 to HcLyso4 genes encode proteins with 144, 144, 161, and 228 amino acids, respectively, and contain a destabilase domain. HcLyso4 also contains SH3b domain in addition to its destabilase domain. Multiple alignments showed that two catalytic residues of Glu and Asp which were necessary for enzyme activity were present in i-type lysozymes. Phylogenetic analysis using CDS sequences of i-type lysozymes showed that these lysozymes can be divided into mollusk and crustacean clades, and that HcLyso1 to HcLyso4 all belong to the mollusk clades. Although there was no positive selection predicted in i-type lysozymes, some branches suffered rapid evolution. HcLyso1 is mainly expressed in hepatopancreas and can be detected in hemocytes. HcLyso2 is primarily expressed in hepatopancreas and can be detected in hemocytes Whereas, HcLyso3 can be detected mainly in hemocytes, hepatopancreas, gills, and mantle. HcLyso4 is expressed in hemocytes and hepatopancreas. qRT-PCR analysis showed that HcLyso1 to HcLyso4 were all nearly down-regulated by Vibrio or Staphylococcus aureus challenge. Moreover, our research indicated that HcLyso1 to HcLyso4 might play a key role in the innate immunity of mussel.

Chemical analysis of a polysaccharide from Cristaria plicata (Leach).

A polysaccharide (MPS) isolated from Cristaria plicata (Leach) consisted of d-glucose. Its structural characteristics were investigated by High Performance Liquid Chromatography (HPLC), infrared analysis, gas chromatography-MS, total acid hydrolysis, methylation analysis, periodate oxidation and Smith degradation. The results indicated that the polysaccharide of C. plicata (Leach) has the weight-average molecular weight of about 2.97 × 106 Da. The structure of the polysaccharide was composed of glucose with α-(1 → 4)-linkages with short exterior chains. The fundamental information obtained from this work is beneficial to the interpretation in the relationship of the polysaccharide structure and its biological functions, and suggests that the polysaccharide from mussel may contribute to be used as a dietary supplement for health foods and therapeutics.

Immunostimulatory activity of the polysaccharides from Hyriopsis cumingii.

The immunostimulatory activity of Hyriopsis cumingii polysaccharides (HCPS) was evaluated by using in vitro cell models and in vivo animal models. The results demonstrated that crude HCPS and its purified fractions (HCPS-1, HCPS-2 and HCPS-3) could promote the splenocyte proliferation, increase the activity of acid phosphatase in peritoneal macrophages and strengthen peritoneal macrophages to devour neutral red in vitro in a dose-dependent manner. Furthermore, HCPS-3 exhibited much stronger immunostimulatory activity in vitro than crude HCPS, HCPS-1 and HCPS-2, probably due to the higher sulfate content of HCPS-3. For assay in vivo, crude HCPS significantly increased the indices of spleen and thymus, the activity of lysozyme in serum and the swelling rate of earlap in delayed-type hypersensitivity in a dose-dependent manner. These results suggested that HCPS might be a potential natural immunomodulator.

A neuroprotective polysaccharide from Hyriopsis cumingii.

A water-soluble polysaccharide from Hyriopsis cumingii (HCp) was isolated by hot-water extraction and ethanol precipitation. We investigated the preliminary neuroprotective activity of HCp on cerebral ischemia/reperfusion (I/R)-induced rat brain tissue injury using the middle cerebral artery occlusion (MCAO) model. The structural analysis showed that the major component of HCp (HP-I) is an α-(1→4)-d-glucan branched with a single α-d-glucose at C-6 every five residues on average and has a weight-average molecular weight of about 2.65 × 10(5). Pharmacological studies revealed that HCp improves and restores nerve function impairment, significantly increases brain tissue total superoxide dismutase activity, lowers malondialdehyde content effectively, and reduces brain levels of caspase-3 mRNA expression in a dose-effect manner, indicating that H. cumingii polysaccharide possesses significant neuroprotective effects.

Prevention of the progression of adjuvant induced arthritis by oral supplementation of Indian fresh water mussel (Lamellidens marginalis) aqueous extract in experimental rats.

AIM OF THIS STUDY: Mussel is well accepted as food all over India. Beside for its nutritive value, people residing in Kosi river basin, Bihar, India, consume a preparation of soup, made from the footpad of molluscan species, with the belief that it gives relief from signs and symptoms of joint pain and related problems. This study was designed to explore the preventive activity of Indian fresh water mussel (Lamellidens marginalis) aqueous extract oral supplementation in experimental arthritis model. MATERIALS AND METHODS: Arthritis was induced in male albino rats by intradermal injection of Freund's complete adjuvant in right hind footpad. Lamellidens marginalis extract (LME1, 500 mg/kg/day and LME2, 1 g/kg/day) peroral supplementation started from the 1st day after adjuvant injection and was continued for the subsequent 13 days. Severity of arthritis was evaluated from paw diameter, ankle diameter, paw weight, urinary hydroxyproline, glucosamine level, serum interleukin-1ß, IL6, IL10, CINC1, TNFα level, lysosomal enzyme levels and from histopathological assessment. RESULTS: Lamellidens marginalis extract supplementation significantly (p<0.05) decreased paw diameter, ankle diameter, and paw weight in treated groups (LME1, 500 mg/kg/day and LME2, 1 g/kg/day) as compared with arthritic group. Urinary hydroxyproline, glucosamine level, serum IL1ß, IL6, CINC1, TNFα, IL10 and lysosomal enzyme levels were restored significantly (p<0.05) in treated groups (LME1, 500 mg/kg/day and LME2, 1 g/kg/day) as compared to arthritic group. Synovial membrane damage and neutrophil infiltration in histopathological examination was restored significantly by LME supplementation as compared to arthritic group. CONCLUSIONS: Thus, it might be concluded that experimental animals supplemented with Lamellidens marginalis extract were protected against the severity of disease progression in adjuvant induced arthritis.

Effects of pearl powder extract and its fractions on fibroblast function relevant to wound repair.

The water soluble matrix (WSM) of pearl powder [Hyriopsis cumingii Lea (Unionidae)] was extracted, and the insoluble residue was demineralized, size-fractionated, and named as MR14 (> 14 kDa), MR3-14 (3-14 kDa), and MR3 (< 3 kDa). The effects of WSM, MR14, MR3-14, and MR3 on primary mouse oral fibroblast proliferation, collagen accumulation, matrix metalloproteinase-2, -9 (MMP-2, -9) activities, and tissue inhibitor of metalloproteinase-1 (TIMP-1) production were tested by MTT assay, chloramine T method, gelatin zymography, and enzyme-linked immunosorbent assay (ELISA), respectively. The results showed that the WSM and MR14 could significantly (p < 0.05) promote fibroblast proliferation; all of the fractions could significantly promote collagen accumulation; MR14 significantly (p < 0.05) inhibited MMP-2 activity; and all of the fractions could significantly promote TIMP-1 production. This study has proved that the mechanism by which pearl powder promotes wound healing is partly due to its ability to stimulate fibroblast mitosis, collagen deposition, and TIMP-1 production, and the major active fraction may be MR14.

[Analysis of complete mitochondrial genome of Cristaria plicata].

The complete mitochondrial genome of Cristaria plicata was obtained using long amplification polymerase chain reaction (LA-PCR). Analysis showed that the total length of sequence was 15 712 bp, including 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, and 26 non-coding regions ranged from 2 bp to 328 bp in size. The nucleotide composition of A, T, C, G were 36.54% 27.22% 23.22% and 13.02%, respectively. Most genes were encoded on the L strand, While ND3-ND5, ND4L, COI-COIII, ATP6, ATP8, tRNAAsp, and tRNAHis were encoded on the H strand. The arrangement of genes in C. plicata was identical to that of Lampsilis ornata, but was different from that of Hyriopsis cum-ingii between COII and 12S rRNA in the same family. Thirteen protein genes contained 3 initiation codons, i.e., I (AUU, AUC), V (GUG), and M (AUA, AUG), all of which had complete stop codons (UAA or UAG), except for the stop codon in ND2 that had an incomplete T. Fifteen tRNAs had typical cloverleaf structure, except for tRNAThr, tRNALys, tRNASer(UCN), tRNAAsp, tRNAArg, tRNATyr, and tRNAMet. Like other freshwater bivalvia, C. plicata contained ATP8 gene, which might be related to the balance of osmotic pressure in the cytoplasm.