Study on bioactivity of extracts from marine sponges in Chinese Sea

The bioactivity screening of fractions from two inter-tidal sponges collected from the north of China Yellow Sea and one sponge collected from the South Chinese Sea was reported in this study. In sponge Hymeniacidon perleve there were 9 fractions out of 15 from CHCl₃ extract with anti Staphylococcus aureus activity, 9 fractions out of 19 from BuOH extract with anti Escherichia coli activity, and three fractions from CHCl₃ extract which had moderate to strong activity in inhibiting Bacillus subtilis, Candida albicans, and Aspergilus niger. The fractions of Reniochalina sp. showed bioactivity against bacteria and fungi. The fractions of Acanthella acuta Schmidt showed bioactivity against S. aureus and fungi. One compound from H. perleve obtained by the bioactively directing isolation was tested for bioactivity against the human hepatoma cell line Qgy7701 (IC₅₀ 10.1 μg/ml), Burkitt's lymphoma cell line Raji (IC₅₀ 9.76 μg/ml) and chronic myelogenous leukemia K562 (IC₅₀ 1.90 μg/ml).     

Aspidosperma (Apocynaceae) plant cytotoxicity and activity towards malaria parasites. Part II: experimental studies with Aspidosperma ramiflorum in vivo and in vitro

Several species of Aspidosperma plants are used to treat diseases in the tropics, including Aspidosperma ramiflorum, which acts against leishmaniasis, an activity that is experimentally confirmed. The species, known as guatambu-yellow, yellow peroba, coffee-peroba andmatiambu, grows in the Atlantic Forest of Brazil in the South to the Southeast regions. Through a guided biofractionation of A. ramiflorum extracts, the plant activity against Plasmodium falciparum was evaluated in vitro for toxicity towards human hepatoma G2 cells, normal monkey kidney cells and nonimmortalised human monocytes isolated from peripheral blood. Six of the seven extracts tested were active at low doses (half-maximal drug inhibitory concentration < 3.8 µg/mL); the aqueous extract was inactive. Overall, the plant extracts and the purified compounds displayed low toxicity in vitro. A nonsoluble extract fraction and one purified alkaloid isositsirikine (compound 5) displayed high selectivity indexes (SI) (= 56 and 113, respectively), whereas compounds 2 and 3 were toxic (SI < 10). The structure, activity and low toxicity of isositsirikine in vitro are described here for the first time in A. ramiflorum, but only the neutral and precipitate plant fractions were tested for activity, which caused up to 53% parasitaemia inhibition of Plasmodium berghei in mice with blood-induced malaria. This plant species is likely to be useful in the further development of an antimalarial drug, but its pharmacological evaluation is still required.     

Development of Chloroplast Microsatellite Markers and Analysis of Chloroplast Diversity in Chinese Jujube (Ziziphus jujuba Mill.) and Wild Jujube (Ziziphus acidojujuba Mill.)

Ziziphus is an important genus within the family Rhamnaceae. This genus includes several important fruit tree species that are widely planted in China and India, such as the Chinese jujube (Ziziphus jujuba Mill.), the wild jujube (Z. acidojujuba), and the Indian jujube (Z. mauritiana). However, information about their domestication based on the chlorotype diversity of Chinese jujube population is lacking. In this study, chloroplast microsatellite (cpSSR) markers were developed and used to investigate the genetic relationships between and domestication of jujube cultivars and wild jujube populations. Primer sets flanking each of the 46 cpSSR loci in non-coding regions of the chloroplast genome sequence of Z. jujuba Mill. cv. ‘Junzao’ were designed. In total, 10 markers showed polymorphisms from 15 samples (9 jujube cultivars and 6 wild jujube individuals), of which 8 loci were due to variations in the number of mononucleotide (A/T) repeats and 2 were due to indels. Six cpSSR markers were used in further analyses of 81 additional samples (63 jujube cultivars, 17 wild jujube samples, and 1 Indian jujube). Using these cpSSR markers, the number of alleles per locus ranged from two to four. In general, the Shannon Index (I) for each cpSSR ranged from 0.159 to 0.1747, and the diversity indices (h) and uh were 0.061 to 0.435 and 0.062 to 0.439, respectively. Seven chlorotypes were found; the Indian jujube showed distinct chlorotypes, and both the Chinese and wild jujube had four chlorotypes and shared two chlorotypes. A dominant chlorotype (G) accounted for 53 of 72 jujube cultivars and 13 of 23 wild jujube individuals. All chlorotypes were highly localized along the Yellow River, from the mid- to the lower reaches, suggesting a wide origin of jujube. These cpSSR markers can be applied to population and evolution studies of Chinese jujube and wild jujube.     

红枣贮藏期果面微生物多样性

以2种成熟度不同的红枣(Zizyphus jujuba Mill.)为试验材料在入库前采用保鲜剂处理,未采用保鲜剂处理的红枣为试验对照,研究红枣不同贮藏时期果面微生物多样性。结果表明:红枣贮藏期果面微生物种群中,细菌是优势类群,占微生物总数80%以上,真菌较少;采摘期红枣果面上就携带微生物,真菌优势菌株属于链格孢(Alternaria),其次为镰刀菌(Fusarium)和木霉(Trichoderma),贮藏期优势菌株属于链格孢(Alternaria)、青霉(Penicillium)、木霉(Trichoderma)和镰刀菌(Fusarium),曲霉(Aspergillus)、毛霉(Mucor)和根霉(Rhizopus)次之。红枣贮藏期果面微生物种群动态失衡是导致贮藏病害发生的主要原因,成熟度较高的红枣果面微生物总数、真菌和细菌的数量明显高于成熟度较低的红枣,使用保鲜剂后有效地降低了红枣贮藏期果面微生物的多样性,贮藏时间越长红枣果面微生物的多样性越明显。     

枣园桃蛀果蛾寄生蜂种类及其与寄主的关系

桃蛀果蛾(Carposina sasakii Matsumura)是我国十分重要的果实性害虫,其中枣受害最严重,因其幼虫在果实内蛀食危害、高度隐蔽性的生活而较难防治。寻找有效天敌,开展生物防治成为必然。2009年与2010年研究调查了我国大枣部分产区陕西、山西、河南与宁夏枣园桃蛀果蛾寄生性天敌,共发现了3种寄生蜂,即中华齿腿姬蜂(Pristomerus chinensis Ashmead)、章氏小甲腹茧蜂(Chelonus(Microchelonus)zhangi Zhang)和金小蜂(Pteromalidae),3种寄生蜂均从桃蛀果蛾的茧中羽化而出,其中中华齿腿姬蜂和章氏小甲腹茧蜂为优势天敌,但它们表现出十分明显的地域性分布。同时,对中华齿腿姬蜂和章氏小甲腹茧蜂羽化、寄生率以及与寄主之间的关系进行了初步研究,结果表明,中华齿腿姬蜂越冬代成虫出蛰活动始于6月中旬,越冬代寄生率较低,第1代成虫羽化在8月下旬至9月上旬,这代寄生蜂具有较高的寄生率,可达25%;章氏小甲腹茧蜂第1代成虫羽化集中在8月中下旬和9月上旬,由于在2009年与2010年对样地进行不施用杀虫剂的管理,该天敌在2010年自然寄生率显著提高,最高时可达64.52%。因此,科学管理,合理保护与利用当地天敌成为实现该害虫生物防治的关键。     

Structural characterization of laminaran and galactofucan extracted from the brown seaweed Saccharina longicruris

Brown seaweed contains several polysaccharides like laminaran, fucoidan and alginate. Laminaran is a β-glucan that has shown anti-apoptotic and anti-tumoral activities, while galactofucan (fucoidan) is a sulfated polysaccharide that has displayed anticoagulant, anti-tumor, anti-thrombosis, anti-inflammatory and antiviral properties. In this study, crude laminaran and galactofucan (fucoidan) were extracted from the brown seaweed Saccharina longicruris at four harvest periods (M05, A05, N05 and J06). The galactofucan M05 and N05 fractions were depolymerized (RDP) over 2 or 4 h to give 4 RDP fractions (M05 RDP 2H, M05 RDP 4H, N05 RDP 2H and N05 RDP 4H) whose molecular weights, monosaccharide compositions and glycosidic linkages were determined by GC-MS. The laminaran fraction gave a molecular weight range from 2900 to 3300 Da and contained between 50.6% and 68.6% d-glucose and an average of 1.3% d-mannitol. The presence of a β-(1,3) linkage between d-glucose in the main chain was observed, with branching at positions 6 and 2. The M05 fraction contained less branching than other laminaran fractions, which might have influenced its conformation in solution and thus its activity. The crude galactofucan fractions displayed a molecular weight range from 638 to 1529 kDa, whereas the RDP fractions had molecular weights <30 kDa. The structure of the galactofucan fractions remained complex after depolymerization, with these also being more sulfated (30-39%) than the crude fractions (13-20%). The crude and RDP fractions contained 3-linked fucopyranose 4-sulfate and 6-linked galactopyranose 3-sulfate moieties, although the galactofucans isolated from M05 and J06 contained less 6-linked galactopyranose 3-sulfate than the A05 and N05 fractions.     

In vitro and in vivo antimalarial activity and cytotoxicity of extracts,fractions and a substance isolated from the Amazonian plant Tachia grandiflora (Gentianaceae)

Tachia sp. are used as antimalarials in the Amazon Region and in vivo antimalarial activity of a Tachia sp. has been previously reported. Tachia grandiflora Maguire and Weaver is an Amazonian antimalarial plant and herein its cytotoxicity and antimalarial activity were investigated. Spectral analysis of the tetraoxygenated xanthone decussatin and the iridoid aglyone amplexine isolated, respectively, from the chloroform fractions of root methanol and leaf ethanol extracts was performed. In vitro inhibition of the growth of Plasmodium falciparum Welch was evaluated using optical microscopy on blood smears. Crude extracts of leaves and roots were inactive in vitro. However, chloroform fractions of the root and leaf extracts [half-maximal inhibitory concentration (IC50) = 10.5 and 35.8 µg/mL, respectively] and amplexine (IC50= 7.1 µg/mL) were active in vitro. Extracts and fractions were not toxic to type MRC-5 human fibroblasts (IC50> 50 µg/mL). Water extracts of the roots of T. grandiflora administered by mouth were the most active extracts in the Peters 4-day suppression test in Plasmodium berghei-infected mice. At 500 mg/kg/day, these extracts exhibited 45-59% inhibition five to seven days after infection. T. grandiflora infusions, fractions and isolated substance have potential as antimalarials.     

Synthesis of a covalent gemcitabine-(carbamate)-[anti-HER2/neu] immunochemotherapeutic and its cytotoxic anti-neoplastic activity against chemotherapeutic-resistant SKBr-3 mammary carcinoma

Gemcitabine is a potent chemotherapeutic that exerts cytotoxic activity against several leukemias and a wide spectrum of carcinomas. A brief plasma half-life in part due to rapid deamination and chemotherapeutic-resistance frequently limit the utility of gemcitabine in clinical oncology. Selective ‘targeted’ delivery of gemcitabine represents a potential molecular strategy for simultaneously prolonging its plasma half-life and minimizing exposure of innocent tissues and organ systems.

Materials and methods

Gemcitabine was combined in molar excess with N-[p-maleimidophenyl]-isocyanate (PMPI) so that the isocyanate moiety of PMPI which exclusively reacts with hydroxyl groups preferentially created a carbamate covalent bond at the terminal C₅-methylhydroxy group of gemcitabine. Monoclonal immunoglobulin with binding-avidity specifically for HER2/neu was thiolated with 2-iminothiolane at the terminal ??-amine group of lysine amino acid residues. The gemcitabine-(carbamate)-PMPI intermediate with a maleimide moiety that exclusively reacts with reduced sulfhydryl groups was then combined with thiolated anti-HER2/neu monoclonal immunoglobulin. Western-blot analysis was utilized to delineate the molecular weight profile for gemcitabine-(carbamate)-[anti-HER2/neu] while cell binding characteristics were determined by cell-ELISA utilizing SKBr-3 mammary carcinoma which highly over-expresses HER2/neu receptors. Cytotoxic anti-neoplastic potency of gemcitabine-(carbamate)-[anti-HER2/neu] between the gemcitabine-equivalent concentrations of 10⁻¹² and 10⁻⁶ M was determined utilizing vitality staining analysis of chemotherapeutic-resistant SKBr-3 mammary carcinoma.

Results

Gemcitabine-(carbamate)-[anti-HER2/neu] was synthesized at a molar incorporation index of 1:1.1 (110%) and had a molecular weight of 150 kDa that was indistinguishable from reference control immunoglobulin fractions. Cell-ELISA detected progressive increases in SKBr-3 mammary carcinoma associated immunoglobulin with corresponding increases in covalent gemcitabine immunochemotherapeutic concentrations. The in vitro cytotoxic anti-neoplastic potency of gemcitabine-(carbamate)-[anti-HER2/neu] was approximately 20% and 32% at 10⁻⁷ and 10⁻⁶ M (gemcitabine-equivalent concentrations) after a 182-h incubation period.

Discussion

The investigations describes for the first time a methodology for synthesizing a gemcitabine anti-HER2/neu immunochemotherapeutic by creating a covalent bond structure between the C₅-methylhydroxy group of gemcitabine and thiolated lysine amino acid residues of monoclonal antibody or other biologically active protein fractions. Gemcitabine-(carbamate)-[anti-HER2/neu] possessed binding-avidity at HER2/neu receptors highly over-expressed by chemotherapeutic-resistant SKBr-3 mammary carcinoma. Alternatively, gemcitabine can be covalently linked at its C₅-methylhydroxy group to monoclonal immunoglobulin fractions that possess binding-avidity for other receptors and membrane complexes uniquely highly over-expressed by a variety of neoplastic cell types. Compared to chemotherapeutic-resistant SKBr-3 mammary carcinoma, gemcitabine-(carbamate)-[anti-HER2/neu] immunochemotherapeutic is anticipated to exert higher levels of cytotoxic anti-neoplastic potency against other neoplastic cell types like pancreatic carcinoma, small-cell lung carcinoma, neuroblastoma, glioblastoma, oral squamous cell carcinoma, cervical epithelioid carcinoma, or leukemia/lymphoid neoplastic cell types based on their reportedly greater sensitivity to gemcitabine and gemcitabine covalent conjugates.     

In vivo antipyretic,antiemetic, in vitro membrane stabilization,antimicrobial, and cytotoxic activities of different extracts from Spilanthes paniculata leaves

Background

The study was conducted to evaluate the in vitro antimicrobial activity, cytotoxic, and membrane stabilization activities, and in vivo antiemetic and antipyretic potentials of ethanolic extract, n-hexane and ethyl acetate soluble fractions of Spilanthes paniculata leaves for the first time widely used in the traditional treatments in Bangladesh.

Results

In antipyretic activity assay, a significant reduction (P < 0.05) was observed in the temperature in the mice tested. At dose 400 mg/kg-body weight, the n-hexane soluble fraction showed the effect (36.7 ± 0.63°C ) as like as the standard (dose 150 mg/kg-body weight) after 5 h of administration. Extracts showed significant (P < 0.001) potential when tested for the antiemetic activity compared to the standard, metoclopramide. At dose 50 mg/kg-body weight, the standard showed 67.23% inhibition, whereas n-hexane and ethyl acetate soluble fractions showed 37.53% and 24.93% inhibition of emesis respectively at dose 400 mg/kg-body weight. In antimicrobial activity assay, the n-hexane soluble fraction (400 μg/disc) showed salient activity against the tested organisms. It exerts highest activity against Salmonella typhi (16.9 mm zone of inhibition); besides, crude, and ethyl acetate extracts showed resistance to Bacillus cereus and Bacillus subtilis, and Vibrio cholera respectively. All the extracts were tested for lysis of the erythrocytes. At the concentration of 1mg/ml, ethanol extract, and n-hexane and ethyl acetate soluble fractions significantly inhibited hypotonic solution induced lysis of the human red blood cell (HRBC) (27.406 ± 3.57, 46.034 ± 3.251, and 30.72 ± 5.679% respectively); where standard drug acetylsalicylic acid (concentration 0.1 mg/ml) showed 77.276 ± 0.321% inhibition. In case of heat induced HRBC hemolysis, the plant extracts also showed significant activity (34.21 ± 4.72, 21.81 ± 3.08, and 27.62 ± 8.79% inhibition respectively). In the brine shrimp lethality bioassay, the n-hexane fraction showed potent (LC₅₀ value 48.978 μg/ml) activity, whereas ethyl acetate fraction showed mild (LC₅₀ value 216.77 μg/ml) cytotoxic activity.

Conclusions

Our results showed that the n-hexane extract has better effects than the other in all trials. In the context, it can be said that the leaves of S. paniculata possess remarkable pharmacological effects, and justify its folkloric use as antimicrobial, antipyretic, anti-inflammatory, and antiemetic agent. Therefore, further research may be suggested to find possible mode of action of the plant part.     

Entamoeba histolytica: Soluble and membrane-associated neutral sphingomyelinase-C and other unidentified esterase activity

Sphingomyelinase (SMase) activity was measured in Entamoeba histolytica particulate and soluble subcellular fractions. The effects on SMase of incubation time, total protein concentration, pH, and several divalent cations were determined. SMase-C and other unidentified esterase activity were detected in soluble and particulate fractions. SMase-C was 94.5-96.0% higher than the unidentified esterase activity. Soluble and insoluble SMase-C specific activities increased with protein dose and incubation time. Soluble and insoluble SMase-C activities were maximum at pH 7.5 and were dependent on Mg²⁺, Mn²⁺, or Co²⁺, and inhibited by Zn²⁺, Hg²⁺, Ca²⁺, and EDTA. SMase-C was active in the pH range of 3-10 and its maximum activity was at pH 7.5. The soluble and insoluble SMases have remarkably similar physicochemical properties, strongly suggesting that E. histolytica has just one isoform of neutral SMase-C that had not been described before and might be essential for E. histolytica metabolism or virulence.     

Repellent activity of alligator pepper, Aframomum melegueta, and ginger, Zingiber officinale, against the maize weevil, Sitophilus zeamais

The repellent activity of alligator pepper, Aframomum melegueta, and ginger, Zingiber officinale (Zingiberaceae), against the maize weevil, Sitophilus zeamais (Coleoptera: Curculionidae), was investigated in four-way olfactometer bioassays. Results showed that vacuum distilled A. melegueta and Z. officinale extracts were repellent towards adult S. zeamais both in the absence and the presence of maize, Zea mays, grains. Bioassay-guided liquid chromatographic fractionation of the distillates showed that fractions containing oxygenated compounds accounted for the repellent activity. Coupled gas chromatography-mass spectrometry (GC-MS), followed by GC peak enhancement and enantioselective GC using authentic compounds, identified 3 major compounds in the behaviourally active fractions of A. melegueta and Z. officinale to be (S)-2-heptanol, (S)-2-heptyl acetate and (R)-linalool in a ratio of 1:6:3, and 1,8-cineole, neral and geranial in a ratio of 5.48:1:2.13, respectively. The identification of these behaviourally active compounds provides the scientific basis for the observed repellent properties of A. melegueta and Z. officinale, and demonstrates the potential for their use in stored-product protection at the small-scale farmer level in Africa.     

Influences of Environmental Factors on Leaf Morphology of Chinese Jujubes

Rainfall and temperature are the primary limiting factors for optimum quality and yield of cultivated jujube (Ziziphus jujuba Mill.). Adaptation to arid and cool environments has been and remains an important goal of many jujube improvement programs. This study summarized the survey results of 116 Chinese jujube varieties grown at 33 sites in China. The objective was to identify the environmental factors that influence leaf morphology, and the implications for breeding and introduction of new jujube varieties. Jujube leaf morphological traits were evaluated for their potential relationships with mean annual temperature (MAT) and mean annual precipitation (MAP). The results showed that many leaf morphological traits had a strong linear relationship with local precipitation and temperature. Longer veins per unit area (VLA) and reduced leaf area and leaf perimeter were typical of arid areas. VLA was inversely related to MAT and MAP at the centers of origin of jujube. There was a positive relationship between leaf shape (perimeter²/area) and both MAT and MAP. These results indicated that leaf vein traits of Chinese jujubes might have resulted from their adaptation to environmental factors in the course of long-term evolution. Principal component analysis allocated the 116 jujube varieties to three different groups, differentiated on the basis of morphological and physiological leaf characteristics. Jujube varieties from the Hebei, Shandong, Henan, southern Shanxi and central Shaanxi provinces were closely related, as were varieties from northwest Shanxi and northeast Shaanxi provinces, and varieties from the Gansu and Ningxia provinces. These close relationships were partially attributed to the frequent exchanges of varieties within each group. Leaf venation characteristics might be used as reference indices for jujube variety introduction between different locations.Influences of Environmental Factors on Leaf Morphology of Chinese Jujubes     

Phytochemical and toxicological investigations of crude methanolic extracts,subsequent fractions and crude saponins of Isodon rugosus

Background

Isodon rugosus is used traditionally in the management of hypertension, rheumatism, tooth-ache and pyrexia. Present study was arranged to investigate I. rugosus for phytoconstituents, phytotoxic and cytotoxic activities to explore its toxicological, pharmacological potentials and to rationalize its ethnomedicinal uses. Briefly, qualitative phytochemical analysis of the plant extracts were carried out for the existence of alkaloids, flavonoids, saponins, oils, glycosides, anthraquinones, terpenoids, sterols and tannins. Plant crude methanolic extract (Ir.Cr), its subsequent fractions; n-hexane (Ir.Hex), chloroform (Ir.Chf), ethyl acetate (Ir.EtAc), aqueous (Ir.Aq) and saponins (Ir.Sp) in different concentrations were tested for phytotoxic and cytotoxic activities using radish seeds and brine shrimps (Artemia salina) respectively. The phytotoxic activity was determined by percent root length inhibition (RLI) and percent seeds germination inhibition (SGI) while the cytotoxicity was obtained with percent lethality of the brine shrimps.

Results

Ir.Cr was tested positive for the presence of alkaloids, glycosides, flavonoids, oils, terpenoids, saponins, tannins and anthraquinones. Among different fractions Ir.Sp, Ir.Chf, Ir.EtAc, and Ir.Cr were most effective causing 93.55, 89.32, 81.32 and 58.68% inhibition of seeds in phytotoxicity assay, with IC₅₀ values of 0.1, 0.1, 0.1 and 52 μg/ml respectively. Similarly, among all the tested samples, Ir.Sp exhibited the highest phytotoxic effect causing 91.33% root length inhibition with IC₅₀ of 0.1 μg/ml. Ir.Sp and Ir.Chf were most effective against brine shrimps showing 92.23 and 76.67% lethality with LC₅₀ values of 10 and 12 μg/ml respectively.

Conclusions

It may be inferred from the current investigations that I. rugosus contains different secondary metabolites and is a potential source for the isolation of natural anticancer and herbicidal drug molecules. Different fractions exhibited phytotoxic and cytotoxic activities, thus providing pharmacological basis for ethnomedicinal uses of this plant.     

Identification and functional analysis of a novel mutation in the SOX10 gene associated with Waardenburg syndrome type IV

Waardenburg syndrome type IV (WS4) is a rare genetic disorder, characterized by auditory–pigmentary abnormalities and Hirschsprung disease. Mutations of the EDNRB gene, EDN3 gene, or SOX10 gene are responsible for WS4. In the present study, we reported a case of a Chinese patient with clinical features of WS4. In addition, the three genes mentioned above were sequenced in order to identify whether mutations are responsible for the case. We revealed a novel nonsense mutation, c.1063C>T (p.Q355*), in the last coding exon of SOX10. The same mutation was not found in three unaffected family members or 100 unrelated controls. Then, the function and mechanism of the mutation were investigated in vitro. We found both wild-type (WT) and mutant SOX10 p.Q355* were detected at the expected size and their expression levels are equivalent. The mutant protein also localized in the nucleus and retained the DNA-binding activity as WT counterpart; however, it lost its transactivation capability on the MITF promoter and acted as a dominant-negative repressor impairing function of the WT SOX10.     

Genome-Wide Characterization of Simple Sequence Repeat (SSR) Loci in Chinese Jujube and Jujube SSR Primer Transferability

Chinese jujube (Ziziphus jujuba), an economically important species in the Rhamnaceae family, is a popular fruit tree in Asia. Here, we surveyed and characterized simple sequence repeats (SSRs) in the jujube genome. A total of 436,676 SSR loci were identified, with an average distance of 0.93 Kb between the loci. A large proportion of the SSRs included mononucleotide, dinucleotide and trinucleotide repeat motifs, which accounted for 64.87%, 24.40%, and 8.74% of all repeats, respectively. Among the mononucleotide repeats, A/T was the most common, whereas AT/TA was the most common dinucleotide repeat. A total of 30,565 primer pairs were successfully designed and screened using a series of criteria. Moreover, 725 of 1,000 randomly selected primer pairs were effective among 6 cultivars, and 511 of these primer pairs were polymorphic. Sequencing the amplicons of two SSRs across three jujube cultivars revealed variations in the repeats. The transferability of jujube SSR primers proved that 35/64 SSRs could be transferred across family boundary. Using jujube SSR primers, clustering analysis results from 15 species were highly consistent with the Angiosperm Phylogeny Group (APGIII) System. The genome-wide characterization of SSRs in Chinese jujube is very valuable for whole-genome characterization and marker-assisted selection in jujube breeding. In addition, the transferability of jujube SSR primers could provide a solid foundation for their further utilization.     

Preparation and properties of 5′-nucleotidases from bovine milk fat globule membranes

The 5′-nucleotidase (5′-ribonucleoside phosphohydrolase, EC 3.1.3.5) from bovine milk fat globule membranes was partially purified. Two separate peaks of activity were obtained from a Sepharose column and the two fractions, designated V and VI in order of elution, were collected and characterized separately. Both V and VI exhibited pH optima between 7.0 and 7.5 for AMP, GMP and CMP in the absence of metal ions. In the presence of Mg²⁺, a second pH optimum at 10.0 was observed with both fractions. Low concentrations of MnCl₂ activated Fraction V but not Fraction VI. HgCl₂ was a potent inhibitor of both fractions. The relative rates of hydrolysis of various 5′-mononucleotides differed comparing the two fractions. Optimum temperature for P_i release was 69 °C for both fractions. Activation energies were 10 400 cal/mole and 9600 cal/mole for Fractions V and VI, respectively. For V, calculated K_m values for AMP, GMP and CMP were 0.94, 2.5 and 1.16 mM, respectively. Calculated K_m values for Fraction VI for AMP, GMP and CMP were 5.0, 3.95 and 1.73 mM, respectively. ATP was a competitive inhibitor of AMP hydrolysis by Fraction V and a noncompetitive inhibitor of AMP hydrolysis by Fraction VI. Both fractions contained chloroform-methanol-extractable phospholipid. The phospholipid distribution pattern of Fraction VI was similar to that of milk fat globule membranes. Fraction V contained only sphingomyelin and phosphatidylcholine. It is proposed that milk fat globule membranes contain two separate 5′-nucleotidases.     

Cuscuta europaea plastid apparatus in various developmental stages: Localization of THF1 protein

It was generally accepted that Cuscuta europaea is mostly adapted to a parasitic lifestyle with no detectable levels of chlorophylls. We found out relatively high level of chlorophylls (Chls a+b) in young developmental stages of dodder. Significant lowering of Chls (a+b) content and increase of carotenoid concentration was typical only for ontogenetically more developed stages. Lower content of photosynthesis-related proteins involved in Chls biosynthesis and in photosystem formation as well as low photochemical activity of PSII indicate that photosynthesis is not the main activity of C. europaea plastids. Previously, it has been shown in other species that the Thylakoid Formation Protein 1 (THF1) is involved in thylakoid membrane differentiation, plant-fungal and plant-bacterial interactions and in sugar signaling with its preferential localization to plastids. Our immunofluorescence localization studies and analyses of haustorial plasma membrane fractions revealed that in addition to plastids, the THF1 protein localizes also to the plasma membrane and plasmodesmata in developing C. europaea haustorium, most abundantly in the digitate cells of the endophyte primordium. These results are supported by western blot analysis, documenting the highest levels of the THF1 protein in “get together” tissues of dodder and tobacco. Based on the fact that photosynthesis is not a typical process in the C. europaea haustorium and on the extra-plastidial localization pattern of the THF1, our data support rather other functions of this protein in the complex relationship between C. europaea and its host.     

Metabolism of dulcitol in Euonymus japonica

Dulcitol-1(6)-¹⁴C was administered to leaves of E. japonica and samples were taken for time periods ranging from 0·5 to 24 hr. For each time period the absolute activity of the glucose, galactose and dulcitol pools was determined. Such studies demonstrated that dulcitol is converted to glucose and galactose. The initial product was glucose, some of which was converted to galactose, glacturonic acid and glucuronic acid. Fractionation of a leaf sample into its pectin, lignin, hemicellulose and α-cellulose components, with subsequent hydrolysis, showed that the dulcitol pool is used in the synthesis of structural carbohydrates. The activity of these fractions was shown to reside in dulcitol, glucose, galactose, galacturonic acid and glucuronic acid residues.