Improvement of phenylethanoid glycosides production by a fungal elicitor in cell suspension culture of Cistanche deserticola

When, on the 15th day of growth, an elicitor from Fusarium solani was added at 40 mg l^–1 to Cistanche deserticola cell suspension cultures, the contents of echinacoside, acteoside and total phenylethanoid glycosides (PeGs) in cultured cells all increased over the next 27 d by over 100% to 15 mg g^–1 dry wt, 9 mg g^–1 dry wt and 57 mg g^–1 dry wt, respectively. The final biomass (1.3 mg dry wt ml^–1) was not affected.     

Pinoresinol and matairesinol accumulation in a Forsythia × intermedia cell suspension culture

A newly established Forsythia × intermedia cell suspension culture was shown to accumulate (+)- and (–)-pinoresinol as well as matairesinol. The influence of the sucrose content of the culture medium and of the cultivation time on pinoresinol and matairesinol accumulation was evaluated. The highest pinoresinol yield was achieved from cells grown in medium containing 6% sucrose for 12 ± 2 days with levels of 0.6–0.8 mg g^–1 dry weight and an average enantiomeric composition of 75 ± 5% (+)-pinoresinol. The highest matairesinol amount was reached in the same medium at the 14th ± 2 culture day with levels of 1.0–2.7 mg g^–1 dry weight. To our knowledge, this is the first report on pinoresinol accumulation in Forsythia × intermedia plants or cell suspension cultures.     

Identification of the site of inhibition of oncogenic ras-p21-induced signal transduction by a peptide from a ras effector domain

We have previously found that a peptide corresponding to residues 35–47 of the ras-p21 protein, from its switch 1 effector domain region, strongly inhibits oocyte maturation induced by oncogenic p21, but not by insulin-activated cellular wild-type p21. Another ras–p21 peptide corresponding to residues 96–110 that blocks ras–jun and jun kinase (JNK) interactions exhibits a similar pattern of inhibition. We have also found that c-raf strongly induces oocyte maturation and that dominant negative c-raf strongly blocks oncogenic p21-induced oocyte maturation. We now find that the p21 35–47, but not the 96–110, peptide completely blocks c-raf-induced maturation. This finding suggests that the 35–47 peptide blocks oncogenic ras at the level of raf; that activated normal and oncogenic ras–p21 have differing requirements for raf-dependent signaling; and that the two oncogenic-ras-selective inhibitory peptides, 35–47 and 96–110, act at two different critical downstream sites, the former at raf, the latter at JNK/jun, both of which are required for oncogenic ras-p21 signaling.     

Utilization of grass carp faeces by the IsopodAsellus aquaticus (L.) in the laboratory

Grass carp (Ctenopharyngodon idella Val) faecal pellets derived fromElodea nutallii consumption were efficiently utilized by the detritivoreAsellus. Immature and adult individuals of 2.1–3.2 mm and 4.9–6.0 mm respectively, exhibited a high mean specific growth (9.03%/day and 3.33%/day) when offered faecal pellets as food during two weeks experimental trials. Individuals of the same groups, when fed onElodea which had been mechanically fragmented to simulate the macerating action of the grass carp pharyngeal teeth, grew at rates (9.57%/day and 4.06%/day) not significantly different from those fed on faecal pellets. However, the growth rates of animals fed on freshElodea (6.40%/day and 2.23%/day) were significantly lower than in the above trials, indicating that the morphological destruction of the plant tissue could be responsible for the higher observed growth. The control trials, including unfed animals, showed negative growth (– 0.49%/day and – 0.48%/day).Grass carp herbivory makes a new food resource available to benthic invertebrates by converting fresh plant material to small plant fragments.     

Inhibition of oncogenic and activated wild-type ras-p21 protein-induced oocyte maturation by peptides from the guanine-nucleotide exchange protein, SOS, identified from molecular dynamics calculations. Selective inhibition of oncogenic ras-p21

In the preceding paper we performed molecular dynamics calculations of the average structures of the SOS protein bound to wild-type and oncogenic ras–p21. Based on these calculations, we have identified four major domains of the SOS protein, consisting of residues 631–641, 676–691, 718–729, and 994–1004, which differ in structure between the two complexes. We have now microinjected synthetic peptides corresponding to each of these domains into Xenopus laevis oocytes either together with oncogenic (Val 12)-p21 or into oocytes subsequently incubated with insulin. We find that the first three peptides inhibit both oncogenic and wild-type p21-induced oocyte maturation, while the last peptide much more strongly inhibits oncogenic p21 protein-induced oocyte maturation. These results suggest that each identified SOS region is involved in ras–stimulated signal transduction and that the 994–1004 domain is involved uniquely with oncogenic ras–p21 signaling.     

Identification, using molecular dynamics, of an effector domain of the ras-binding domain of the raf-p74 protein that is uniquely involved in oncogenic ras-p21 signaling

By comparing the average structures, computed using molecular dynamics, of the ras-binding domain of raf (RBD) bound to activated wild-type ras-p21 and its homologous inhibitory protein, rap-1A, we formerly identified three domains of the RBD that changed conformation between the two complexes, residues 62–76, 97–110, and 111–121. We found that one synthetic peptide, corresponding to RBD residues 97–110, selectively inhibited oncogenic ras-p21-induced oocyte maturation. In this study, we performed molecular dynamics on the Val 12-ras-p21-RBD complex and compared its average structure with that for the wild-type protein. We find that there is a large displacement of a loop involving these residues when the structures of the two complexes are compared. This result corroborates our former finding that the RBD 97–110 peptide inhibits only signal transduction by oncogenic ras-p21 and suggests that oncogenic p21 uses this loop to interact with raf in a unique manner.     

A new species of armored sea robin, Paraheminodus kamoharai (Teleostei: Peristediidae), from the Philippines

A new peristediid, Paraheminodus kamoharai, is described from three specimens (106.9–114.8mm SL) collected from the Sulu Sea, the Philippines. The new species is easily distinguished from its congeners (Paraheminodus laticephalus and P. murrayi) in having 33 bony plates in the upper lateral row, the 24–25th to 30th bony plate each in the upper lateral row with a forwardly directed spine, 17–19 gill rakers on the lower limb, a long slender rostral projection (43.4–47.0% of head length), and short upper (41.1–42.3%) and lower jaws (36.5–37.2%).     

Studies on the production of useful chemicals,especially fatty acids in the marine diatom Nitzschia conspicua Grunow

A local marine diatom, Nitzschia conspicua Grunow, was cultured in enriched synthetic seawater using flasks (agitated by magnetic stirring) and a 1.2 l fermenter. Lipids, fatty acids, proteins, carbohydrates and ash of the flask cultures were determined at various stages of growth (day 3, 5, 7, 10, 13, 15 and 17). The fermenter culture was harvested during the stationary phase for similar chemical analyses. N. conspicua attained a higher biomass concentration during the stationary phase when cultured in the fermenter (188 mg dry weight l^–1) than in flasks (140–151 mg dry weight l^–1). However, both systems showed similar specific growth rates based on chlorophyll-a concentration. Appreciable amounts of the essential fatty acids 20:4 (0.6–4.7% total fatty acids) and 20:5 (1.9–4.7% total fatty acids) are present in this diatom. Maximal amounts of these fatty acids were produced after 7 days' growth (i.e. 2 days after the end of the exponential phase). Lipids, fatty acids, proteins, carbohydrates and ash varied with culture age in N. conspicua.author for correspondence     

Biocontrol of the shore fly Scatella tenuicosta with Hypoaspis miles and H. aculeifer in peat pots

Hypoaspis miles Berlese and H. aculeiferCanestrini (Acari: Laelapidae) were usedagainst Scatella tenuicosta Collin(Diptera: Ephydridae) at the rates of 7, 14and 36 mites per 0.3-l peat pot (1000, 2000 and5000 per net-m² of peat) with mint (Mentha piperita L.) as the crop. The predatorswere applied either one day (preventivecontrol) or nine days after (curative control)exposing the pots to the flies in thegreenhouse. Based on the number of flies thatemerged from the pots, the control efficacy of preventive H. aculeifer treatments at therate of 36 mites per pot was 84–100% and 92–97% (range over four replicate blocks)after two and three weeks, respectively, fromthe application. The control efficacy ofcurative treatments was 93–100% and 83–94% after two and three weeks, respectively,from the application. Preventive treatmentswith H. miles applied at the rate of 36per pot controlled the flies by 92–100% and 68–97% two and three weeks, respectively, afterthe application. Curative treatments with 36H. miles per pot were less effective (–53–68% and –16–65%, respectively). All treatments with lower mite rates with eitherspecies were ineffective. Due to moreconsistent reduction of fly numbers by Hypoaspis aculeifer, the species was concluded to be a better biocontrol agent ofshore flies in short-term greenhouse cropsgrown in peat pots, probably due to its higherpredation capacity in comparison to H. miles.     

New Substrates for Enteropeptidase: I. Biologically Active Hepta-, Octa-, and Nonapeptides

Enteropeptidase (enterokinase, EC 3.4.21.9) hydrolyzes peptide bonds formed by carboxyl groups of Lys or Arg residue if less than four negatively charged amino acid residues are in positions P ₂–P ₅ of its substrate. We determined the kinetic parameters of three substrates of this type: human angiotensin II (AT) (DR VYIHPF) and the Hb(2–8) (LTAEEK A) and Hb(1–9) (MLTAEEK AA) peptides of the cattle hemoglobin -chain. The K _m values for all the substrates (10^–3 M) were one order of magnitude higher than those of the typical synthetic substrates of enteropeptidase or chimeric proteins with the –DDDDK– full-size linker (K _m 10^–4 M). The k _cat values for AT and Hb(2–8) were also close and low (30 min^–1). The general hydrolysis efficiency of such substrates is no more than 1% of the corresponding value for the typical peptide and protein substrates of the enteropeptidase. However, the elongation of Hb(2–8) peptide by one amino acid residue from both its N- and C-termini results in a dramatic increase in the catalytic efficiency of the hydrolysis: the k _cat value for Hb(1–9) is 1510 min^–1, which means that it is hydrolyzed only three times less effective than the chimeric protein with the full-size linker.     

Inhibition of Oncogenic and Activated Wild-Type ras-p21 Protein-Induced Oocyte Maturation by Peptides from the ras-Binding Domain of the raf-p74 Protein, Identified from Molecular Dynamics Calculations

In the preceding paper we found from molecular dynamics calculations that the structure of the ras-binding domain (RBD) of raf changes predominantly in three regions depending upon whether it binds to ras-p21 protein or to its inhibitor protein, rap-1A. These three regions of the RBD involve residues from the protein–protein interaction interface, e.g., between residues 60 and 72, residues 97–110, and 111–121. Since the rap-1A–RBD complex is inactive, these three regions are implicated in ras-p21-induced activation of raf. We have therefore co-microinjected peptides corresponding to these three regions, 62–76, 97–110, and 111–121, into oocytes with oncogenic p21 and microinjected them into oocytes incubated in in insulin, which activates normal p2l. All three peptides, but not a control peptide, strongly inhibit both oncogenic p21- and insulin-induced oocyte maturation. These findings corroborate our conclusions from the theoretical results that these three regions constitute raf effector domains. Since the 97–110 peptide is the strongest inhibitor of oncogenic p21, while the 111–121 peptide is the strongest inhibitor of insulin-induced oocyte maturation, the possibility exists that oncogenic and activated normal p21 proteins interact differently with the RBD of raf.     

Synthesis of chitooligomer-based glycoconjugates and their binding to the rat natural killer cell activation receptor NKR-P1

NKR-P1 protein is an important activating receptor at the surface of the rat natural killer cells. GlcNAc and chitooligomers were identified as strong activation ligands in vitro and in vivo. Their clustering brings about increase of their affinity to the NKR-P1 by 3–6 orders. Here we describe novel methodology for preparation of neoglycoproteins based on BSA carying the chitooligomers (n = 2–5). Further on we developed novel methodology of the coupling of glycosylamines via aromatic-SCN activated linker both to protein or synthetic cores. Inhibition studies of chitooligomer glycoconjugates with the NKR-P1 receptor show that our neoglycoproteins are very strong ligands with high binding affinity (–log IC₅₀ = 13–15). In analogy with our previous observations with GlcNAc clustered on protein or PAMAM backbones the synthetic chitooligomer clusters should provide considerably better ligands in the in vivo antitumor treatment.     

Ontogenesis of auditory fovea representation in the inferior colliculus of the Sri Lankan rufous horseshoe bat,Rhinolophus rouxi

Summary This report describes the ontogenesis of tonotopy in the inferior colliculus (IC) of the rufous horseshoe bat (Rhinolophus rouxi). Horseshoe bats are deaf at birth, but consistent tonotopy with a low-to-high frequency gradient from dorsolateral to ventromedial develops from the 2nd up to the 5th week. The representation of the auditory fovea is established in ventro-mediocaudal parts of the IC during the 3rd postnatal week (Fig. 3). Then, a narrow frequency band 5 kHz in width, comprising 16% of the bat's auditory range, captures 50–60 vol% of the IC (Fig. 3c). However, foveal tuning is 10–12 kHz (1/3 octave) lower than in adults; foveal tuning in females (65–68 kHz) is 2–3 kHz higher than in males (62–65 kHz). Thereafter, foveal tuning increases by 1–1.5 kHz per day up to the 5th postnatal week, when the adult hearing range is established (Figs. 4, 5). The increase of sensitivity and of tuning sharpness of single units also follows a low-to-high frequency gradient (Fig. 6).Throughout this development the foveal tuning matches the second harmonic of the echolocation pulses vocalised by these young bats. The results confirm the hypothesis of developmental shifts in the frequency-place code for the foveal high frequency representation in the IC.Abbreviations BF best frequency - CF constant frequency - FM frequency modulation - IC inferior colliculus - IHC inner hair cell; - OHC outer hair cell - RR Rhinolophus rouxi     

The effect of N-terminal substitutions on the biological activity of MSH fragments

In order to study the role of N-terminal substitutions of peptide sequences related to the active site of α-melanotropin, [Glp⁵]α-MSH(5–10), [Glp⁵, -Phe⁷]α-MSH(5–10), [Sar⁵, -Phe⁷]α-MSH(5–10), [Nle⁴, -Phe⁷]α-MSH(4–10), [N-carbamoyl]α-MSH(5–10), and formyl and acetyl derivatives of α-MSH(5–10), [Gly⁵]α-MSH(5–10) and [Gly⁵, -Phe⁷]α-MSH(5–10), were synthesized in solution. The N-terminal acylations enhance by 2 to 10 times the melanin-dispersing activity of the unsubstituted sequences. Alkylation of the N-terminus does not change the biological activity of the parent peptide, suggesting the necessity of a carbonyl group for increasing the hormonal effect.     

Cleavage Specificities of Individual Members of Kallikrein Family of Proteins on Synthetic Peptide Containing the Bradykinin Sequence

We have analyzed the effect on bond specificity of various isolated members of the mouse kallikrein family of proteins on a synthetic peptide containing the bradykinin sequence. The cleavage pattern shows the selected specificity of these proteases toward the synthetic peptide. The Phe–His bond (positions 11–12) in the synthetic peptide was favorably cleaved by most of the members in this family, including gamma nerve growth factor. On the other hand, the Lys–Arg bond (position 3–4) was found to be susceptible only to -NGF. The combination of these cleavages could result in the degradation of bradykinin in vivo.     

In vitro bactericidal activity of the N-terminal fragment of the frog peptide esculentin-1b (Esc 1–18) in combination with conventional antibiotics against Stenotrophomonas maltophilia

In this study the bactericidal effect of the N-terminal fragment of the frog skin peptide esculentin-1b [Esc(1–18)] in combination with clinically used antimicrobial agents was evaluated against Stenotrophomonas maltophilia, either in standard conditions (phosphate buffer) or in the presence of human serum. A synergistic bactericidal effect was observed after a 24 h incubation when combinations of Esc(1–18) and amikacin or colistin were used against clinical strains of S. maltophilia with or without resistance to these antibiotics, both in buffer and in the presence of serum. An indifferent effect was observed when the peptide was combined with levofloxacin or ceftazidime. A synergistic effect was also observed at earlier time points when the peptide was used in combination with colistin. Sequential exposure of bacterial cells to Esc(1–18) and amikacin or colistin, or vice versa, indicated that while Esc(1–18) and colistin cooperated in enhancing the bactericidal effect of their combination, when Esc(1–18) was combined with amikacin, the peptide had a major role in initiating the bactericidal effect, while amikacin was required for the subsequent effector phase. Altogether, the results obtained indicate that exposure of S. maltophilia to sub-bactericidal concentrations of Esc(1–18) increases its susceptibility to amikacin or colistin and may also render resistant strains susceptible to these antibiotics.     

Chronology of Development in the Starfish <Emphasis Type="Italic">Asterina pectinifera</Emphasis> from Vostok Bay,Sea of Japan

The development of the starfish Asterina (= Patiria) pectinifera (Muller et Troschel) from fertilization to metamorphosis took 27–28 days at 22°C and a salinity of 33–33.4‰. The embryonic development was completed by the release of swimming ciliary blastula from egg envelopes 13 h after fertilization. The larvae passed into the stage of gastrula and reached the stage of dipleurula in 35 h and the stage of bipinnaria in 3.5 days. At the stage of brachiolaria, by the 12th day of development, two lateral brachioles and one medioventral brachiole with papillae developed in the larvae. The attachment disk and the primordia of five radial canals of the juvenile starfish became visible by the 15th and 18th days, respectively. By the 24th–25th day, a differentiated primordium of a juvenile starfish had developed in the brachiolaria. The size of the larvae prior to settlement was 1765.4 ± 51.5 µm. Metamorphosis was completed one day after settlement.Original Russian Text Copyright ¢ 2005 by Biologiya Morya, Kashenko.     

Growth curves,morphology and ultrastructure of ten Paracoccidioides brasiliensis isolates

The yeast phase of ten P. brasiliensis isolates were studied to characterize their growth pattern, morphology and ultrastructure. Growth curves were determined after counts of total and viable fungi units (FU) during 20 days. Three growth patterns were observed: slow, reaching approximately 10–30× 10⁶ FU/tube (Pb 18, Pb 265 and PB 2); intermediate, reaching 60–150×10⁶ FU/tube (IVIC Pb 9, IVIC Pb 267, Pb SN, Pb Vitor and Pb Campo Grande) and fast, reaching 180–370×10⁶ FU/tube (Pb 2052 and Pb 192). The highest percentage of viable cells occurred on the 6th day of culture for Pb 192, Pb Campo Grande, Pb 2052 and IVIC Pb 9; on the 8th day for Pb Vitor, Pb SN, Pb 18 and IVIC Pb 267; on the 10th day for Pb 265 and on the 12th day of culture for Pb 2. Mean generation times varied from approximately 21.2 (Pb 2052) to 102.6 hours (Pb 265). The isolates showed similar morphology, except IVIC Pb 267 which did not present a typical yeast-phase at 35°C and the two fast-growing isolates (Pb 2052 and Pb 192) that presented smaller cell sizes and less tendency to clump. The ultrastructure of the isolates was similar: the cell walls presented a width of 0.1 to 0.2 °; the mitochondria presented few cristae and had equivalent patterns of distribution and morphology; the endoplasmic reticulum was scanty, presenting narrow cisternae; the vacuoles, empty or filled with electrondense material, were numerous and two to five nuclei with pores were constantly observed.     

The influence of cyclophosphamide on antitumor immunity in mice bearing late-stage tumors

Spleen cells from mice bearing late-stage methylcholanthrene-induced tumor did not show any tumor activity when mixed with tumor cells in Winn's assay. Treatment of these mice with cyclophosphamide (CY) induced a tumor-inhibitory activity in spleen, occurring on day 7 after treatment, reaching its maximum on day 11 and disappearing by day 21. This antitumor activity could not be induced in control, tumor-free or T-deficient tumor-bearing mice. CY-induced tumor-inhibitory activity was immunologically specific, and mediated by Thy-1⁺, L3T4^–, Ly-2⁺ cells. Contrary to spleen cells from untreated tumor-bearing mice, spleen cells from CY-treated tumor-bearing mice did not suppress the antitumor activity of immune spleen cells in Winn's assay. However, in contrast to immune spleen cells, CY-induced tumor-inhibitory cells did not manifest antitumor activity when transferred systemically (i. v.) into T-cell-deficient tumor-bearing mice. Even more, spleen cells from CY-pretreated mice, harvested 7–15 days after the drug administration, partially suppressed the antitumor activity of concomitantly transferred spleen cells from specifically immune mice. Nevertheless, CY-pretreated mice manifested concomitant immunity, i.e. these mice exhibited higher resistance to a second inoculum of the same tumor than did nontreated mice or even mice with excised primary tumor.     

Antitumor effect of heat-killed Lactobacillus plantarum L-137 through restoration of impaired interleukin-12 production in tumor-bearing mice

 We have previously reported that heat-killed Lactobacillus plantarum L-137 is a potent inducer of interleukin-12 (IL-12) in vivo as well as in vitro in mice. In order to develop effective usage of L. plantarum L-137 for tumor immunotherapy, we examined its antitumor effect against DBA/2 mice inoculated with syngenic P388D1 tumor cells in different treatment schedules. Daily injection of L. plantarum L-137 from the day of tumor inoculation induced a steep increase in plasma IL-12 only after the first injection but not after subsequent injections, and had no effect on tumor growth and survival time. In contrast, daily injection of L. plantarum L-137 from the 7th day after tumor inoculation exerted a marked antitumor effect but such an effect was not evident in mice treated with L. plantarum L-137 twice a week from the 7th day. IL-12 production was considerably impaired at the first injection but steeply increased after the third injection in the mice injected daily with L. plantarum L-137 from the 7th day. Our results suggest that daily administration of L. plantarum L-137 is required to exert an antitumor effect at the late stages of tumor development when IL-12 production is considerably impaired. Received: 15 July 1999 / Accepted: 28 January 2000