Thin layer chromatographical detection of tyrosine produced from <Emphasis Type="SmallCaps">l</Emphasis>-[U-<Superscript>14</Superscript>C]phenylalanine by ruminal microbes

Summary.  Thin layer chromatographical detection of tyrosine (Tyr) synthesized from l-[U-¹⁴C]phenylalanine (Phe) (1 mM) by rumen bacteria (B) and protozoa (P) collected from fistulated Japanese Goat was carried out. About 16 and 12% of the added Phe was converted to Tyr by B and P, respectively. Large amount of radioactivity in ether fractions indicated an abundant production of aromatic acids from Phe. Small amount of radioactivity found in CO₂ fractions implied an occurrence of considerable decarboxylation reaction(s) by rumen bacteria and protozoa. Received July 18, 2001 Accepted December 3, 2001     

Biosynthesis of Threonine from Homoserine by Mixed Rumen Microorganisms: An In Vitro Study

The biosynthesis of threonine (Thr) by using the main biosynthetic pathway involving homoserine (Hser) was quantitatively investigated by mixed rumen bacteria (B), protozoa (P), and their mixture (BP) in an in vitro system. Rumen contents were collected from fistulated goats to prepare the microbial suspensions and were incubated anaerobically at 39°C for 12 h with or without Hser (2 mm) as a substrate. Thr and other related compounds produced in both the supernatants and hydrolysates of the incubation were analyzed by HPLC. During a 12-h incubation period, 84.2%, 58.1%, and 92.0% of Hser disappeared in B, P, and BP suspensions, respectively. Rumen bacteria and the mixture of rumen bacteria and protozoa were demonstrated for the first time to produce Thr from Hser, and the production of Thr from Hser in BP (371.9 and 297.2 μmol/g MN) (MN, microbial nitrogen) was about 13.0% and 9.1% higher than that in B alone (329.2 and 272.5 μmol/g MN) during 6- and 12-h incubations, respectively. On the other hand, mixed rumen protozoa were unable to synthesize Thr from Hser. Other metabolites produced from Hser were found to be glycine (Gly) and 2-aminobutyric acid (2AB) in B and BP. In P, Gly and 2AB were not found. The results mentioned above indicated the abilities of rumen bacteria and the mixture of rumen bacteria and protozoa to synthesize Thr de novo from Hser and appeared as first-time report. Received: 24 May 2000/Accepted: 4 August 2000     

In vitro metabolism of phenylalanine by ruminal bacteria,protozoa, and their mixture

An in vitro study was conducted to examine the metabolism of phenylalanine (Phe) by mixed rumen bacteria (B), mixed rumen protozoa (P), and a combination of the two (BP). Rumen microorganisms were collected from fistulated goats fed lucerne cubes (Medicago sativa) and a concentrated mixture twice a day. Microbial suspensions were anaerobically incubated at 39 degrees C for 12 h. Phe and some other related compounds in both supernatants and microbial hydrolysates of the incubations were analysed by HPLC. The net degradation rate (&mgr;mol/g microbial nitrogen) of Phe in B was about 1.5-fold higher than that in P. Phe was converted mainly into phenylacetic acid (PAA) and unknown compound(s) that presumably involved tyrosine in B, P, and BP during the 12 h incubation period. Small amounts of benzoic acid (BZA), and traces of phenylpropionic acid (PPR) and phenyllactic acid (PLA) were also produced from Phe. PAA production in B was found to be higher than that in P, whereas it was significantly higher in BP. Although BZA production was less than one-tenth that of PAA production, it was higher in P than in B and BP. PPR was detected in both B and BP, but not in P. PLA was detected only in B. The production of unknown compound(s) was higher in B than in P and BP.     

Aromatic amino acid biosynthesis and production of related compounds from p-hydroxyphenylpyruvic acid by rumen bacteria,protozoa and their mixture

Summary. Aromatic amino acid biosynthesis and production of related compounds from p-hydroxyphenylpyruvic acid (HPY) by mixed rumen bacteria (B), protozoa (P), and their mixture (BP) in an in vitro system were quantitatively investigated. Microbial suspensions prepared from mature, fistulated goats fed Lucerne (Medicago sativa) cubes and a concentrate mixture were anaerobically incubated at 39°C for 12 h. Tyrosine (Tyr), phenylalanine (Phe), tryptophan (Trp) and other related compounds in both supernatants and hydrolyzates of all incubations were analyzed by HPLC. Large amounts of Tyr (27.0, 47.0 and 50.8% of disappeared HPY in B, P and BP, respectively) were produced from 1 mM HPY during a 12-h incubation period. The formation of Tyr in P was 1.8 and 1.6 times higher than those in B and BP, respectively. Appreciable amounts of Phe (3–12% of the disappeared HPY) and Trp (2–10% of the disappeared HPY) were also produced from HPY in B, P, and BP. Phe synthesis in B and P was almost similar but Trp synthesis in B was 1.8 times higher than that in P. The biosynthesis of both Phe and Trp from HPY in BP was higher than those in B plus P. A large amount of p-hydroxyphenylacetic acid (about 45% of the disappeared HPY) was produced from HPY in B which was 1.9 times higher than that in P. p-Hydroxybenzoic acid produced from HPY in P was 1.6 times higher than that in B. Considerable amounts of phenylpropionic acid, phenyllactic acid, and phenylpyruvic acid (2–6% of the disappeared HPY) were produced only in B. Received March 21, 2001 Accepted July 4, 2001     

Studies on the possibility of histidine biosynthesis from histidinol, imidazolepyruvic acid, imidazoleacetic acid, and imidazolelactic acid by mixed ruminal bacteria, protozoa, and their mixture in vitro

The possibility of histidine (His) synthesis using a main biosynthetic pathway involving histidinol (HDL) and also the recycling capability of imidazolic compounds such as imidazolepyruvic acid (ImPA), imidazoleacetic acid (ImAA), and imidazolelactic acid (ImLA) to produce His were investigated using mixed ruminal bacteria (B), protozoa (P), and a mixture of both (BP) in an in vitro system. Rumen microorganisms were anaerobically incubated at 39 degrees C for 18 h with or without each substrate (2 mM) mentioned. His and other related compounds produced in both the supernatants and hydrolyzates of the incubation were analyzed by high-performance liquid chromatography. B, P, and BP suspensions failed to show His synthesizing ability when incubated with HDL. His was synthesized from ImPA by B, P, and BP. Expressed in units "per gram of microbial nitrogen (MN)", ImPA disappearance was greatest in B (72.7 micromol/g MN per hour), followed by BP (33.13 micromol/g MN per hour) and then P (18.6 micromol/g MN per hour) for the 18-h incubation period. The production of His from ImPA in B (240.0, 275.9, and 261.2 micromol/g MN in 6, 12, and 18 h incubation, respectively) was about 3.5 times higher than that in P (67.3, 83.8, and 72.7 micromol/g MN in 6, 12, and 18 h incubation, respectively). Other metabolites produced from ImPA were ImLA, ImAA, histamine (HTM), and urocanic acid (URA), found in all microbial suspensions. ImLA as a substrate remained without diminution in all microbial suspensions. Although ImAA was found to be degraded to a small extent (3.4-6.3%) only after 18 h incubation, neither His nor other metabolites were detected on the chromatograms. These results have been demonstrated for the first time in rumen microorganisms and suggest that His may be an essential amino acid for rumen microorganisms.     

Effects of salinomycin and vitamin B(6) on the in vitro synthesis of lysine from the stereoisomers of 2,6-diaminopimelic acid by mixed rumen protozoa,bacteria, and their mixture

An in vitro study was conducted to examine the effects of salinomycin (SL) and vitamin B(6) (pyridoxine hydrochloride) (B(6)) on the production of lysine from the three stereoisomers of 2,6-diaminopimelic acid (DAP-SI) by mixed rumen protozoa (P), mixed rumen bacteria (B), and their mixture (PB). P, B, and PB were isolated from the rumen of goats given a concentrate mixture and lucerne cubes, separately incubated for 12 h with and without DAP-SI (5 mM) as a substrate and SL (5 &mgr;g/ml) and/or B(6) (10 &mgr;g/ml) as additives. In P suspensions, SL and B(6) reduced the amount of DAP-SI by 2.1 times (p<0.001, where p is probability) and 19.9% (p<0.05), respectively, and also increased the production of lysine by 2.4 times (p<0.001) and 26.8% (p <0.05), respectively, during 12 h incubation. In B suspensions, the reductions of DAP-SI with a single addition of SL or B(6) were 8.5% (p<0.001) and 2.7%, respectively, and lysine production increased by 54.3 and 32.9% (p<0.001), respectively, during 12 h incubation. In PB suspensions, the reductions of DAP-SI were 21.9 and 11.7% (p<0.001) with a single addition of SL or B(6), respectively, and the production of lysine increased by 81.4 and 39.4% (p<0.001), respectively, during 12 h incubation. When SL and B(6) were added together to the P, B, and PB suspensions, lysine production further increased by 12.3, 21.3, and 12.4% more than the cases of adding SL only during 12 h incubation, respectively. SL and B(6) were demonstrated to enhance the production of lysine from DAP-SI by mixed rumen protozoa, mixed rumen bacteria and their mixture in this study.     

Degradation of tryptophan and related indolic compounds by ruminal bacteria,protozoa and their mixture in vitro

Summary.  In vitro experiments were conducted to examine the degradation of d- and l-isomers of tryptophan (Trp) and 10 related indolic compounds by mixed rumen bacteria (B), protozoa (P) and a combination of the two (BP). The analyses were carried out by HPLC. d-Trp (1.0 mM) was not degraded by rumen microorganisms during the 24-h incubation period. The net degradation of 1 mM l-Trp was 46.5%, 8.7% and 80.0% by B, P and BP suspensions, respectively. Trp was degraded into indoleacetic acid, indolelactic acid and indole by rumen bacteria and protozoa, and into skatole, p-cresol and indolepropionic acid by rumen bacteria only. Of them, indoleacetic acid was the major product of Trp found in B (15.4%) and P (3.1%), and skatole in BP (43.2%). This is the first report of the production of indolelactic acid and p-cresol from Trp by rumen microbes. Starch, d-glucose, salinomycin and monensin inhibited the production of skatole and indole from Trp, and skatole from indoleacetic acid by rumen bacteria. Received August 2, 2001 Accepted June 21, 2002 Published online November 14, 2002 Acknowledgements The authors are extremely grateful to Dr. H. Ogawa, Professor, the University of Tokyo and Dr. T. Hasegawa, Associate Professor, Miyazaki University, for inserting a permanent fistula in goats. The present study was financially supported by research grants from Kyowa Hakko Kogyo Co. Ltd. and Daiichi Seiyaku Co., Japan. Nazimuddin Mohammed thanks the Ministry of Education, Science, Sports and Culture of Japan (Monbusho) for the award of a research studentship from 1996. Authors' address: Dr. Nazimuddin Mohammed, Laboratory of Agricultural Production Technology, Faculty of Agriculture, Field Science Center, Tokyo University of Agriculture and Technology, Saiwai-cho 3-5-8, Fuchu-shi, Tokyo 183-8509, Japan     

Studies on the utilization of methionine sulfoxide and methionine sulfone by rumen microorganisms in vitro

Summary.  An in vitro experiment was conducted to test the ability of mixed rumen bacteria (B), protozoa (P), and their mixture (BP) to utilize the oxidized forms of methionine (Met) e.g., methionine sulfoxide (MSO), methionine sulfone (MSO₂). Rumen contents were collected from fistulated goats to prepare the microbial suspensions and were anaerobically incubated at 39°C for 12 h with or without MSO (1 mM) or MSO₂ (1 mM) as a substrate. Met and other related compounds produced in both the supernatants and hydrolyzates of the incubation were analyzed by HPLC. During 6- and 12-h incubation periods, MSO disappeared by 28.3 and 42.0%, 0.0 and 0.0%, and 40.6 and 62.4% in B, P, and BP suspensions, respectively. Rumen bacteria and the mixture of rumen bacteria and protozoa were capable to reduce MSO to Met, and the production of Met from MSO in BP (156.6 and 196.1 μmol/g MN) was about 17.3 and 14.1% higher than that in B alone (133.5 and 171.9 μmol/g MN) during 6- and 12-h incubations, respectively. On the other hand, mixed rumen protozoa were unable to utilize MSO. Other metabolites produced from MSO were found to be MSO₂ and 2-aminobutyric acid (2AB) in B and BP. MSO₂ as a substrate remained without diminution in all-microbial suspensions. It was concluded that B, P, and BP cannot utilize MSO₂; but MSO can be utilized by B and BP for producing Met. Received December 28, 2001 Accepted May 21, 2002 Published online October 14, 2002 Acknowledgements The authors are extremely grateful to Professor H. Ogawa, the University of Tokyo, Japan and Dr. Takashi Hasegawa, Miyazaki University, Japan for inserting permanent rumen fistulae in goats. We would like to thank MONBUSHO for the award of a research scholarship to Mamun M. Or-Rashid since 1996–2001. Authors' address: Shaila Wadud, Laboratory of Animal Nutrition and Biochemistry, Division of Animal Science, Miyazaki University, Miyazaki 889-2192, Japan, Fax. +81-985-58-7201, E-mail: rafatkun@hotmail.com     

In Vitro Metabolism of the Stereoisomers of 2,6-Diaminopimelic Acid by Mixed Rumen Protozoa and Bacteria

Formation of lysine from stereoisomers (SI) of 2,6-diaminopimelic acid (DAP) and the epimerization between the three SI of DAP (DAP-SI) by rumen protozoa and bacteria were examined. Mixed rumen protozoa (P) and bacteria (B) were isolated from the rumen of goats given a concentrate and hay cubes and incubated separately with and without a mixture and a single one of the three DAP-SI. In P suspensions, mixed DAP-SI decreased by 10.59% as a whole and converted mainly to lysine by 8.41% during 12 h incubation. When meso-, L- and D-DAP were added singly to the media, the results showed that each DAP-SI interconverted and produced lysine. This means that mixed rumen protozoa have an ability to synthesize lysine from not only meso-DAP but also from D- and L-DAP, though probably via meso-DAP, and hence have DAP epimerase activities for the reversal conversion of each DAP-SI. This is the first discovery to show the interconversion of DAP-SI and synthesis of lysine from them by protozoa. In B suspensions, mixed DAP-SI decreased by 10.92% as a whole and converted to lysine by 4.20% during 12 h incubation. When a single DAP-SI was added to the media, meso-, L- and D-DAP were interconverted and then converted to lysine by the rumen bacteria as well as the protozoa. This also means that mixed rumen bacteria have DAP epimerase activities to interconvert DAP-SI and have an ability to synthesize lysine from not only meso-DAP but also from L- and D-DAP, and this is also the first finding in rumen bacteria. Received: 16 March 1996 / Accepted: 14 May 1996     

Plantlet regeneration from petiole explants of the african horned cucumber,Cucumis metuliferus

Plantlet regeneration in Cucumis metuliferus from several explant sources, including cotyledons, leaves, hypocotyls and petioles, was evaluated on Murashige and Skoog's medium containing various combinations of auxin (IAA, NAA, 2,4-d) and cytokinin (BA, kinetin, zeatin), Callus development was obtained within 4 to 5 weeks on all growth regulator combinations which were tested at concentrations ranging from 1.0 M to 4.0 M of each. The response was similar when the tissues were incubated under light or in continuous darkness. Differentiation of callus to form adventitious buds or shoot primordia occurred only with petiole explants on medium containing NAA/BA or 2,4-d/BA at 2.0/1.0 M; none of these calluses, however, differentiated further to form shoots. When the differentiated calluses derived from petiole explants which had been initiated on 2,4-d/BA at 2.0/1.0 M were transferred onto medium with 2.0 M zeatin, formation of shoots occurred within 2 to 3 weeks. The frequency of shoot formation was 14.6%. Subculture of these shoots onto MS medium without growth regulators gave rise to plantlets of normal appearance. Regeneration in C. metuliferus requires callus initiation on an appropriate growth regulator regime followed by transfer to a medium containing the cytokinin, zeatin, and can be achieved within 10–12 weeks.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA napthaleneacetic acid     

Glycoside hydrolases of rumen bacteria and protozoa

Sixteen strains of rumen bacteria and 21 protozoal preparations were screened for glycoside hydrolase and phosphatase activity, using 22 nitrophenyl glycoside substrates. The range and level of bacterial enzyme activities were species dependent, although, the glycosidases associated with plant cell wall breakdown were most active in the cellulolytic and hemicellulolytic species. Alkaline phosphatase occurred widely in the organisms examined, but was most active in the twoBacteroides ruminicola strains.A wide range of enzyme activities was also detected in the holotrich and Entodiniomorphid ciliates isolated from the rumen or cultured in vitro. The glycosidases involved in cellulose and hemicellulose breakdown were detected in all of the protozoa examined, and, with the exception ofEntodinium spp., were most active in the Entodiniomorphid protozoa; -l-arabinofuranosidase, an essential hemicellulolytic glycoside hydrolase, was particularly active in this latter group of ciliates.     

In Vitro Catabolism of Histidine by Mixed Rumen Bacteriaand Protozoa

An in vitro study was conducted to examine the metabolism of histidine (His) by mixed rumen bacteria (B), mixed rumen protozoa (P), and a combination of the two (BP). Rumen microorganisms were collected from fistulated goats fed with lucerne cubes (Medicago sativa) and a concentrate mixture twice a day. Microbial suspensions were anaerobically incubated with or without 2 mm each of His, or histamine (HTM), or 1 mm urocanic acid (URA) at 39°C for 12 h. His and other related compounds in both supernatant and microbial hydrolysates were analyzed by HPLC. After 6- and 12-h incubations, the net degradation of His was 26.1% and 51.7% in B, 13.5% and 20.9% in P, and 21.7% and 46.0% in BP, respectively. The rate of the net degradation of His in B (98.0 μmol/g microbial nitrogen/h) was about 2.6 times higher than that of P during a 12-h incubation period. His was found to be degraded into urocanic acid (URA), imidazolelactic acid (ImLA), imidazoleacetic acid (ImAA), and histamine (HTM). Of these degraded His was mainly converted into URA in all microbial suspensions. The production of ImLA and ImAA was higher in B than in P suspensions, whereas the production of HTM was higher in P than in B suspensions. From these results, the existence of diverse catabolic routes of His in rumen microorganisms was indicated. Received: 23 May 2000 / Accepted: 31 July 2000     

Biosynthesis of methionine from homocysteine, cystathionine and homoserine plus cysteine by mixed rumen microorganisms in vitro

This study quantitatively investigated the biosynthesis of methionine (Met) and the production of related compounds from homocysteine (Hcys), cystathionine (Cysta), and homoserine (Hser) plus cysteine (Cys) by rumen bacteria (B) or protozoa (P) alone and by a mixture of these bacteria and protozoa (BP). Rumen contents were collected from fistulated goats to prepare the microbial suspensions and were anaerobically incubated at 39 degrees C for 12 h. Hcys, Cysta, and Hser plus Cys were catabolized by all rumen microbial fractions to different extents. B, P, and BP converted Hcys to Met with 2-aminobutyric acid (2AB) and methionine sulfoxide. The Met-producing ability of B (83.2 micromol g(-1) microbial nitrogen; MN) from Hcys was about 3.6 times higher than that of P in a 6-h incubation period. The ability of BP, during the same incubation period, was about 30.0% higher than that of B. Hcys, Met, and 2AB were formed when Cysta was incubated with B, P, or BP. Rumen microbial fermentation of Hser plus Cys led to the formation of Cysta, Met (through Hcys), and 2AB. Thus the results indicated that a trans-sulfurylation pathway for Met synthesis was operating in the rumen bacteria and protozoa. The results mentioned above have been demonstrated for the first time in B, P, and BP in the present study.     

Effects of salinomycin and vitamin B(6) on in vitro metabolism of phenylalanine and its related compounds by ruminal bacteria,protozoa and their mixture

An in vitro study was conducted to examine the effects of salinomycin (SL) and vitamin B(6) (B(6)) on the production of phenylalanine (Phe) from phenylpyruvic acid (PPY) and phenylacetic acid (PAA) and of PAA from Phe and PPY by mixed rumen bacteria (B), mixed rumen protozoa (P) and their mixture (BP). Rumen microorganisms were collected from fistulated goats fed lucerne cubes (Medicago sativa) and a concentrate mixture (3 : 1) twice a day. Microbial suspensions were anaerobically incubated at 39 degrees C for 12 h. Phe and some other related compounds in both supernatants and microbial hydrolysates of the incubations were analyzed by HPLC. When PPY was used as a substrate, it completely disappeared without additives and converted mainly to Phe and PAA on the average by 396 and 178, 440 and 189, and 439 and 147 &mgr;M in B, P and BP, respectively, during the 12 h incubation period. The rate of disappearance showed no significant differences between the microbial suspensions with and without SL and B(6) during the incubation period. The production of Phe from PPY with SL was enhanced (p<0.05) by 40, 20 and 19% in B, P and BP, respectively, while PAA production from PPY with SL was inhibited (p<0.05) by 35, 37 and 38% in B, P and BP, respectively, during the 12 h incubation period. On the other hand, with B(6), the production of Phe and PAA from PPY tended to be enhanced by 14 and 17, 9 and 11, and 7 and 22% in B, P and BP, respectively, during the 12 h incubation period. When PAA added as a substrate was incubated in the incubation medium without any additives, it disappeared by 483, 462 and 507 &mgr;M and converted mainly to Phe on the average by 231, 244 and 248 &mgr;M in B, P and BP, respectively. The disappearance of PAA with SL was inhibited (p<0.05) by 16, 15 and 20%, in B, P and BP, respectively, whereas the disappearance of PAA with B6 was almost the same as that without B(6) in B and BP suspensions but tended to be enhanced by more than 9% in P suspensions during the 12 h incubation period. The production of Phe from PAA with SL tended to be inhibited by 12, 11 and 8% in B, P and BP, respectively, during the 6 h incubation period, but the inhibition was weakened during the 12 h incubation period, whereas Phe production from PAA with B(6) tended to be enhanced by 13, 16 and 8% in B, P and BP, respectively. When Phe was added as a substrate, the net Phe disappearance without additives was 549, 365 and 842 &mgr;M and converted mainly to PAA on the average by 254, 205 and 461 &mgr;M in B, P and BP, respectively. The net disappearance of Phe with SL was inhibited (p<0.05) by 38, 28 and 46%, whereas the net disappearance of Phe with B(6) was enhanced (p<0.05) by 9, 8 and 7% in B, P and BP, respectively. The production of PAA from Phe with SL was inhibited (p<0.05) by 73, 54 and 76% in B, P and BP, respectively. On the other hand, with B(6), PAA production from Phe was enhanced (p<0.05) by 19, 18 and 20% in B, P and BP, respectively. Based on these results, it seems that SL inhibited Phe disappearance and enhanced the synthesis of Phe from PPY, though not from PAA, and accumulated free Phe in the medium, whereas B(6) also enhanced Phe synthesis both from PPY and PAA, which could provide additional amino N for animals.     

Somatic embryogenesis and plant regeneration from cell suspensions of Exacum affine

Somatic embryos were produced in seven cultivars of Exacum affine Balf. using flower buds and peduncles as explants. Flowering plants were produced from five of the cultivars, and no visible mutations were detected. The best medium for callus induction and growth was MS supplemented with 9.0 M 2,4-dichlorophenoxyacetic acid and either zero or 0.089 M BA. Callus suspensions were made by passing the callus through a 100 m sieve. The best embryo regeneration was achieved on growth regulator-free medium. Callus and embryos could be grown in liquid medium.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - BA 6-benzyladenine - SD standard deviation     

Induction of microspore embryogenesis in Camellia japonica cv. Elegans

Three methods of microspore culture were tested for the induction of microspore embryogenesis in Camellia japonica L. cv. Elegans. Culture was performed on 17 different media consisting of Murashige and Skoog (MS) and N6 basal media with different combinations of carbon, growth regulators, serine and glutamine. Microspore suspensions plated over solid MS medium containing 4.5 M 2,4-dichlorophenoxyacetic acid and 0.5 M kinetin, with sucrose (MS6) or glucose (MS9) were seen as the best culture conditions for induction of embryogenesis. The development of microspore derived proembryos was obtained in MS medium supplemented with 2.2 M N⁶-benzyladenine (MS10) and reached the highest level when the microspores were cultured in MS6 inducing medium. The development of microspore-derived embryos ceased at the maturation stage.Abbreviations BA N⁶-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid     

Suspension feeding in ciliated protozoa: Feeding rates and their ecological significance

The quantitative uptake of suspended particles has been studied in 14 species of ciliated protozoa in terms of the maximum rate of water cleared at low particle concentrations and of the maximum ingestion rate at high particle concentrations. The results, supported by data from the literature, show that ciliates which feed on larger particles (> 1–5m) compare favorably with metazoan suspension feeders with respect to the ability to concentrate dilute suspensions of particles. Species specialized on smaller food particles (0.2–1m), the size range of most bacteria in natural environments, require a higher concentration of particles. Bacterial population densities which can sustain ciliate growth are found in sediments, waters rich in organic material, and in the early successional stages of decomposing organic material. This is not the case in open waters in general where bacterivorous ciliates cannot play a role as grazers of bacteria.     

Membrane leakage in Bacillus subtilis 168 induced by the hop constituents lupulone,humulone, isohumulone and humulinic acid

Summary The bactericidal activity of the hop constituents lupulone, humulone, isohumulone and humulinic acid against Bacillus subtilis 168 is shown to result from primary membrane leakage. This process leads to complete inhibition of active transport of -methyl-d-glucopyranoside and several amino acids into whole bacteria and isolated membrane vesicles within 5 min at 37°C at the corresponding minimal inhibitory concentrations of 1, 2, 25 and 250 g/ml, respectively. Inhibition of the respiratory chain, of protein, RNA and DNA synthesis seems to be a secondary event.     

Purification and characterization of flavone-specific β-glucuronidase from callus cultures of Scutellaria baicalensis Georgi

-Glucuronidase from callus cultures of Scutellaria baicalensis Georgi was purified to apparent homogeneity by fractionated ammonium-sulfate precipitation and chromatography on diethylaminoethyl-cellulose, hydroxylapatite and baicalin-conjugated Sepharose 6B. A 650-fold purification was obtained by this purification system. When subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified protein migrated as a single band with a molecular mass of 55 kDa. We determined that the native enzyme has a molecular mass of 230 kDa using gel-filtration chromatography. These results suggested that the enzyme exists as a homotetramer composed of four identical 55-kDa subunits. The enzyme showed a broad pH optimum between 7.0 and 8.0. The K _m values were 9 M, 10 M, 30 M and 40 M for luteolin 3 -O--d-glucuronide, baicalin, wogonin 7-O--d-glucoronide and oroxlin 7-O--d-glucuronide, respectively. The enzyme was most active with flavone 7-O--d-glucuronides.Abbreviations BA N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - pI isoelectric point - R _t retention time     

Clonal propagation of Camptotheca acuminata through shoot bud culture

The chinese tree Camptotheca acuminata produces the anti-cancer and anti-retroviral drug camptothecin. Methods were developed for the clonal propagation of this important medicinal plant through shoot bud culture. Shoot buds were excised from 25 to 30 day old seedlings, presoaked for 48 h in three different liquid media containing either BA (2.22–17.4 M), kinetin (2.32–18.58 M), or thidiazuron (0.1–10 M) and were subsequently cultured on semi-solid medium of the same composition. Multiple shoots only developed from the 6-benzyladenine presoaked explants with the maximum number of shoots initiated from buds presoaked in and grown on B5 medium containing 17.4 M 6-benzyladenine. Individual shoots were removed from clusters and rooted on B5 supplemented with indole-3-butyric acid (4.9–19.6 M). The lowest concentration of indole-3-butyric acid (4.9 M) gave the highest percentage of rooting (82%) and the shortest root initiation period (18 d). Over 90% of the in vitro rooted plantlets survived transfer to soil.Abbreviations BA 6-benzyladenine - B5 Gamborg's B5 medium (Gamborg et al., 1968) - CPT camptothecin - 2,4-d 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - kinetin 6-furfurylaminopurine - LS Linsmaier & Skoog medium (Linsmaier & Skoog, 1965) - MS Murashige & Skoog (Murashige & Skoog, 1962) - NAA I-naphthaleneacetic acid - PGR plant growth regulator - TDZ thidiazuron - WPM woody plant medium (Lloyd & McCown, 1981)