Rapid 2,2′-bicinchoninic-based xylanase assay compatible with high throughput screening**

High-throughput screening requires simple assays that give reliable quantitative results. A microplate assay was developed for reducing sugar analysis that uses a 2,2-bicinchoninic-based protein reagent. Endo-1,4--d-xylanase activity against oat spelt xylan was detected at activities of 0.002 to 0.011 IU ml^–1. The assay is linear for sugar concentrations from 0 to 86 g ml^–1 and can also be used to assay protein concentrations (0 to 143 g ml^–1) on the same plate. A variety of temperatures and pH conditions can be used and, after incubation, the assay requires only one detection reagent and one heating step.     

Factors effecting adventitious shoot regeneration from leaf explants of quince (Cydonia oblonga)

A procedure for adventitious shoot regeneration from leaf explants of quince (Cydonia oblonga Mill.) using thidiazuron (TDZ) was developed. Excised leaves of cultures grown on Murashige and Skoog (MS) medium containing 5 M benzyladenine (BA) and 0.9% Gibco Phytagar were used. Several experiments were conducted to determine optimum concentrations of thidiazuron, -naphthaleneacetic acid (NAA) and sucrose. When the medium contained 1.5 M TDZ and 2.5 M NAA, 85% of the discs regenerated shoots with an average of eight shoots per leaf disc. An incubation period of three weeks in the dark was necessary for optimum shoot regeneration. Leaves excised from four to six-week-old cultures gave a higher percent shoot regeneration than leaves from cultures older than six weeks. Regeneration percentages were significantly reduced when sucrose concentration in the medium was less than 3%. A significantly higher percentage of shoots regenerated when leaf discs were placed on the regeneration medium abaxial side down as compared to the adaxial side.Regenerated shoots were cultured on MS medium containing 5 M BA and rooted on half-strength MS medium containing 10 M NAA. Rooted plantlets were acclimatized to greenhouse conditions for evaluation of any somaclonal variation. The importance of these findings are discussed in relation to in vitro improvement of plants.Abbreviations BA benzyladenine - MS Murashige & Skoog (1962) salt mixture - NAA -naphthaleneacetic acid - TDZ thidiazuron (N-phenyl-N'-1,2,3,-thiadiazol-5-ylurea) Approved for publication by the Director, West Virginia Agric. and For. Expt. Sta. as Scientific Article No.2346     

Effect of anti-regucalcin antibody on neutral phosphatase activity in rat liver cytosol: Involvement of endogenous regucalcin

The effect of anti-regucalcin monoclonal antibody on neutral phoshatase activity in rat liver cytosol was investigated. Phosphotyrosine, phosphoserine, and phosphothreonine were used as the substrate toward phosphatase asssy. Liver cytosolic phosphatase activity with three phosphoaminoacids was significantly increased in the presence of anti-regucalcin antibody (100 and 200 ng/ml) in the enzyme reaction mixture with calcium chloride (0.1 mM) or EGTA (1.0 mM). The effect of anti-regucalcin antibody was completely abolished in the presence of exogenous regucalcin (1.0 M), indicating the involvement of endogenous regucalcin. The anti-regucalcin anti body- increased phosphatase activity was not significantly altered in the presence of trifluoperazine (20 M), an antagonist of calmodulin, or akadaic acid (10 M), an inhibitor of protein phosphatase, although these inihibitors caused a slight decrease in liver cytosolic phosphatase activity. The effect of endogenous regucalcin might be not related to calmodulin, and it was insensitive to okadaic acid. The present findings suggest that endogenous regucalcin is involved in the regulation of protein phasphatase in rat liver cytoplasm.     

Aging of plasmodial heterokaryons in Didymium iridis

Summary Isogenic diploid plasmodia of different ages were produced by crossing clonal gamete populations of the myxomycete Didymium iridis. These plasmodia were then used to produce age heterokaryons to study the timing of death in comparison to the senescence of the two original homokaryons. A majority of the age heterokaryons died concurrently with the oldest nuclear component which indicates that the control of aging may be a dominant or active process in the true slime molds. Age heteroplasmons using transfilter contact between two plasmodia across 1 pore size Nucleopore membrane filters were also produced which indicates that senescence may not be a cytoplasmic infectious particle.     

The contribution of size-fractionated plankton to biomass and primary production of phytoplankton in saline–alkaline ponds

Primary productivity, biomass and chlorophyll-a of size fractionated phytoplankton (<0.22 m, <3 m, <8 m, <10 m, <40 m, <64 m, <112 m and <200 m) were estimated in 6 ponds and 5 experimental enclosures. The results showed that the planktonic algae less than 10 m are important in the biomass and production of phytoplankton in saline–alkaline ponds. The production of size fractionated phytoplankton corresponding to <112 m, <10 m and <3 m in saline–alkaline ponds were 10.5 ± 6.6 , 8.6 ± 5.4 and 0.33 ± 0.1 mgC l^–1 d^–1, respectively. Mean community respiration rate was 1.80 ± 0.73, 1.69 ± 0.90 and 1.38 ± 1.12 mgC l^–1 d^–1, respectively. The average production of phytoplankton corresponding to micro- (10–112 m), nano- (3–10 m) and pico- (<3 m) were 1.61, 8.30 and 0.33 mgC l^–1 d^–1, respectively. The ratio of those to the total phytoplankton production was 15%, 79% and 3%, respectively. The mean respiration rate of the different size groups was 0.11, 0.31 and 1.38 mgC l^–1 d^–1; the ratio of those to total respiration of phytoplankton was 6%, 17% and 77%, respectively. The production of size-fractionated phytoplankton corresponding to <200 m, <10 m and <3 m in enclosures was 2.19 ± 1.63, 2.08 ± 1.75 and 0.22 ± 0.08 mgC l^–1 d^-1, respectively. Mean community respiration rates were 1.25 ± 1.55, 1.17 ± 1.42 and 0.47 ± 0.32 mgC l^–1 d^–1, respectively. The average production of phytoplankton corresponding to micro- (10–200 m), nano- (3–10 m) and pico- (<3 m) plankton was 0.11, 1.86 and 0.22 mgC l^–1 d^–1, respectively. The ratio of those to the total production of phytoplankton was 5%, 85% and 10%, respectively. The mean respiration rate of different size groups were 0.08, 0.72 and 0.46 mgC l^–1 d^–1, the ratio of those to total respiration of phytoplankton was 6%, 57% and 37%, respectively. The concentrations of chlorophyll-a of the phytoplankton in the corresponding size of micro- (10–112 m), nano- (3–10 m) and pico- (<3 m) plankton in the experimental ponds were 19.3, 98.2 and 11. 9 g l^–1, respectively. The ratio of those to the total chlorophyll-a was 15%, 76% and 9%, respectively. The concentrations of chlorophyll-a of phytoplankton micro- (10–200 m), nano- (3–10 m) and pico- (<3 m) plankton in enclosures were 1.7, 34.3 and 3.0 g l^–1, respectively. The ratio of those to the total chlorophyll-a was 4%, 88% and 8%, respectively.     

Somatic embryogenesis in callus cultures of Rosa hybrida L. cv. Landora

Callus was initiated from immature leaf and stem segments of rose (Rosa hybrida cv. Landora) and subcultured every four weeks on a basal medium of half-strength Murashige & Skoog (1962) salts plus 30 g l^-1 sucrose (1/2 MS) and supplemented with 2.2 M BA, 5.4 M NAA and 2.2–9.0 M 2,4-D. Embryogenic callus and subsequently somatic embryos were obtained from 8-week-old callus culture on 1/2 MS+2.2 M BA+0.05 M NAA+0.3 M GA₃+200–800 mg l^-1 L-proline. Long-term cultures were established and maintained for up to 16 months by repeated subculture of embryogenic callus on L-proline deficient medium. About 12% of cotyledonary stage embryos taken from cultures cold-stored at 8±1°C for 4 days germinated on 1/2 MS+2.2 M BA+0.3 M GA₃+24.7 M adenine sulphate.Abbreviations BA benzyladenine - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid     

Plant regeneration via somatic embryogenesis in Styrian pumpkin: cytological and biochemical investigations

Somatic embryo formation was induced from cotyledon explants of Styrian pumpkin (Cucurbita pepo L. subsp. pepo var. styriaca Greb.) by using a solid MS medium supplemented with 16.11M NAA and 4.44M BA or 26.85M NAA and 13.32M BA. The callus proliferation was more efficient on medium supplemented with 26.85M NAA and 13.32M BA. In contrast, the embryogenic response was higher on medium with lower concentrations of growth regulators (16.11M NAA and 4.44M BA). The time needed for embryo induction did not depend on medium composition. Embryos in globular stage were transferred to three different maturation media, containing 2.89M GA₃ in combination with 0.54M NAA, 11.42M IAA and growth regulator-free medium. The germination rate was the highest when embryos were cultured on medium with 11.42M IAA. Plantlets grown on this medium achieved maturity suitable for transplantation into soil within 9 to 10weeks. The regenerated plants were successfully transferred into field and developed fertile flowers and set fruits. Biochemical analysis showed significant lower total glutathione levels among in vitro grown plantlets compared to seedlings grown in soil. When the plantlets were transferred into soil, they reached a normal size within a month and the glutathione concentration was comparable to seed-derived plants at the same developmental stage. Transmission electron microscopy was used to investigate possible differences in the ultrastructure of cells from callus cultures, and leaf cells of regenerated and seed-derived plants. Differences in the ultrastructure were found within chloroplasts which contained only single thylakoids, large starch grains and small plastoglobuli in callus cells in comparison to leaf cells, which possessed a well developed thylakoid system, small starch grains and large plastoglobuli.     

Possible modulation of phosphorylation of acetylcholine receptor-enriched membrane preparations

During phosphorylation of acetylcholine receptor (AChR)-enriched membrane preparations fromTorpedo fuscomaculata, phosphate is incorporated into a single protein, with a molecular weight corresponding to that of one of the receptor subunits (37,000 daltons). This protein also seems to contain the receptor binding site. ATP binds to four protein species, one of which corresponds to a different subunit of the receptor (molecular weight 45,000). Phosphorylation of these membrane preparations is affected by several factors, known to be involved in postsynaptic events. Ca²⁺ (10 M) inhibits the reaction, whereas cGMP (20 M), causes stimulation. Furthermore it has been shown that the agonists, acetylcholine, and carbamylcholine (10 M and 1 M) stimulate the phosphorylation reaction, while the antagonists, tubocurarine, hexamethonium, and decamethonium (1 M), cause inhibition.     

Mercury distribution in estuarine environments from Argentina: the detoxification and recovery of salt marshes after 15 years

Total Hg contents from abiotic (surface sediments and suspendedparticulate matter) and biological (crabs, fishes and halophytes)compartments from Bahía Blanca estuary and Mar Chiquita CoastalLagoon, Argentina, have been monitored since the 1980's. At BahíaBlanca estuary, high Hg concentrations were recorded during the early1980's in surface sediments (0.34 ± 0.22 g/g) andsuspended particulate matter (0.19 ± 0.10 g/g). Fishspecies, Mustelus schmitti (0.89 ± 0.29 g/g), Paralichthys brasiliensis (0.85 ± 0.18 g/g) and Micropogonias furnieri (0.37 ± 0.11 g/g) also presentedhigh Hg concentrations. The large industrial nucleus located within theestuary has been identified as the main metal source for this environment.Hg contents from the same area during 1996–1998 were significantlylower: surface sediments (0.164 ± 0.023 g/g), suspendedparticulate matter (0.048 ± 0.0017 g/g), fish Micropogonias furnieri (0.13 ± 0.02 g/g) and crab Chasmagnathus granulata (0.334 ± 0.071 g/g). This trendof environmental detoxification is probably related with (i) thetechnological changes incorporated by the local industry, (ii) a mostadequate management of industrial effluents, and (iii) the removal ofgreat sediment volume by dredging and refill.During the 1980's Mar Chiquita Lagoon Hg concentrations reached 0.08± 0.01 g/g in surface sediments and 0.09 ±0.025 g/g in suspended particulate matter, and 0.14 ±0.04 g/g in the fish Basilichthys bonariensis and 0.22 ±0.08 g/g in Paralichthys brasiliensis, and 0.08 ±0.01 g/g in the crab C. granulata, Hg concentrations werelower than at Bahía Blanca. Remote Hg sources for this Coastal Lagoonand atmospheric and stream transport of Hg is proposed as major Hgsources, since no Hg point sources exists nearby. Mercury concentrationsrecorded in the 1996–1998 period were lower than those recorded inthe previous decade: surface sediments (0.019 ± 0.004 g/g), suspended particulate matter (0.030 ± 0.008 g/g), halophyte Spartina densiflora (0.013 ± 0.008 g/g) or crab C. granulata (0.011 ± 0.009 g/g).Both Hg bioaccumulation and biomagnification processes were verified inBahía Blanca estuary and in Mar Chiquita Coastal Lagoon. This apparentrecovery of both estuarine environments deserves to be carefully analyzed,in order to fully understand the foundations of these processes.     

Somatic embryogenesis and plant development from anther culture of Vitis vinifera (L.)

Anthers from Frumoasa alba (White beauty), Otilia, Valerien, Mission and Siegfried Rebe (FS₄) cultivars were cultured at the uninucleate stage of the microspore on Murashige and Skoog (1962) and Nitsch and Nitsch (1969) media supplemented with 2,4-dichlorophenoxyacetic acid (4.9 M) and benzyladenine (4.4 M). The primary calli were subcultured on MS medium with 6.6 M BA and 1.1 M indolylacetic acid, in order to induce their growth and plant regeneration. After seven months, vegetative buds were obtained with Frumoasa alba (2.7%), Otilia (0.3%), Valerien (4.5%), embryogenic callus was obtained with Mission and plant regeneration with Siegfried Rebe. Long term embryogenesis was maintained in Mission cv. for four years, by selection and regular transfer of the embryogenic areas of anther-derived calli. The embryogenic calli have the ability to generate abnormal somatic embryos with one, two or three cotyledons and cup or trumpet-shaped with fused cotyledons. In parallel with the embryogenic process, organogenesis with buds, leaf and shoot differentiation was regularly observed.     

Calpain-PKC Inter-Relations in Mouse Hippocampus: A Biochemical Approach

In previous studies, we isolated and identified a -calpain/PKC complex from rabbit skeletal muscle. Here, we have used specific purification procedures in order to study the interactions between -calpain and PKC in mouse hippocampus, a brain structure implicated in memory processes. We observed that -calpain and conventional PKCs (, II and ) are co-eluted after anion exchange chromatography. In contrast to our previous results obtained on skeletal muscle, -calpain and PKC isoenzymes were dissociated after gel filtration chromatography. Furthermore, -calpain induced the proteolytic conversion of PKC, II, and into PKM, II, and with a preferential hydrolysis of PKC, a specific isoenzyme of the nervous system. Although the -calpain/PKC interactions in the hippocampus are quite different from skeletal muscle, our results however, point out the functional importance of these inter-relations. Moreover, as PKC has been involved in the biochemical events underlying learning and memory, the preferential relationship between -calpain and PKC promotes the importance of the role that -calpain could play in the cellular mechanisms of memory formation.     

Plant regeneration from leaf protoplasts of apple

Protoplasts were isolated from young leaves or etiolated shoot apices. For initiation of divisions the protoplasts were embedded in sodium alginate and cultivated in MS or MI medium supplemented with 2.2 M BA, 2.6 M NAA and 2.2 M 2,4-dichlorophenoxyacetic acid. The protoplasts of all seven lines tested developed to protocalluses at high frequencies. No genotypic differences were observed. When BA was used in combination with NAA in the regeneration experiments, only a few protocalluses (highest frequency 3%) exhibited shoot organogenesis. When BA was replaced with thidiazuron, the percentage of protocalluses that developed shoots increased in two of three tested lines to 7% and 56%, respectively. Shoot development was achieved under light conditions. The shoots were then rooted and transferred into soil.Abbreviations ABA abscisic acid - BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - FW fresh weight - GA₃ gibberellic acid - IBA indole-3-butyric acid - MES 2-N-morpholinoethane sulphonic acid - NAA -naphthaleneacetic acid     

Der Entwicklungscyclus mit Sexualphase bei der marinen Diatomee Coscinodiscus asteromphalus

Zusammenfassung Die Kultur der großen marinen Diatomee Coscinodiscus asteromphalus wird beschrieben. Die Synchronisation der vegetativen Stadien aus dem Entwicklungscyclus mit Sexualphase wird durch Messung der Valvendurchmesser charakterisiert. Die Art entwickelt sich von Stadien mit 200 m Valvendurchmesser (V.-D.), die nicht sexuell induzierbar sind, zu Stadien mit 80–90 m V.-D. mit einem Optimum der Induzierbarkeit und weiter zu Stadien mit 55–60 m V.-D. Bei dieser Größe ist keine weitere mitotische Zellteilung mehr möglich. Entwicklungsstadien mit 200–190 m, 140–130 m und 100–90 m. V.-D. zeigen bei 24°C und bei 18°C die gleiche Generationszeit im mitotischen Entwicklungscyclus von 1 bzw. 0,6 Zellteilungen pro Tag. Der Valvendurchmesser verringert sich bei dieser Art um 1,5 m bei 24°C und 1,4 m bei 18°C während einer Zellteilung.

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The life cycle with sexual phase in the marine diatom Coscinodiscus asteromphalus I. Culture and synchronisation of developmental stages

Summary Culture-conditions for the large marine centric diatom Coscinodiscus asteromphalus are described. The cells grow in a defined medium in a light-dark regime of 14: 10 h. Synchronization of different stages of the sexual life cycle is characterized by measuring the valve diameter (v.d.) of the cells. The cells develop from stages with 200 m v. d. (not sexually inducible) to stages with 80–90 m v. d. (optimum for sexual induction), and further to stages with 55–60 m v. d., where no following mitotic cell division is possible. The length of the pervalvar axis does not change during this development. Different stages (200–190m, 140–130 m and 100–90 m v. d.) grow with the same doubling time during their mitotic life cycle: 1 cell division per day at 24° C and 0.6 cell divisions per day at 18°C. During one cell division the valve diameter of this species decreases by about 1.5m at 24°C and by 1.4 m at 18°C. Therefore, the development from stages with 200 m v.d. to stages with 60 m v. d. takes between 90 days at 24°C and 165 days at 18°C.

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Teile einer Habilitationsschrift der Naturwissenschaftlichen Fakultät der Universität Marburg (Lahn).     

Trois nouveaux Nadejdolepis Spasskii & Sasskaya, 1954 (Cestoda: Hymenolepididae) parasites de Charadrii (Aves) de Tasmanie

Three species of Nadejdolepis from Tasmania, Australia, are described and illustrated. N. burgessi n. sp., a parasite of Charadrius ruficapillus, is 4-6 mm long, with rostellar nitiduloid hooks 63-66 m long, a short evaginated cirrus 13-16 m long with a short collar of thin spines 1 m long, a narrow and tubular sclerotinoid vagina 40-50 long and 3-4 m in diameter with a little ampulla 3-5 m in diameter at the proximal end, and a membranous atrial segment with smooth, short (1 m) and compact spines which are sometimes difficult to observe. N. smithi n. sp., a parasite of 40-50 long and 3-4 m in diameter with a little ampulla 3-5 m in diameter at the proximal end, and a membranous atrial segment with smooth, short (1 m) and compact spines which are     

Uptake and Retention of Selenite and Selenomethionine in cultured K-562 cells

The selenium uptake and retention have been studied in K-562 cells exposed to selenite or selenomethionine. In the uptake experiments the cells were exposed to two doses of selenite (5 or 50 M) or selenomethionine (10 or 50 M). In the retention study the cells were treated for 2 h with the above mentioned doses of the selenocompounds before being observed at different times. The selenium uptake in cells exposed to selenite 5 M began to saturate at 8 h, but increased again between 48 and 96 h. In cells exposed to selenite 50 M the selenium uptake never reached a maximum, however, at 48 and 96 h the cell viability decreased strongly. The two doses of selenite showed different retention patterns, with a relatively small cellular decrease of selenium after treatment with selenite 5 M compared to treatment with 50 M of selenite. The selenium uptake in cells exposed to selenomethionine 10 M or selenomethionine 50 M began to saturate at 24 h and 48 h, respectively. The retention patterns were similar for both selenomethionine doses with a continuous decrease of the selenium concentration during the whole observation period. The results indicated a more controlled uptake and retention pattern of selenomethionine compared to selenite.     

Minimal growth in vitro conservation of coffee (Coffea spp.)

We have studied the influence of low concentrations of 6-benzyladenine on growth limitation, in order to preserve coffee germplasm through a microcutting collection. Concentrations of 0 M, 1.3 M and 4.4 M were compared in four species: Coffea congensis, C. canephora, C. liberica and C. racemosa. After six months, microcutting behaviour varied between the different treatments, and a species effect was observed. The slow growing species (C. liberica and C. congensis) needed 1.3 M; the others coffee species (C. canephora and C. racemosa) exhibited moderate caulogenesis on 6-benzyladenine-free medium. Zero and low concentrations did not affect survival rates. In conclusion 1.3 M seems most appropriate for conserving all four species.Abbreviation BA 6-benzyladenine     

Feeding in the rotifer Brachionus calyciflorus

Summary The laboratory feeding behavior of Brachionus calyciflorus varies depending upon the type of food cell available in suspension. When feeding on the yeast Rhodotorula glutinis, rotifers show a continuous increase in ingestion with increased cell density between 0.01 and 1000 g dry weight ml^-1. Effective clearance rates drop from ca. 50 l animal^-1 h^-1 to less than 0.5 l animal^-1 h^-1 over this food density range. When feeding on Englena gracilis, B. calyciflorus ingestion rates are constant between 1.0 and 100 g ml^-1 of available food, averaging close to 25 ng animal^-1 h^-1. The decrease in clearance rate is more striking than with R. glutinis, dropping from 45 l animal^-1 h^-1 at 0.1 g ml^-1 to 0.13 l animal^-1 h^-1 at 100 g ml^-1. Differences between the patterns obtained with the two food types indicate fundamental dissimilarities in the feeding behavior of this rotifer species when presented with these different foods.     

Estimating the age of Antarctic larval fish from otolith microstructure using light and electron microscopy

Larval fish of Antarctica have very narrow rings on their otoliths (<1 m) that may not be resolved with light microscopy. In this study, age data from the otoliths of larval Nototheniidae (Gobionotothen gibberifrons and Lepidonotothen larseni), determined using light and scanning electron microscopy, are compared. Rings 0.4 m wide were observed on otoliths viewed under electron microscopy; however, light microscopy could only resolve rings 0.5 m wide. Scanning electron microscopy is more time consuming and costly than light microscopy but has greater resolving power and is recommended to validate ring counts made using light microscopy in otolith studies with Antarctic larval fish.     

Development of shoots and roots in anther-derived callus of Azadirachta indica A. Juss.—a medicinal tree

Callus originated in microsporangial wall layers and connective tissues of anthers containing uninucleate microspores on Nitsch's or Murashige and Skoog's medium supplemented with growth regulators. A higher percentage of cultures (43) produced callus on Nitsch's medium containing 10 M indole-3-acetic acid + 1 M 6-benzyladenine. After 13–15 weeks, green nodular structures and prominent roots developed in 25% of the cultures on Murashige and Skoog's medium + 10 M -naphthaleneacetic acid + 1 M kinetin. Multiple shoots were induced in this anther-derived callus when subcultured on Murashige and Skoog's medium augmented with 4.44 M 6-benzyladenine + 0.53 M -naphthaleneacetic acid along with 18.75 M polyvinylpyrrolidone. The excised shoots formed roots after subculturing on Murashige and Skoog's medium + 4.90 M indole-3-butyric acid + 18.75 M polyvinylpyrrolidone, thus developing complete plantlets. Examination of callusing anthers also revealed two- to multi-celled pollen masses with intact exine.Abbreviations BA 6-benzyladenine - CW coconut water - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - HCl hydrochloric acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - KMnO₄ potassium permanganate - MS Murashige & Skoog's medium - NAA -naphthaleneacetic acid - NB Nitsch's medium - PVP polyvinylpyrrolidone     

The Effect of Phenylalanine on DOPA Synthesis in PC12 Cells

DOPA synthesis from phenylalanine was studied in PC12 cells incubated with m-hydroxybenzylhydrazine, to inhibit aromatic L-amino acid decarboxylase. DOPA synthesis rose with increasing concentrations of either phenylalanine or tyrosine; maximal rates (~55 pmol/min/mg protein for tyrosine; ~40 pmol/min/mg protein for phenylalanine) occurred at a medium concentration of ~10 M for either amino acid. The K_m for either amino acid was about 1 M (medium concentration). At tyrosine concentrations above 30 M, DOPA synthesis declined; inhibition was observed at higher concentrations for phenylalanine (300 M). These effects were most notable in the presence of 56 mM potassium. Measurements of intracellular phenylalanine and tyrosine suggested the K_m for either amino acid is 20–30 M; maximal synthesis occurred at 120–140 M. In the presence of both phenylalanine and tyrosine, DOPA synthesis was inhibited by phenylalanine only at a high medium concentration (1000 M), regardless of medium tyrosine concentration. The inhibition of DOPA synthesis by high medium tyrosine concentrations was antagonized by high medium phenylalanine concentrations (100, 1000 M). Together, the findings indicate that for PC12 cells, phenylalanine can be a significant substrate for tyrosine hydroxylase, is a relatively weak inhibitor of the enzyme, and at high concentrations can antagonize substrate inhibition by tyrosine.