Occurrence of colloid-containing follicles in the pars distalis of pituitary glands from aging guinea pigs

Summary Colloid-containing follicles in the pars distalis of pituitary glands from guinea pigs at various ages ranging from 5 days to 36 months were examined by the periodic acid-Schiff (PAS) reaction, immunohistochemistry, and electron microscopy. The follicles storing PAS-positive colloid were first detected in 6-month-old animals, in which only a few follicles were present and mean diameters of colloid deposits were small: 4.3±1.0 m in males and 4.1±0.4 m in females. Thereafter, the follicles gradually increased in number and size with age. The largest number of follicles was observed in the senile groups: 410.5±92.3 in males, 454.7±84.7 in females. Mean diameters of colloid masses in the senile groups were more than 2 times larger than those in 6-month-old animals: 10.0±0.1 m in males, 9.7±0.1 m in females. These findings suggest that the formation of colloidcontaining follicles in the guinea-pig pars distalis is an aging phenomenon. The follicular lumina were mainly surrounded by thin cytoplasmic processes or cell bodies of folliculo-stellate cells immunoreactive for S-100 protein. The lining folliculo-stellate cells showed aggregations of intermediate-sized filaments, numerous lysosomes and colloid-like inclusions. Granulated cells in contact with colloid were occasionally encountered. Intracellular cavities storing colloid-like and fibrous materials were detected in the syncytial formation of GH cells.     

Effect of DNA concentration on transgenesis rates in mice and pigs

A retrospective analysis of transgenesis rates obtained in seven pronuclear microinjection programs was undertaken to determine if a relationship existed between the amount of DNA injected and transgenesis rates in the pig. Logistic regression analysis showed that as the concentration of DNA injected increased from 1 to 10ng/l, the number of transgenics when expressed as a proportion of the number liveborn (integration rate) increased from 4% to an average of 26%. A similar relationship was found when the number of molecules of DNA injected per picolitre was analysed. No evidence was obtained to suggest either parameter influenced integration rate in mice when the same constructs were injected. The number of transgenics liveborn when expressed as a proportion of ova injected (efficiency rate), increased as DNA concentration increased up to 7.5ng/l and then decreased at 10ng/l for both species suggesting that at this concentration DNA (or possible contaminants) may have influenced embryo survival. The relationship between efficiency and the number of molecules injected per picolitre was complex suggesting that the concentration at which DNA was injected was a better determinant of integration and efficiency rates. In conclusion, the present study suggests that transgenes need to be injected at concentrations of between 5 and 10ng/l to maximise integration and efficiency rates in pigs.     

Effects of acute opiate-peptide administration on pro-opiomelanocortin cells of the intermediate lobe of the rat pituitary

Summary The effects of acute injections of synthetic opiate peptides into the lateral cerebral ventricle of young adult male rats on cells of the intermediate lobe of the pituitary were studied. Met-enkephalin (100/g) injected into anesthetized rats, or 20 g beta-endorphin administered via a previously implanted cannula to unanesthetized animals, will lead to cell degranulation and often to expanded Golgi zones and prominent regions of rough endoplasmic reticulum in secretory cells when tissue is fixed 45–60 min after peptide administration. Treatment of animals with the opiate antagonist naloxone hydrochloride prior to enkephalin injection appeared to prevent the cellular changes elicited with peptide alone. Observations suggest that opiate peptides administered to the cerebrospinal fluid may stimulate release of pro-opiomelanocortin-peptide from pituitary cells.     

Effect of estradiol treatment on male mice synaptonemal complexes: difference of sensitivity between neonates and adults

The effect of estrogen on pachytene spermatocytes was studied with the assistance of the synaptonemal complex analysis under electron microscopy. Male NMRI mice were injected with estradiol benzoate from birth onwards and allotted to different groups according to the dose administered: 1) three injections of either 12.5 g or 25 g or 50 g on d0, d5 and d10; 2) single injections of 50 g either on d0 or on d5 or on d10; 3) double injections of 50 g on d0 and d5; and 4) daily injection at the dose of 0.5 g/g BW from d0 to d27. Animals were sacrificed on day 28, 60 and 90. Adult male mice were treated daily with E2B (0.5 g/g BW) for one (from d30 to d60) or two months (from d30 up to d90) to test the age-related sensitivity to estrogen. A number of different SC anomalies were observed at each harvest time. Among all the anomalies, pairing failure (asynapsis) was predominant followed in decreasing order of importance by SC breakage (fragmentation of SCs), and heterotelomeric associations resulting either in quadrivalent-like figures or in trivalents. In E2B treated neonates the frequency of SC anomalies, which was less than 2% in controls, varied from 3.6 to 27% of pachytene cells regardless of the harvest time. In E2B treated adult mice, the SC anomalies were rare (<4%), but significantly different from controls in which the frequency of SC aberrations did not exceed 1% of pachytene cells. The prevalence of anomalies appeared to be independent of the TW decrease. Our observations suggest that estrogens act indirectly on SCs. Different mechanisms of action are discussed.Abbreviations BW body weight - E2B estradiol benzoate - d day - Gr(s) or gr(s) group(s) - LE(s) lateral element(s) - n number of examined mice - NAC number of abnormal cells - NPC number of cells at pachytene stage - SC(s) synaptonemal complex(es) - SD standard deviation - TW testicular weight     

Hormones andCuscuta development: In vitro induction of haustoria by cytokinin and its inhibition by other hormones

Cytokinins induced haustoria formation in excised 10-mm segments ofCuscuta vine, the subapical 25-to-50-mm region being most responsive, producing a mean of 4–6 haustoria per segment. The order of effectiveness of cytokinins continuously applied (72 h) was 6-benzylaminopurine (BA) isopentenyladenine (iP) zeatin (Z). Ribosides of BA and Z were as effective as the bases, whereas riboside of iP ([9R]iP) was half as effective as iP. Haustoria induction was influenced by weather and seasonal conditions at the time of vine collection; materials obtained on warm, sunny days responded better than those obtained on rainy, cloudy, or cool days. Haustoria were induced equally well all around the segment, and no thigmostimulus was needed for induction. p ]A 10-min pulse of 100 M BA induced half as many haustoria as a 60-min pulse or continuous application of BA. White light inhibited haustoria induction elicited by a short (30-min) pulse of BA, whereas a longer (120-min) BA application overcame this light inhibition. Auxins (IAA or NAA, 1–10 M), gibberellin (GA₃, 1–10 M), ethylene (as ethrel, 10–100 M), and abscisic acid (ABA, 100 M) were individually inhibitory (60–80%) with respect to haustoria induction when given continuously with 50 M BA. A 60-min pulse of auxins (10 M), GA₃ (100 M), or ethrel (10 M), given at various time intervals during or after a 60-min pulse of 100 M BA, showed that inhibition was maximal (70–95%) between 4 and 16 h of BA application and negligible (GA₃) or much reduced (auxin, ethrel) at 20 h, indicating a commitment to haustoria formation by this time.     

Corticosteroids inhibit the generation of lymphokine-activated killer activity in vitro

Summary In phase-I clinical trials of adoptive immunotherapy using lymphokine-activated killer (LAK) cells plus recombinant interleukin-2 (rIL-2) (Cetus) for the treatment of malignant glioma, we observed that blood mononuclear cells (MNC) from patients dependent on dexamethasone for management of cerebral edema produced substantially less LAK activity as compared to MNC of normal blood donors or glioma patients not receiving steroid therapy. Therefore, we examined the in vitro effects, brought about by therapeutically attainable concentrations of various corticosteroids, on the proliferative response, production of interferon (IFN-), and induction of LAK activity from blood MNC of normal donors. Incubation in media containing rIL-2 (1000 U/ml) with either dexamethasone, hydrocortisone, methylprednisolone, or prednisolone profoundly affected all of these parameters. First, while 0.01 g/ml of either dexamethasone or hydrocortisone caused a slight enhancement of the mitogenic response of lymphocytes to phytohemagglutinin, a dose-dependent decline occurred as concentrations increased to 10 g/ml. The addition of prednisolone and methylprednisolone elicited a dose-dependent inhibition of lymphocyte proliferation over the entire concentration range tested. At 0.1 g/ml or higher, dexamethasone, hydrocortisone, methylprednisolone and prednisolone significantly (P<0.02) inhibited the production of IFN-: respectively 18.9%, 4.4%, 2.2%, and 12.3% of the IFN- produced by MNC in the absence of steroids. All four corticosteroids inhibited the induction of LAK activity. Compared to MNC that had been incubated with 1000 U/ml rIL-2 alone, MNC cultured with rIL-2 and 10 g/ml either dexamethasone or prednisolone demonstrated significantly lower cytotoxicity (P<0.05) for the natural-killer-cell-resistant cell line, Daudi. Culturing MNC with hydrocortisone had a more dramatic result, causing a significant decline (P<0.01) in lytic activity at both 1.0 g/ml and 10 g/ml, while incubation with methylprednisolone produced a significant drop (P<0.02) in LAK-mediated cytotoxicity at 0.1 g/ml as well as 1.0 g/ml and 10 g/ml. When cytotoxicity was expressed as lytic units per million effectors, a dose-response decline in lytic activity was once again apparent, with hydrocortisone, methylprednisolone and prednisolone showing significant inhibition (P<0.05) at both 1.0 g/ml and 10 g/ml and dexamethasone at 10 g/ml (P<0.01). These results indicate that corticosteroids commonly used in the management of cerebral tumors and other malignancies inhibit induction of LAK activity in vitro, and this may explain why it is often difficult to generate LAK activity from blood MNC of patients who are receiving chronic steroid therapy.     

Localization of 3H-dihydrotestosterone in the pituitary gland of the rhesus monkey

Summary The uptake and retention of radiolabelled dihydrotestosterone by the pituitary gland was examined in the rhesus monkey. Two animals were given an intravenous injection of 1.0g/kg ³H-dihydrotestosterone (DHT) alone while one monkey received both the labelled androgen and 100g/kg of unlabelled steroid. One and a half hours later, they were sacrificed. The pituitary glands were removed and processed for autoradiography and immunocytochemistry. Autoradiographic localization of DHT was discernible in the partes nervosa, intermedia and distalis, albeit the highest concentration of radiolabelled cells was noted in the pars distalis. Immunocytochemical staining with antibodies to rat PRL, human TSH and ovine LH revealed a population of steroid-concentrating cells that contained TSH and a second group that contained LH. None of the cells that reacted with the anti-PRL serum were radiolabelled.     

Characteristic induction of 70 000 Da-heat shock protein and metallothionein by zinc in HeLa cells

The synthesis of a 70 000 dalton-heat shock protein (hsp70) is one of several heat shock proteins induced in HeLa cells during the incubation in medium containing zinc sulphate. The synthesis of hsp70 was increased in the presence of 200 M zinc sulphate and above, but not at 100 M zinc sulphate. On the other hand, the synthesis of metallothionein was activated in the presence of 100 M zinc sulphate and above. Uptake of zinc into the cells depended on the concentration of zinc sulphate in the medium. The separation of intracellular zinc into three fractions by gel filtration chromatography; high molecular, metallothionein, and low molecular fractions, showed that zinc in the low molecular weight and metallothionein fractions was elevated in the presence of 100 M zinc sulphate in the medium, whereas increase in the zinc content of the high molecular weight fraction occurred at 200 M zinc sulphate and above. Inhibition of cell growth and cellular protein synthesis was also observed at 200 M zinc sulphate and above, but not at 100 M. From these findings, since the induction of hsp70 synthesis and inhibition of cell growth occurred concomitantly with the increase of zinc in the high and low molecular weight fractions, hsp70 seemed not to function in the detoxification of zinc, but it may participate in the repair of zinc-induced damage.     

Intrastriatal Administration of Methylmercury Increases In Vivo Dopamine Release

Mercury is a neurotoxin that exists in a number of physical and chemical forms, producing different effects in the brain. In the present work, we have studied the effects of intrastriatal administration of different doses (40 M, 400 M, and 4 mM) of organic mercury (methylmercury, MeHg) on the dopaminergic system of rat striatum, in conscious and freely-moving animals, using microdialysis coupled to Liquid Chromatography. In previous works, we have discussed the effects of chronic and acute administration of MeHg on striatal dopaminergic system assessing changes in both release and metabolism of striatal dopamine (DA). In the present study we report that the intrastriatal administration of different doses of MeHg (40 M, 400 M, and 4 mM) produced significant increases (907 ± 31%, 2324 ± 156%, and 9032 ± 70% of basal levels, respectively for the different doses) in DA release from rat striatal tissue associated with significant decreases in extracellular levels of its main metabolites dihydroxyphenylacetic acid (DOPAC) and homovallinic acid (HVA) using the dose of 4 mM MeHg (35 ± 3% and 48 ± 1%, respectively), whereas non-significant changes in metabolite levels were observed with the doses of 40 M and 400 M MeHg. We explain these effects as a result of stimulated DA release and/or decreased DA intraneuronal degradation.     

Messung von radioaktiv markierten Gewebeschnitten

Summary The amount of tritium activity in labeled histological sections mounted on slides can be measured in a liquid szintillation spectrometer. An efficiency of about 20% is obtained, dependence on section thickness (tested on 3 , 5 , and 10 , sections) does not exist. After washing out the szintillation fluid autoradiography and staining of the sections as usual is possible.     

Quantitative observations of the effect of sex-steroids on the postnatal development of LH-cells

Summary The effects of sex-steroids on the LH-cell development in neonatal rats were studied. The cells were stained immunohistochemically by applying anti-HCG serum. On the second day after birth some of the animals of both sexes were gonadectomized and simultaneously injected with testosterone or estradiol (50 or 200 g). The remaining animals were either gonadectomized or injected with either one of the sex-steroids. The LH-cell numbers in each group were determined on the 12th day of age from serially cut histological sections of the pituitary. In castrated males the number of LH-cells was about twice that of the intact animals. In the so-called sex-zone, LH-cells tended to be hypertrophied in castrates. These alterations in the appearance of LH-cells did not occur after ovariectomy. In gonadectomized animals injected with sexsteroids (200 g), the cells were markedly reduced in number and size, both in males and females. Testosterone injection (50 g) into intact newborn animals also suppressed the numerical development of LH-cells, especially in females. These alterations were particularly evident in the sex-zone in both sexes. Thus the present findings show that sex-steroids may be involved in sexual differences in morphological development of LH-cells in newborn rats.     

Adventitious in vitro plantlet formation from immature floral stems of Crinum macowanii

Adventitious shoots and plantlets were regenerated in vitro from floral stem explants of Crinum macowanii (Bak.) (bush lily). The length (age) of the floral stem as well as the orientation and position of the explant disc in the floral stem were the most important factors affecting shoot regeneration. The highest number of shoots were regenerated when immature floral stems of 70–100 mm were used as starting material, using the middle or basal parts of the stem, and orientating the discs with their proximal ends on the medium. Combinations of kinetin (4.65 M) and either indoleacetic acid (0.57 M) or naphthaleneacetic acid (0.54 M), or a combination of benzyladenine (4.44 M) and 2,4-dichlorophenoxyacetic acid (0.45 M) resulted in the highest numbers of shoots being regenerated. Although a slight degree of callus formation was noticed on the cut-edges of the discs, shoot formation did not occur via callus, but directly from the floral stem epidermis. Unrooted shoots were rooted on MS-medium containing 0.17 M sucrose.Abbreviations BA benzyladenine - IAA indoleacetic acid - IBA indolebutyric acid - NAA naphthaleneacetic acid - 2,4-d 2,4 dichlorophenoxyacetic acid     

Cytoplasmic effects on the tissue culture response of callus from winter wheat mature embryos

A simple and reliable method was established for the maintenance of a permanent stock of several Medicago truncatula genotypes selected from a general seed stock by their in vitro culture amenability and embryogenic capacity. In the first step, multiple shoots were induced from the cotyledon axillary meristem meristem of pre-germinated seeds in Murashige and Skoog medium supplemented with cytokinins (9.3 M zeatin, 22.2 M benzylaminopurine or 4.5 M thidiazuron). In the second step, the induced shoots were allowed to develop in growth-regulator-free medium. Benzylaminopurine at 22.2 M supported the best combination of shoot quality and number of shoots produced. Rooting of microshoots depended on the cytokinin used for shoot induction and was faster for zeatin-treated shoots. In this work a propagation system was devised where the addition of growth regulators was restricted to the induction phase therefore reducing the risks of epigenetic and somaclonal variation.     

The effect of antibiotics on the inhibition of callus induction and plant regeneration from cotyledons of sugarbeet (Beta vulgaris L.)

Callus induction and plantlet regeneration from cotyledonary expiants of sugarbeet was observed utilizing two media formulations, MS and a modified MS termed RVIM both supplemented with 1.0 g/ml BAP as the sole growth regulator. Callus induction was genotype dependent The USDA line 8787 produced the highest response for callus induction followed by Betaseed 4587 and the USDA line C600. This order was conserved on both media formulations. Shoot induction was consistently higher averaging 32% from the RVIM formulation over the 3 genotypes compared to 25% from MS. The antibiotics geneticin, gentamycin, hygromycin, kanamycin and phleomycin were screened with the modified RV system utilizing Betaseed 4587. Callus growth was inhibited by levels of 50 g/ml geneticin, 150 g/ml gentamycin, 10 g/ml hygromycin, 150 g/ml kanamycin and 20 g/ml phleomycin. The results indicate that the concentrations of antibiotics used to inhibit callus induction will be sufficient for use as selectable markers in transformation experiments with Beta vulgaris.Abbreviations B₅ basal medium (Gamborg et al, 1968) - BAP N⁶-Benzylaminopurine - IBA Indole-3-butanoic acid - MS basal medium (Murashige and Skoog 1968) - RVIM modified MS basal medium (Freytag et al, 1988) - MES (2[N-Morpholino] ethanesulfonic acid     

Somatic embryogenesis and plant regeneration from the immature cotyledonary tissues of cultivated tea (Camellia sinensis (L).O. Kuntze)

Embryogenic callus development, plant regeneration, and plant recovery were achieved from immature cotyledon explants of cultivated tea, when cultured on MS basal medium. The somatic embryo induction frequency was influenced when the medium was supplemented with 1 M auxin (NAA, NOA, 2,4-D, TPB, and PBOA) in combination with cytokinin (0.5 M BA, KIN) or 10% CM. The highest somatic embryo induction frequency was obtained using PBOA + BA or PBOA + KIN treatments. All auxins except 2,4-D stimulated rhizogenesis using 0.8% and l.5% agar concentrations, and differentiation of a characteristic swelling and friable callus from the exposed surface of the explant that remained nonembryogenic. Conversely, the novel auxins TPB and PBOA at 1 M concentration with 3% or 6% agar, produced somatic embryo induction, while at 0.8% and 1.5% produced nonembryogenic callus. Explants isolated proximal to the zygotic embryonal axis showed a greater somatic embryo induction frequency than did the distal explants. The embryogenic competence was maintained through repetitive embryogenesis for a period of over 18 months. The somatic embryos developed into plantlets when incubated on hormone-free medium. The conversion frequency was increased by 50% in MS medium containing 1 M Brassin and 0.8% agar. Concentration of agar at 3% and 6% decreased the conversion frequency and promoted anomalous plantlet development. The normal plantlets were treated with 1 M IAN, 1 M Brassin and 10 Phloroglucinol in liquid MS medium for 15 d, where profuse lateral roots were induced favoring a high rate of plant recovery.     

A comparison of BA,zeatin and thidiazuron for adventitious bud formation from Picea glauca embryos and epicotyl explants

Picea glauca (white spruce) zygotic embryos and one-week-old-seedling epicotyl explants were placed on either Woody Plant Medium (WPM) or half-strength Schenk & Hildebrandt (1/2S&H) medium supplemented with varying levels of benzyladenine (BA) (0.1, 1.0, 10, 50, 100 M), zeatin (10, 50, 100 M) or thidiazuron (TDZ) (0.01, 0.1 M). In addition to differences in the number of buds induced at three months on the two media, buds induced on WPM were visually more uniform, less vitrified and elongated faster. On 1/2S&H supplemented with BA, maximum bud induction from embryos occurred on 1.0 M BA with 0.01 M TDZ with higher BA concentrations inhibitory to bud induction. In contrast, on WPM there was little difference in the number of buds induced from embryos placed on 10, 50 and 100 M BA with or without TDZ. One-week-old-seedling epicotyl explants required higher BA levels on 1/2S&H, as bud induction at three months was greatest at 10 M BA. On WPM, as with the embryos, there were only minor differences in the number of buds induced from epicotyl explants on the various BA levels. Zeatin was more effective at inducing buds than BA with both media. From embryos, bud induction was greatest on 50 or 100 M zeatin without TDZ and 50 or 100 M zeatin with or without TDZ on 1/2S&H and WPM respectively. From epicotyl explants on 1/2S&H, there was little difference in the number of buds induced with the zeatin concentrations used, while with WPM, 50 and 100 M zeatin induced the greatest number of buds. Interestingly, with BA, the epicotyl explants needed a higher level than the embryos for maximal response, while with zeatin, the level was the same for both embryos and epicotyl explants. Long-term (six month) survival was higher on WPM than with 1/2S&H. Additionally, embryos had a higher percentage of genotypes surviving at six-months when compared with epicotyl explants. For overall survival and development of the buds, 50 M zeatin with 0.01 M TDZ was the best treatment tested.Abbreviations BA benzyladenine, 1/2S&H-half-strength Schenk & Hildebrandt medium - TDZ thidiazuron - WPM woody plant medium     

Mitochondria in fine afferent nerve fibres of the knee joint in the cat: a quantitative electron-microscopical examination

The distribution of mitochondria, their content and concentration (expressed as the ratio of the mean volume of mitochondria and the surface of the sensory axon) were determined in group-III and-IV nerve fibres innervating the knee joint capsule in the cat. Mitochondria mainly accumulated in axonal swellings (beads) and end bulbs of the terminal branches. Between single nerve fibres, marked differences in the content and the concentration of mitochondria were obtained in proximal portions (inside of the perineurium) and in distal portions (unmyelinated sensory endings). In group-III nerve fibres, the mitochondrial concentration ranged from 0.005 to 0.030 m³/m² (proximal portion) and from 0.016 to 0.080 m³/m² (distal portion). In unmyelinated group-IV nerve fibres, the values also showed a broad variation ranging from 0.001 to 0.011 m³/m² (proximal portion) and from 0.003 to 0.019 m³/m² (distal portion). The wide range of mitochondrial concentrations may reflect different energy consumption during receptive processes: nerve fibres with a low mechanical threshold and a high probability of excitatory events may be rich in mitochondria, whereas fibres with a high mechanical threshold and a low probability of excitatory events may be poor in mitochondria.     

Adventitious shoot formation and plant regeneration from tissues of tomatillo (Physalis ixocarpa Brot.)

Cultured hypocotyl explants of tomatillo (Physalis ixocarpa Brot.), were evaluated with regard to their morphogenic responses to combinations of benzyladenine (BA, 0–5 M) with either naphthaleneacetic acid (NAA, 0–50 M) or 2,4-dichlorophenoxyacetic acid (2,4-D, 0–50 M). The induction of shoots or roots was dependent on the cytokinin/auxin combination.Hypocotyl explants failed to form shoots when they were grown on media containing either a cytokinin or an auxin alone. The highest frequency of shoot formation was observed on media containing 12.5–25 M BA and 5 M NAA. Likewise the highest frequency of root formation was observed on media supplemented with 1 M BA and 1 M NAA. Complete plants were regenerated and transferred to soil, where they reached maturity.     

Beauveria bassiana protoplast regeneration and transformation using electroporation

Summary Regeneration of Beauveria bassiana GK2016 protoplasts demonstrated three phases: (1) enlargement, (2) formation of a chain of budding cells, and (3) development of a true germ tube from the original protoplast. Regeneration frequencies of up to 80% were obtained when using the stabilizer ammonium sulphate. Using electroporation, protoplasts were transformed to methyl 1,2-benzimidazole carbamate (MBC) resistance with the Neurospora crassa MBC-resistant -tubulin gene. A transformation efficiency of one of three transformants per 5 g vector DNA was obtained. The MBC phenotype was stable and transformants grew in the presence of 5 g MBC/ml. Southern DNA-DNA hybridization analysis demonstrated that integration of the vector into the chromosomal DNA had occurred. Correspondence to: G. G. Khachatourians     

β-Carotene production by Flavobacterium multivorum in the presence of inorganic salts and urea

Flavobacterium multivorum, a non-fermenting Gram-negative bacteria, normally produces zeaxanthin (3R, 3 R-, -carotene-3, 3 diol) as its main carotenoid. The effect of supplementation of various inorganic salts and urea on the growth, total carotenoid production, and proportion of -carotene (, -carotene), -cryptoxanthin (, -caroten-3-ol), and zeaxanthin produced by F. multivorum was investigated. Urea and several salts, such as calcium chloride, ammonium chloride, lithium chloride, and sodium carbonate, improved total carotenoid production by 1.5- to 2.0-fold. Urea and sodium carbonate had an unexpectedly strong positive effect on -carotene production at the expense of zeaxanthin formation. The effect was found to be independent of incubation time, and -carotene represented 70% (w/w) of the total carotenoid content. The cumulative effect of urea and sodium carbonate was further studied using response surface methodology. An optimum medium was found to contain 4,000 and 4,070 mg l^–1 urea and sodium carbonate, respectively. The maximum -carotene level was 7.85 g ml^–1 culture broth, which represented 80% (w/w) of the total carotenoid produced. Optimization resulted in 77- and 88-fold improvements in the volumetric and specific -carotene levels, respectively, accompanied by a simultaneous decrease in the zeaxanthin level as compared to the control medium. The carotenoid production profile in the optimized medium indicated that -carotene was produced maximally during the late exponential phase at 0.41 g ml^–1 h^–1. It is possible that this organism could be an excellent commercial source of either -carotene or zeaxanthin, depending on initial culture conditions.