S-100 protein stimulates cellular proliferation

Summary S-100 protein (S-100p) is a small, acidic, calcium-binding protein that is present (predominantly) in the cytoplasm of many types of cells including those of neuroectodermal origin, such as glial cells, schwann cells and melanocytes. In human melanoma cells S-100p is abundant relative to the small quantities expressed by normal melanocytes. We investigated the possibility that this protein may be a growth factor. Purified S-100p from bovine brain or human melanoma cells was added exogenously to human melanoma cells and peripheral blood lymphocytes (PBL) and their growth in the presence of different concentrations of S-100p was determined using a [³H]dT-uptake proliferation assay. The growth of melanoma cells was stimulated by S-100p at concentrations of 1.95–31.25 g/ml. Slight inhibition of cell proliferation occurred at high concentrations (125 g/ml). Maximum stimulation of PBL was at 31.25 g/ml. PBL were not inhibited even at high concentrations of S-100p (125 g/ml). PBL stimulation by S-100p did not require the presence of monocytes/macrophages. Though stimulation by S-100p is not restricted to a specific cell type, when released by melanoma cells it may function as an autocrine tumor growth factor. Other cells, such as PBL, coming in contact with S-100p are also stimulated to proliferate.     

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.     

In vitro plant regeneration via somatic embryogenesis from root culture of some rhizomatous irises

A method for plant regeneration of Iris via somatic embryogenesis is described. Root and leaf pieces from in vitro-grown plants of several genotypes of rhizomatous Iris sp. were cultured in vitro. Callus induction occurred only on root cultures incubated under low light intensity (35 mol m^-2 s^-1) on two induction media containing 2,4-D (4.5 or 22.5 M), NAA (5.4 M) and kinetin (0.5 M). Somatic embryos developed after transfer of callus onto four regeneration media containing 9 or 22 M BA, or 5 M kinetin and 2 M TIBA or 9 M BA and 4 M TIBA. Plantlets could be obtained from these somatic embryos. Genotypic differences were found both in callus induction and somatic embryo formation, with I. pseudacorus responding better than I. versicolor or I. setosa. Cytological analysis performed on root tips of 80 regenerated plants revealed that two of the I. pseudacorus regenerants were tetraploid.Abbreviations 2,4-D dichlorophenoxy acetic acid - NAA naphthaleneacetic acid - BA 6-benzyladenine - TIBA 2,3,5-triiodobenzoic acid - IBA indolebutyric acid     

Developmental aspects of long-term callus culture ofVicia faba L.

Summary Vicia faba callus line (VFS 1), isolated from expiants of immature embryo, grew satisfactorily onMurashige andSkoog complete medium with 1.38 M 2,4-D, or with 0.92 M 2,4-D to which 1.0 M kinetin was added. It also grew well on the B 5 modified medium containing 2.3 M 2,4-D and 25.0 M kinetin. On the last of these media the cultures grew more uniformly and without necrosis. They also showed diminishing variation in polyploidy in favour of diploids and corresponding aneuploids (hypodiploids).After being cultured for nearly three years on MS containing 1.38 M 2,4-D, 8–33% of cultures of VFS 1 were able to regenerate roots when transferred to either MS half strength with 5.37 M NAA, or to a medium without 2,4-D, or else to media with the addition of kinetin only (in various concentrations).     

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     

Arsenic concentrations in water and fish from Chautauqua Lake,New York

Synopsis Arsenic persists in Chautauqua Lake, New York waters 13 years after cessation of herbicide (sodium arsenite) application and continues to cycle within the lake. Arsenic concentrations in lake water ranged from 22.4–114.81 g l^–1, = 49.0 ag l^–1. Well water samples generally contained less than 10 g l^–1 arsenic. Arsenic concentrations in lake water exceeded U.S. Public Health Service recommended maximum concentrations (10 g l^–1) and many samples exceeded the maximum permissible limit (50 g l^–1). Fish accumulated arsenic from water but did not magnify it. Fish to water arsenic ratios ranged from 0.4–41.6. Black crappie (Pomoxis nigromaculatus) contained the highest arsenic concentrations (0.14–2.04 g g^–1 ), X = 0.7 g g^–1) while perch (Perca flavescens), muskellunge (Esox masquinongy) and largemouth bass (Micropterus salmoides) contained the lowest concentrations (0.02–0.13 g g^–1). Arsenic concentrations in fish do not appear to pose a health hazard for human consumers.     

Evaluation of radioprotective activities of Rhodiola imbricata Edgew – A high altitude plant

The present study reports the radioprotective properties of a hydro-alcoholic rhizome extract of Rhodiola imbricata (code named REC-7004), a plant native to the high-altitude Himalayas. The radioprotective effect, along with its relevant superoxide ion scavenging, metal chelation, antioxidant, anti-lipid peroxidation and anti-hemolytic activities was evaluated under both in vitro and in vivo conditions. Chemical analysis showed the presence of high content of polyphenolics (0.971 ± 0.01 mg% of quercetin). Absorption spectra analysis revealed constituents that absorb in the range of 220–290 nm, while high-performance liquid chromatography (HPLC) analysis confirmed the presence of four major peaks with retention times of 4.780, 5.767, 6.397 and 7.577 min. REC-7004 was found to lower lipid oxidation significantly (p < 0.05) at concentrations viz., 8 and 80 g/ml respectively as compared to reduced glutathione, although the optimally protective dose was 80 g/ml, which showed 59.5% inhibition of induction of linoleic acid degradation within first 24 h. The metal chelation activity of REC-7004 was found to increase concomitantly from 1 to 50 g/ml. REC-7004 (10–50 g/ml) exhibited significant metal chelation activity (p < 0.05), as compared to control, and maximum percentage inhibition (30%) of formation of iron-2,2-bi-pyridyl complex was observed at 50 g/ml, which correlated well with quercetin (34.9%), taken as standard. The reducing power of REC-7004 increased in a dose-dependent manner. The absorption unit value of REC-7004 was significantly lower (0.0183± 0.0033) as compared to butylated hydroxy toluene, a standard antioxidant (0.230± 0.091), confirming its high reducing ability. Superoxide ion scavenging ability of REC-7004 exhibited a dose-dependent increase (1–100 g/ml) and was significantly higher (p < 0.05) than that of quercetin at lower concentrations (1–10 g/ml), while at 100 g/ml, both quercetin and REC-7004 scavenged over 90% superoxide anions. MTT assay in U87 cell line revealed an increase in percent survival of cells at doses between 25 and 125 g/ml in case of drug + radiation group. In vivo evaluation of radio-protective efficacy in mice revealed that intraperitoneal administration of REC-7004 (maximally effective dose: 400 mg/kg b.w.) 30 min prior to lethal (10 Gy) total-body -irradiation rendered 83.3% survival. The ability of REC-7004 to inhibit lipid peroxidation induced by iron/ascorbate, radiation (250 Gy) and their combination [i.e., iron/ascorbate and radiation (250 Gy)], was also investigated and was found to decrease in a dose-dependent manner (0.05–2 mg/ml). The maximum percent inhibition of formation of MDA-TBA complex at 2 mg/ml in case of iron/ascorbate, radiation (250 Gy) and both i.e., iron/ascorbate with radiation (250 Gy) was 53.78, 63.07, and 51.76% respectively and were found to be comparable to that of quercetin. REC-7004 (1 g/ml) also exhibited significant anti-hemolytic capacity by preventing radiation-induced membrane degeneration of human erythrocytes. In conclusion, Rhodiola renders in vitro and in vivo radioprotection via multifarious mechanisms that act in a synergistic manner.     

A temporal study at the ultrastructural level of the developing pro-oocyte of Drosophila melanogaster

The first pro-oocyte in developing pupal germaria of females grown at 25 ° has been followed at 6 h intervals from its formation at 129 h post-ovipostion until Stage 1, to provide an unambiguous temporal order. EM autoradiographs were made of sectioned germaria, scanned at lower magnification for location of the pro-oocyte(s) within the most posterior 16-cell cyst and photographed at higher magnification to show the presence of label indicating DNA replication and synaptonemal complex indicating synapsis in the same pro-oocyte nucleus. Label, detected at 132 h and at all subsequent intervals up to and including 162 h, delimited an S-phase of 30 h and identified this period as premeiotic interphase. Extensive SCs (av. length 50 m/genome) measured in serial sections at 132 h provide irrefutable evidence that synapsis in Drosophila begins close to both pro-oocyte formation and initiation of premeiotic interphase. Measurements of SCs at 6 h intervals during interphase reveal a sharp increase between 132 and 138 h, a peak length (75 m/genome) at 144 h, a decrease and subsequent plateau ( 60 m/genome) from 150–162 h and a further drop (R~50 m/genome) at Stage 1. Maximal extension of SC at 144 h coincides with maximal genome response to heat (Grell and Day, 1974) and with midpremeiotic-S. Spherical nodules, detected at 1/genome between 138 and 150 h would, on the questionable assumption that they are the sites of recombination, provide proof of recombination during early interphase, as genetic evidence strongly implies. Evidence contrary to interpretations of fibrillar material within the nucleus as either precursor of the central region of the SC or sagittal sections of the central element of the SC, is presented. No structure corresponding to the polytene chromocenter was observed.     

The bactericidal effects of Lactobacillus acidophilus,garcinol and Protykin® compared to clarithromycin,on Helicobacter pylori

Chronic infection with Helicobacter pylori causes peptic ulcers, gastric cancer and lymphoma. We evaluated the inhibitory effects of the probiotic Lactobacillus acidophilus DDS-1J, the antibiotic clarithromycin and the natural antioxidants garcinol and Protykin^® (containing 50% trans-resveratrol) on Helicobacter pylori strain ATCC 49503. The findings of this study indicate that Lactobacillus acidophilus DDS-1J exerts a growth inhibitory effect on H. pylori at a ratio of 1:1 or higher in vitro. In the case of clarithromycin, garcinol and resveratrol, the bactericidal effect is time and concentration dependent. Clarithromycin completely inhibited growth at 62.5 g/ml at 6 h and at 31.5 g/ml at 12 h. For garcinol the highest concentration needed for complete inhibition was 31.5 g/ml at 6 h and 3.9 g/ml after 12 h incubation. For resveratrol, significant inhibition was noted at 1000 g/ml at 12 h only. The bactericidal effect of garcinol was reduced by the addition of resveratrol at all concentrations 125 g/ml at 6 and 12 h. We conclude from this study that Lactobacillus acidophilus DDS-1J inhibits H. pylori at 1:1 and higher ratios. Also, between the two antioxidants, garcinol is much more potent than resveratrol as a bactericidal agent against H. pylori, and that resveratrol may antagonize this effect. Finally, our study showed equivalent or better bactericidal activity of garcinol compared to clarithromycin against H. pylori at 6 and 12 h incubation, indicating a potential role for this antioxidant in treatment for H. pylori infection.     

Effect of aldosterone on incorporation of amino acids into renal medullary proteins

Summary Studies on the effects of pretreatment with aldosterone on the incorporation of³H leucine or³H methionine into proteins in renal slices were carried out in Joklik-modified minimal essential medium. Administration of aldosterone (2 g/100 g body wt) to adrenalectomized rats increased³H leucine incorporation into trichloroacetic acid insoluble fractions of crude homogenates of cortical slices by 15.5±0.4% and of medullary slices by 53.5±1.3%. No increase in isotope incorporation was observed in slices of renal papilla or spleen prepared from the same rats. Aldosterone had no effect on the³H-leucine content of the trichloroacetic acid-soluble fractions of all three renal zones and the spleen. The dose of aldosterone that elicited a half-maximal increase in³H-methionine incorporation into proteins of renal medullary slices (0.45 g of aldosterone/100 g body wt) was indistinguishable from that needed to elicit a halfmaximal increase in the urinary K⁺/Na⁺ ratio (0.35 g of aldosterone/100 g body wt). Dexamethasone, a potent glucocorticoid, at a dose of 0.8 g/100 g body wt did not augment³H-leucine incorporation into renal medullary proteins but was effective at 8 g/100 g body wt. Spirolactone (SC-26304), a potent anti-mineralocorticoid, abolished the effect of aldosterone on amino acid incorporation into medullary proteins when administered at a 100-fold higher dosage [i.e., 80 gvs. 0.8 g (per 100 g body wt)]. These results imply that the action of aldosterone on amino acid incorporation is mediated by the mineralocorticoid rather than the glucocorticoid pathway, presumably the mineralocorticoid receptors. Moreover, pretreatment of the rats with actinomycin D (70–80 g/100 g body wt) erased the effect of aldosterone (0.8 g/100 g body wt) on amino acid incorporation into medullary proteins.In paired experiments with³H and³⁵S methionine, aldosterone (0.8 g/100 g body wt) increased methionine incorporation into trichloroacetic acid precipitable proteins of subcellular fractions of the renal medulla. The effect of aldosterone on incorporation of methionine into medullary cytosol proteins was analyzed further by polyacrylamide gel electrophoresis at pH 8.3 in tris-glycine buffer. The gel profiles indicate that aldosterone significantly increased methionine incorporation into at least one protein (independent of the isotope) with a molecular weight of 31,000. This increase was inhibited by either pretreatment of the rat with actinomycin D (70–80 g/100 g body wt or SC-26304 (80 g/100 g body wt). Dexamethasone (0.8 g/100 g body wt) did not increase incorporation of methionine into the medullary cytosol proteins resolved by polyacrylamide gel electrophoresis.     

Specific induction of encystment ofLagenidium giganteum zoospores by concanavalin A and derivatives of chitin and chitosan

Summary Zoospores of the mosquito pathogenic fungusLagenidium giganteum preferentially attach to and encyst on the cuticular surface of the immature stages of many species of mosquitoes as the initial step in the infection process. Recognition by zoospores of specific chemical or physical signals on the cuticular surface triggers attachment. A number of compounds likely to be present on the surface of mosquito larvae were evaluated for efficacy in eliciting zoospore encystment. Free amino acids and oligomers, a number of phenolic and polyphenolic compounds and most carbohydrates did not induce encystment at concentrations less than 500 g/ml. Colloidal chitin and chitin films were also ineffective as was O-carboxy-methylchitin; however, glycol chitin and glycol chitosan induced rapid encystment at concentrations at or below 1 g/ml. Zoospores also attached to and encysted in great numbers on fibers of oxycellulose, but not on cellulose. Concanavalin A was the only lectin which induced encystment at concentrations less than 10 g/ml, which suggests that a glycoprotein with terminal mannose and/or glucose residues is involved in encystment. A number of phenols were metabolized by peroxidase on the zoospore surface. Addition of hydrogen peroxide to zoospore suspensions reduced the time needed to induce zoospore encystment by some phenols; however, there was no consistent relationship between the presence or absence of this synergistic effect and the ability ofL. giganteum peroxidase to metabolize a given substrate. The sterol-binding compound amphotericin B induced immediate encystment at 3.5 g/ml, suggesting that sterols, which are required for the induction of zoosporogenesis, were present on the zoospore membrane.     

First survey on the natural occurrence of Fusarium mycotoxins in Bulgarian wheat

Wheat for human consumption (140 samples) was collected after harvest from all regions of Bulgaria. The 1995 crop year was characterized by heavy rainfall in the spring and summer months. The internal mycoflora of wheat samples was dominated by Fusarium spp. and Alternaria spp., and storage fungi were rarely present. The samples were analysed for contamination with Fusarium mycotoxins deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-AcDON), 15-acetyldeoxynivalenol (15-AcDON), T-2 Toxin (T-2), diacetoxyscirpenol (DAS), and zearalenone (ZEA), using enzyme immunoassay methods. DON and ZEA were the predominant toxins, with a contamination frequency of 67% and 69%, respectively. The average levels of these toxins in positive samples were 180 g/kg (DON) and 17 g/kg (ZEA), maximum concentrations were 1800 g kg^–1 and 120 g kg^–1, respectively. Acetyl derivatives of DON, namely 3-AcDON and 15-AcDON, were found in 2.1 % and 0.7% of the samples, at at maximum level of about 100 g kg^–1. Only one sample was positive for T-2 (55 g/kg), DAS was not detected. This is the first report about the natural occurrence of a range of Fusarium mycotoxins in wheat for human consumption in Bulgaria.Abbreviations 3-AcDON 3-acetyldeoxynivalenol - 15-AcDON 15-acetyldeoxynivalenol - DAS diacetoxyscirpenol - DON deoxynivalenol - EIA enzyme immunoassay - T-2 T-2 toxin - ZEA zearalenone     

In vitro culture of Aloe Barbadensis Mill.: Micropropagation from vegetative meristems

A method for rapid and highly effective plant micropropagation from vegetative meristems was established for Aloe barbadensis Mill. Plant micropropagation was achieved culturing apices on medium containing 1.1 M 2,4-dichlorophenoxyacetic acid and 2.3 M kinetin for 15–30 days. High morphogenetic ability was maintained by transferring explants (after 60 days) on media containing 0.11 M 2,4-dichlorophenoxyacetic acid and 2.2 M 6-benzylaminopurine.     

The spermatozoon of Oikopleura dioica Fol (Larvacea,Tunicata)

Summary The spermatozoon of Oikopleura dioica is about 30 m long, with a spherical head, about 1 m wide, a 3 m long and 1 m wide midpiece, and a 25 m long tail with a tapered end piece. The head contains a nucleus with the chromatin volume limited to about 0.1 m³. A small acrosome is found in an anterior inpocketing, and a flagellar basal body in a posterior inpocketing of the nucleus. The midpiece contains a single mitochondrion with the flagellar axoneme embedded in a groove along its medial surface. The flagellar axoneme has the typical 9 + 2 substructure, and the basal body the typical 9+0 substructure. A second centriole and special anchoring fibres are absent.     

Resistance to Spodoptera exigua in Apium prostratum

Two Apium accessions were compared with the commercial cultivar Tall Utah 52–70R (A. graveolens [L.]) for resistance to Spodoptera exigua (Hübner)(Lepidoptera: Noctuidae). Oviposition rate was not significantly different between the three genotypes. In all accessions, eggs were usually placed on the upper half of the plants. Implications of this oviposition pattern on S. exigua management in celery are discussed. The wild species A. prostratum ssp prostratum var filiform (A230) showed a significantly higher resistance to S. exigua than 52–70R. The levels of carcinogenic and mutagenic linear furanocoumarins in the commercial cultivar 52–70R (1.41 g/g in the petioles; 5.85 g/g in the leaves) and in the plant accession A. nodiflorum (5.40 g/g in the petioles; 2.99 g/g in the leaves) were far below the concentration reported to produce acute contact dermatitis (18.0 g/g). The levels of furanocoumarins in A. prostratum petioles (186.14 g/g) and leaves (326.45 g/g) were 10 and 18 times higher, respectively, than the concentration known to cause contact dermatitis. However, resistance in A. prostratum was primarily due to non-preference and the linear furanocoumarins did not induce non-preference. Therefore, the resistance shown by this plant accession does not appear to be furanocoumarin-based and may be suitable for transfer to commercial celery for use in S. exigua management.     

Development of high frequency multiple shoot formation in Persian clover (<Emphasis Type="Italic">Trifolium resupinatum</Emphasis> L.)

An efficient and reliable micropropagation system for Persian clover (Trifolium resupinatum L.) was developed using different explants and media. Node, hypocotyl and cotyledonary node explants were cultured on Murashige and Skoog (MS) medium supplemented with combinations of either 6-benzyladenine (BA) and indole-3-butyric acid (IBA) or BA, Kinetin (KIN) and IBA. Direct multiple shoots developed within 6weeks in all explants in most media tested. The best shoot multiplication capacity was obtained from cotyledonary node explants on MS medium containing 7.1M BA and 1M IBA or 14.1M BA and 1M IBA. Elongated shoots were rooted on either MS medium alone or combination with different concentrations of indole-3-butyric acid (IBA), indole-3-acetic acid (IAA) and -naphthaleneacetic acid (NAA). High rooting was achieved in half strength MS medium containing 8M IBA.     

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.     

A procedure for axenic isolation of the marine microalga Isochrysis galbana from heavily contaminated mass cultures

Isochrysis galbana, one of the most widely usedmarine microalgae in the rearing of finfish and shellfish larvae, is masscultured frequently in outdoor tanks. Under prolonged and repeated culture,severe contamination occurs. Axenic isolation of I.galbanafrom such cultures was best achieved by using a ternary procedure involvingpercoll-gradient centrifugation, treatment with antibiotics, and growth on agarmedium. Protozoa and other algae were removed most effectively by isolation ofI. galbana at the 30–40% density layer on apercoll-gradient. Removal of bacteria was accomplished using a mixture of 5antibiotics (250 g mL^–1 ampicillin, 50g mL^–1 gentamycin, 100 gmL^–1 kanamycin, 500 gmL^–1 neomycin, 50 gmL^–1 streptomycin). Axenic colonies were isolated fromasolid medium prepared from 1% purified agar. The ternary procedure isconsideredapplicable to the isolation of other axenic single-celled microalgae fromheavily contaminated cultures.     

Eubacteria have 3 growth modes keyed to nutrient flow

Aerobic growth of Escherichia coli and Paracoccus denitrificans has been studied in chemostat, fed batch, and recycling fermentor modes under carbon and energy limitation. Two abrupt drops or discontinuities in molar growth yield, Y, have been found that occur over relatively short ranges in the value of specific growth rate.Before the first discontinuity, Y is constant and maximal. After the first discontinuity, at a doubling time of 33 h, Y becomes constant again and independent of until the second discontinuity appears at a doubling time of about 50 h, corresponding to a of about 0.014. At this point, Y drops to a lower value that is constant at doubling times longer than 100 h, corresponding to a of about 0.007.The second discontinuity is associated in Paracoccus with elevated levels of guanosine tetraphosphate (ppGpp) that impose stringent regulation as has been found previously with Bacillus and Escherichia species. It is thus likely that the stringent response generally occurs in bacteria in vivo at a doubling time of about 50 h. The cause of the first discontinuity is unknown. All experiments indicate that Pirt-type calculations relating , Y, and maintenance energy demand are no longer valid. In chemostat experiments, the intercept of the relationship between specific substrate utilization and specific growth rate is defined as maintenance. However, this intercept most probably is caused by stringent regulation at low dilution rates. Three regions of bacterial growth rates are defined by this study, corresponding to doubling times of 0.5 to 15 h, 33 to 50 h, and >100 h. Some growth behavior in each region is unique to that region.Abbreviations ppGpp guanosine 5 diP 3 diP - pppGpp guanosine 5 triP 3 diP - SPR substrate provision rate (mol/l h)