A decrease in intracellular zinc level precedes the detection of early indicators of apoptosis in HL-60 cells

Low extracellular zinc concentrations have been associated with the induction of apoptosis. To assess the relationship between intracellular zinc concentration and the rate of apoptosis, cells were grown in media containing 0.5, 25, or 50 M zinc and analyzed by flow cytometry or fluorescence microscopy. Cells grown in 0.5 M zinc medium over 48 h showed a successive decrease in intracellular zinc concentration measured by the zinc-specific fluorophore, zinquin. After 18 h in 0.5 M zinc medium, rhodamine 123 retention decreased. However, the addition of 10 M zinc to the 0.5 M medium before 16 h in culture restored rhodamine retention in the cells. After 30 h there was an increase in the number of cells cultured in 0.5 M zinc medium that bound annexin V-FITC. These data indicated that decreased intracellular zinc concentration preceded early markers of apoptosis, with alterations in mitochondrial transmembrane potential preceding the loss of polarity in the cell membrane.     

Effects of mefluidide and dicamba on in vitro growth and embryogenesis ofDactylis glomerata (orchard grass)

The effects of N-(2,4-dimethyl-5-(((trifluoromethyl)sulfonyl)amino)phenyl)acetamide (mefluidide) and 3,6-dichloro-o-anisic acid (dicamba) on in vitro growth and somatic embryogenesis ofDactylis glomerata L. (orchard grass) were studied using suspension cultures and explanted leaf bases. All experiments employed modified Schenk and Hildebrandt medium amended with concentrations of dicamba ranging from 15 to 120 M (SH-15 to SH-120) and of mefluidide ranging from 1 to 100 M. SH medium without either growth regulator was used for embryo germination. Embyro production in suspension cultures with SH-30 medium plus 3 g/L casein hydrolysate was significantly reduced by 1 M mefluidide. Only 15% of these embryos germinated and produced plants compared to 84% from controls. Growth, as measured by dry weight, was significantly reduced by 50 or 100 M mefluidide. The number of embryos formed on leaf sections was significantly reduced by 20 or 25 M mefluidide. Embryos that formed with 10 M or more mefluidide were callused on both SH-15 and SH-30 media. Shoot formation was inhibited from individual embryos and embryo/callus masses that developed on either SH-15 or SH-30 medium containing 5 M or more mefluidide. Radicle emergence was significantly reduced with 10 M mefluidide regardless of 15 or 30 M dicamba. Histological examination revealed that mefluidide inhibited both shoot and root meristem development with shoot development being the more sensitive. Inhibition of both was independent of dicamba concentrations. Shoot formation was also reduced from embryos that had developed on SH-30 medium without mefluidide when transferred to medium containing mefluidide without dicamba.     

Ultrastructural changes in the mitochondria of brown adipose cells during the hibernation cycle of Citellus lateralis

Summary The mitochondrial structure in the brown adipose cells of the golden mantled squirrel, Citellus lateralis, was examined throughout the year in biopsy samples. The mitochondria showed remarkable and apparently reversible changes in size and internal structure related to the physiologic activity of the animal. In the active animal the size of the largest mitochondria was 2.4 m × 1.5 m; during hibernation it increased to 7 m × 2.5 m; and during arousal it reached 11.2m × 5.3 m. The cristae of the mitochondria in the brown adipose cells of the animals in hibernation phase formed loops, whorls and mesh-like interconnections. During the arousal phase they underwent further configurational changes. The most remarkable structure was associated with mitochondria of most unusual proportions which by dissolution gave rise to a new generation. This was a common finding during arousal but did not occur in any other phase of the hibernation cycle. The new mitochondria were virtually indistinguishable from those of brown adipose cells of any active animal.Supported by a grant from the Medical Research Council of CanadaThe author is grateful to colleagues, Dr. G. Dempster and Dr. W.A. Spencer, for many valuable suggestions in the course of the work     

Presynaptic Modulation of K+-Evoked [3H]Dopamine Release in Striatal and Frontal Cortical Synaptosomes of Normotensive and Spontaneous-Hypertensive Rats

Regional differences in presynaptic [³H]dopamine ([³H]DA) release and its modulation by D₂ DA-receptors between the frontal cortex and striatum obtained from Wystar-Kyoto (WKY) and spontaneous-hypertensive rats (SHR) have been evaluated using superfused synaptosomes. Synaptosomal tritium content was significantly lower in the frontal cortex than in the striatum in both SHR and WKY (45% and 48%, respectively), but no differences in tritium content were obtained between strains. However, the 15 mM K⁺-evoked [³H]DA overflow was lower in the SHR as compared to WKY rats in both brain regions (striatum 23%, frontal cortex 21). Concentration-response curves for quinpirole (1nM-10 M)-mediated inhibition of 15mM K⁺-evoked [³H]DA release showed no differences between SHR and WKY. These results suggest that SHR has less ability to release [³H]DA as compared to WKY rats, but SHR did not show differences in the autoregulation of such release in both the frontal cortex and striatum.     

Induction of precocious melanogenesis of pigment cells in cultures of neuroretinal cells of chick embryo by amphotericin B

Summary When dissociated neuroretinal cells of the 9-day-old chick embryo were cultured, the cells formed monolayer sheets of somewhat flattened epithelial cells within 15 days after inoculation. During 15 to 30 days, numerous foci of non-pigmented epithelial cells were formed. During 30 to 50 days, melanin appeared in the cells of these foci. When amphotericin B (1 g/ml) was added to the culture medium on day 25 of culture, brown pigments appeared precociously, i.e. within the first two days, in the cells. The brown pigments were identified as melanins by histochemical and electron-microscopic methods. Induction of melanogenesis required continuous treatment with amphotericin B. With the precocious appearance of melanins, tyrosinase activity increased rapidly. This rapid increase in tyrosinase activity was inhibited by the addition of phenylthiourea or diethyl-dithiocarbamate. It was not enhanced by iodoacetamide, but was blocked by a low concentration of cycloheximide or actinomycin D. These findings indicate that amphotericin B induces de novo synthesis of tyrosinase rather than activation of pre-existing tyrosinase.     

The formation of free axonal sprouts from a dorsal root ganglion-nerve preparation maintained in organotypic culture,and the effects of demecolcine

Summary Nerve-ganglion preparations from rat dorsal spinal nerve roots were maintained in organotypic culture for 20 h. Free axonal sprouts formed at the cut tips. Clear and dense-core vesicles, mitochondria and smooth endoplasmic reticulum accumulated in the axons for a distance of 500 m behind the cut, as has previously been described in dorsal roots sectioned in vivo. Sprouting did not occur in dorsal roots maintained in culture without their ganglia attached. Sprouting was also prevented by demecolcine (3 × 10^-7 M) which reduced the number of microtubules in non-myelinated, small myelinated and large myelinated axons to respectively 45, 30 and 20% of control values. The sprouts contained several types of vesicle including small clear vesicles, large and small dense-core vesicles and flattened vesicles. The possible relevance of the vesicles to transmitter mechanisms in these neurones is discussed.I.R.D. is supported by the Medical Research Council; P.K. thanks the Mental Health Trust for a project grant     

In vitro regeneration of silktree (Albizzia julibrissin) from excised roots

Root segments (1 cm long) were excised from 15–20 day old seedlings of silktree (Albizzia julibrissin) grown on B5 medium. About 50% of the control (no growth regulators added) root explants formed shoot buds within 15 days after placement on the culture medium. After 30 days, there were about 4 shoots per control explant. Addition of low levels of various auxins (0.5 M) did not influence the formation of shoot buds from the explants. Higher concentrations (5M), however, decreased shoot regeneration. Kinetin and 2iP did not influence shoot regeneration at the concentrations tested (1 & 10 M). Addition of benzyladenine, Zeatin, or thidiazuron to the culture medium increased both the percentage of explants that formed shoots and the number of shoots per explant. Thidiazuron was highly effective in stimulating shoot formation at low concentrations (<1 M). At 0.05 M thidiazuron, 95% of the explants produced shoots and about 10 shoots were formed per explant. Compared to TDZ, higher concentrations (10 M) of benzyladenine and Zeatin were required to enhance shoot formation. Upon excision and transfer to B5 medium, regenerated shoots developed into normal rooted plantlets.Abbreviations BA Benzyladenine - IAA Indoleacetic acid - IBA Indolebutyric acid - NAA Naphthaleneacetic acid - TDZ Thidiazuron - 2ip Isopentenyladenine     

Architecture of the media of the arterial vessels in the dog brain: A scanning electron-microscopic study

Summary The architecture of the media of arterial vessels in dog brain was investigated using scanning electron microscopy. The arrangement and shape of the circularly-oriented smooth muscle cells varied with vessel diameter: The arteries (>100 m in diameter) had 4–10 layers of spindle-shaped smooth muscle cells; the muscular arterioles (30–100 m), 2–3 layers of spindle-shaped smooth muscle cells; the terminal arterioles (10–30 m), a compact layer of spindle-shaped smooth muscle cells with more dominant nodular or rod-like processes and thin lateral processes; and the precapillary arterioles (5–15 m), a less compact layer of branched smooth muscle cells.Longitudinally-oriented muscles were observed in the medio-adventitial border. The distribution and arrangement of these muscles varied with vessel size: in the large arteries (> 300 m in diameter), at the branching sites only; in the small arteries (100–300 m), at both the branching and non-branching sites; in the muscular arterioles, at both the branching and non-branching sites in a reticular arrangement with some muscle cells having an asteroid appearance; in the terminal aterioles, only asteroid-like muscle cells were found at the branching and non-branching sites.     

Production of C2 toxin by Alexandrium tamarense CI01 using different culture methods

The dinoflagellate Alexandrium tamarense CI01 wasgrown in three types of cultures: batch culture, semi-continuous culture andtemporary culture, to investigate the effects of different culture methods oncell growth and the productivity of C2 toxin (C2, a paralytic shellfish toxin).In the batch cultures, cells grew in three phases: a short lag phase, anexponential phase with a specific growth rate () of 0.78day^–1 and a relatively long stationary phase. Themaximum toxin productivity was 1.2 mol L^–1 or77 fmol cell^–1 in 9 days. In the semi-continuouscultures, cells grewin response to the dilution cycles, with values being 0.64, 0.32 and 0.35day^–1 for one-day, two-day and three-day cycles,respectively. The toxin yield was about one half of that of the batch cultures.A "temporary" culture method was used to maintain the metabolically activecellsremoved from the semi-continuous cultures, in a nutrient-depleted condition, toachieve a high toxin productivity of 1.0 molL^–1 in 4 days. Thus,the semi-continuous culture method provided an efficient means to generateamounts of metabolically active algal cells. The temporary culture offered aneffective way to produce C2. The highest yields of C2 were obtained in3–4days when the temporary cultures were aerated.     

“In vitro” filbert (Corylus avellana L.) micropropagation from shoot and cotyledonary node segments

Plantlets were successfully regenerated from shoot and cotyledonary node segments excised from 20 day old filbert seedlings. In both cases the optimum initiation and elongation of shoot buds was obtained after 15 days culture in 1/2K(h) medium plus BAP (25 M) followed by 20 days culture in the same medium in presence of a reduced BAP concentration (0.5 or 2.5 M).Maximum of functional roots were readily formed after 5 days of submersion of the basal end of shoots in 1/2K(h) liquid medium plus IBA (50 M), then transferred to a fresh 1/2K(h) solid medium for a further 15 days. Following these two consecutive steps, root initiation and development was achieved in 80% of the explants.The histological origin of neoformed organs was studied.Abbreviations BAP 6-benzylaminopurine - IBA indole-3-butyric acid - 1/2K(h) half-strength Cheng's (1975) nedium - K(h) full-strength Cheng's medium     

In vivo restitution of airway epithelium

Epithelial shedding occurs in health and, extensively, in inflammatory airway diseases. This study describes deepithelialisation, reepithelialisation and associated events in guinea-pig trachea after shedding-like epithelial denudation in vivo. Mechanical deepithelialisation of an 800-m wide tracheal zone was carried out using an orotracheal steel probe without bleeding or damage to the basement membrane. Reepithelialisation was studied by scanning- and transmission electron microscopy and light microscopy. Nerve fibres were examined by immunostaining. Cell proliferation was analysed by [³H]-thymidine autoradiography. Immediately after epithelial removal secretory and ciliated (and presumably basal) epithelial cells at the wound margin dedifferentiated, flattened and migrated rapidly (2–3 m/min) over the denuded basement membrane. Within 8–15 h a new, flattened epithelium covered the entire deepithelialised zone. At 30 h a tight epithelial barrier was established and after 5 days the epithelium was fully redifferentiated. After completed migration an increased mitotic activity occurred in the epithelium and in fibroblasts/smooth muscle beneath the restitution zone. Reinnervating intraepithelial calcitonin gene-related peptide-containing nerve fibres appeared within 30 h. We conclude that (1) reproducible shedding-like denudation, without bleeding or damage to the basement membrane, can be produced in vivo; (2) secretory and ciliated cells participate in reepithelialisation by dedifferentiation and migration; (3) the initial migration is very fast in vivo; (4) shedding-like denudation may cause strong secretory and exudative responses as well as proliferation of epithelium, and fibroblasts/smooth muscle. Rapid restitution of airway epithelium may depend on contributions from the microcirculation and innervation.     

Regeneration from long-term cell suspension cultures of tepary bean (Phaseolus acutifolius)

Plant regeneration has been achieved from long-term cell suspension cultures established from leaf derived callus of tepary bean (Phaseolus acutifolius). The proportion of densely cytoplasmic cells in suspension culture increased when cultured in the L-6 medium with 54 M NAA and 2 M KN. Filtration of the cells at each of five consecutive subcultures resulted in the isolation of a plant regenerating cell line (TB 686), which is being maintained in L-6 medium with 4.5 M 2,4-D and 2.3 M zeatin. Differentiated green cell aggregates were obtained when cells from maintenance medium were transferred to the same medium with 10 M BA. Embryo-like structures developed from these aggregates on L-6 medium with 2.3 M zeatin, 0.69 M GA₃ and 1.5 M NAA. Plantlets regenerated from these structures when they were cultured on L-6 medium with 7.0 M NAA and 1.0 M KN. Plant regeneration from the cell line remained relatively constant for 270 days. Regenerated plants were grown to maturity in the greenhouse.Abbreviations BA Benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ Gibberellic acid - IPA Isopentenyladenine - KN Kinetin - NAA Naphthaleneacetic acid - AA Amino acid medium (Toriyama and Hinita, 1985) The research was sponsored by United States Agency for International Development, Washington D.C., Cooperative Agreement DAN-4137-A-00-4053-00     

Somatic embryogenesis through pseudo-bulblet transverse thin cell layer of Lilium longiflorum

Somatic embryogenesis was achieved directly from pseudo-bulblettransverse thin cell layers (tTCL) of Lilium longiflorum.Embryo-like structures (globular embryos) were obtained from different sizeexplants of pseudo-bulblet tTCLs after 45 days culture on Murashige and Skoog,1962 (MS) medium containing 5.4 M naphthalene acetic acid(NAA)and 1.1 M thidiazuron (TDZ). The embryo-like structures werethen isolated and mass proliferated on MS medium, containing 5.4M NAA and 0.4 M TDZ, every 45 days. A0.8–1.0 mm thick explant was shown to be optimal forobtaining the highest number of embryo-like structures. For plant regenerationthese structures were transferred to hormone-free MS medium with 30g/l sucrose. All of these structures formedplantlets after 90 days culture.     

Thidiazuron-induced in vitro shoot formation from roots of intact seedlings of Albizzia julibrissin

Seedlings of silktree (Albizzia julibrissin Durrazz.) were grown in vitro on MS-media containing B5 vitamins, 3% sucrose, 0.25% phytagel and various concentrations (0.1–10 M) of thidiazuron (TDZ). Addition of TDZ to the culture medium greatly reduced shoot and root elongation but did not influence shoot production from the cotyledonary node or apex. Within 8–10 days the seedling roots split open, formed large masses of callus, and developed green patches which eventually grew into normal shoots while still within the culture medium containing TDZ at 0.1–1.0 M. Such callus and shoot formation did not occur in control cultures lacking TDZ. At higher TDZ concentrations (2.5–10 M), the green patches formed in the callus did not further develop into shoots. Addition of other cytokinins (kinetin, benzylaminopurine, zeatin) to the culture medium also induced some shoot formation from the roots, but higher concentrations than TDZ were required to induce regeneration. Isopentenyladenine failed to induced shoot formation. Following excision and transfer to MS media with or without 4.9 M IBA, the shoots induced by kinetin or benzylaminopurine rooted 4–7 days earlier than those induced by TDZ, but all excised shoots developed into normal rooted plantlets within 3 weeks.Abbreviations TDZ thidiazuron     

Modulation of brain mitochondrial membrane permeability and synaptosomal Ca2+ transport by dopamine oxidation

Effects of dopamine on the membrane permeability transition, thioredoxin reductase activity, production of free radicals and oxidation of sulfhydryl groups in brain mitochondria and the Ca²⁺ uptake by Na⁺-Ca²⁺ exchange and sulfhydryl oxidation in brain synaptosomes were examined. The brain mitochondrial swelling and the fall of transmembrane potential were altered by pretreatment of dopamine in a dose dependent manner. Depressive effect of dopamine on mitochondrial swelling was reversed by 10 g/ml catalase, and 10 mM DMSO. The activities of thioredoxin reductase in intact or disrupted mitochondria were decreased by dopamine (1-100 M), 25 M Zn²⁺ and 50 M Mn²⁺. Dopamine-inhibited enzyme activity was reversed by 10 g/ml SOD and 10 g/ml catalase. Pretreatment of dopamine decreased Ca²⁺ transport in synaptosomes, which was restored by 10 g/ml SOD and 10 mM DMSO. Dopamine (1-100 M) in the medium containing mitochondria produced superoxide anion and hydrogen peroxide, while its effect on nitrite production was very weak. The oxidation of sulfhydryl groups in mitochondria and synaptosomes were enhanced by dopamine with increasing incubation times. Results suggest that dopamine could modulate membrane permeability in mitochondria and calcium transport at nerve terminals, which may be ascribed to the action of free radicals and the loss of reduced sulfhydryl groups.     

Organization of heterogeneous mitochondrial DNA molecules in mitochondrial nuclei of cultured tobacco cells

Summary The molecular size of mitochondrial DNA (mtDNA) molecules and the number of copies of mtDNA per mitochondrion were evaluated from cultured cells of the tobacco BY-2 line derived fromNicotiana tabacum L. cv. Bright Yellow-2. To determine the DNA content per mitochondrion, protoplasts of cultured cells were stained with 4,6-diamidino-2-phenylindole (DAPI), and the intensity of the fluorescence emitted from the mitochondrial nuclei (mt-nuclei) was measured with a video-intensified photon counting microscope system (VIM system). Each mitochondrion except for those undergoing a division contained one mt-nucleus. The most frequently measured size of the DNA in the mitochondria was between 120 and 200 kilobase pairs (kbp) throughout the course of culture of the tobacco cells. Mitochondria containing more than 200 kbp of DNA increased significantly in number 24 h after transfer of the cells into fresh medium but their number fell as the culture continued. Because division of mitochondria began soon after transfer of the cells into fresh medium and continued for 3 days, the change of the DNA content per mitochondrion during the culture must correspond to DNA synthesis of mitochondria in the course of mitochondrial division. By contrast, the analyses of products of digestion by restriction endonucleases indicated that the genome size of the mtDNA was at least 270 kbp. Electron microscopy revealed that mtDNAs were circular molecules and their length ranged from 1 to 35 m, and 60% of them ranged from 7 to 11 rn. These results indicate that the mitochondrial genome in tobacco cells consists of multiple species of mtDNA molecules, and mitochondria do not contain all the mtDNA species. Therefore, mitochondria are heterogeneous in mtDNA composition.Abbreviations DAPI 4, 6-diamidino-2-phenylindole - mtDNA mitochondrial DNA - mt-genome mitochondrial genome - mt-nucleus mitochondrial nucleus - ptDNA proplastid DNA - pt-nucleus proplastid nucleus - VIM system video-intensified photon counting microscope system     

Development of a new coulter counter system: Measurement of the volume,internal conductivity,and dielectric breakdown voltage of a single guard cell protoplast ofVicia faba

Summary Two intracellular microelectrodes were used to study electrotonic interaction between cultured embryonic (16- to 20-day-old) chick myocardial cells reaggregated into small spheresin vitro. Under different culture conditions, reaggregates with two types of functional membrane properties were produced: (i) highly differentiated reaggregates, and (ii) reverted reaggregates. In the highly differentiated state, the cells had high stable resting potentials and produced rapidly-rising tetrodotoxin (TTX)-sensitive action potentials in response to electric field stimulation. In the reverted state, the cells exhibited slowrising spontaneous action potentials having prominent pacemaker potentials and TTX-insensitive upstrokes. These states resemble electrophysiological properties of the highly differentiated (18 daysin ovo) and less fully differentiated (3 daysin ovo) intact embryonic chick heart, respectively. Both types of reaggregates had similar ultrastructural appearance, with many elongated cells and intercalated disc-like structures; gap-like junctions were not seen. The highly differentiated cells had input resistances of about 5 M, and exhibited only little electrotonic interaction in response to intracellular current injection either when the cells were at rest or during the action potential plateau. Intracellular stimulation produced propagating action potentials which triggered contraction of the entire reaggregate. Large hyperpolarizing current pulses applied during the action potential plateau caused premature repolarization which also propagated to the other impaled cell. In the reverted reaggregates, electrotonic interaction was weak or absent in about 52% of the impaled cell pairs, moderate in 30%, and strong in 18% (encountered only at interelectrode distances of less than 100 m). The difference in degree of electrotonic interaction may be due to the state of differentiation with respect to the membrane electrical properties.     

Scanning electron microscopy of shoot differentiationin vitro from leaf explants of the African violet

The shoot differentiation process from leaf explants ofSaintpaulia ionantha Wendl. Gypsy Trail culturedin vitro was investigated via scanning electron microscopy. From 16 combinations of -naphthaleneacetic acid (NAA) and 6-benzyladenine (BA), the optimum concentration for direct shoot formation without callus formation for the cultivar tested was estimated as 0.5 M NAA and 0.5M BA. The first cell divisions were observed after 5 days, in culture and were restricted to cells adjacent to the basal cells of glandular hairs. Meristematic domes were formed after 15 days and were investigated at 20 days. The origin of shoot formation was restricted to epidermal cells adjacent to basal cells of glandular hairs.     

Ultrastructure of the main excretory duct epithelium of the female mouse submandibular gland with special reference to sexual dimorphism

The fine structure of the main excretory duct epithelium (MEDE) of female mouse submandibular gland was investigated by scanning and transmission electron microscopy and the results compared with the previously established structure of male mouse MEDE. A comparative analysis of the subepithelial capillaries of both sexes was also performed. In this pseudostratified epithelium, principal cell-types were observed: types-I,-II,-III and basal cells. This differed significantly from male MEDE, where type-II and-III are absent and type-I cells are the most numerous. The latter cell-type had abundant mitochondria, a few lipid-containing granules, lysosomes in the infra-nuclear cytoplasm and well-developed basal infoldings. These cells were also characterized by abundant glycogen granules throughout the cytoplasm, many profiles of strands of smooth endoplasmic reticulum in the apical region, and lysosomes in the infra-nuclear region. Type-II cells were the second most numerous. Their most characteristic features were the presence of tubular vesicles which appeared to be invaginated from the plasma membrane, RER, SER, free ribosomes, a few peroxisomes with nucleoids, and primary lysosomes in extremely light cytoplasm. They had many mitochondria throughout the cytoplasm, except in the apical region, a few lipid-containing granules and no basal infoldings. Type-III cells were very few and were characterized by well developed basal infoldings, abundant free ribosomes, RER, SER, vesicles containing moderately dense material, and many lipid-containing granules. They also had many mitochondria throughout the cytoplasm, except apically. Basal cells had a large nucleus and the cytoplasm had few organelles. In the male continuous capillaries predominated in the subepithelial network, and capillary density per 200 m of epithelium (3.76±1.54) was lower than in the female, as was the number of fenestrae per 10 m of available endothelium (4.46±1.71). In the female, fenestrated capillaries predominated, and the capillary density per 200 m of epithelium was 6.76 (±1.54), and the number of fenestrae per 10 m of available endothelium was 4.91 (±1.77).     

Mitochondrial protein synthesis in aerobic and micro-aerobic continuous cultures ofCandida parapsilosis

Candida parapsilosis grows oxidatively under aerobic conditions and fermentatively in micro-aerobic (0.2 M oxygen) continuous culture. The amount of unsaturated fatty acids and sterol in cells and mitochondria, and the aerobic cytochromes are decreased in micro-aerobic cultures. In aerobic cells mitochondrial protein synthesis accounts for the formation of more than one-third of the proteins of the organelles; in micro-aerobic cultures, this declines to 5%. At the same time, minor but significant differences in the nature of the protein products formed by the mitochondrial system are evident in micro-aerobic and aerobic cells.