Chirality differences in amino acid retention and release from the acid-extractable pool of cultured mammalian cells

In previous work, no chiral differences were found between D and L enantiomers of Leu in their ability to displace one another from the acid-extractable pool in mammalian cells. Recent evidence suggested otherwise. Our aim is to examine whether, in physiological range, D-amino acids have an equivalent ability to displace L-amino acids from the acid-extractable pool of HeLa cells, and vice versa. In the millimolar range, D-Leu and L-Leu have similar uptake and displacement properties with regard to the acid-extractable pool in HeLa cells, despite only the latter isomer being incorporated into protein. Below millimolar concentrations however, a distinct difference was found in the displacement of tritium-labelled L-Leu from the pool by unlabelled D-Leu compared with unlabelled L-Leu. Thus, unlabelled L-Leu in the external medium at 10⁻⁴ or 10⁻⁵ M displaced an equivalent amount of label from the pool as D-Leu introduced at a concentration approx. one order of magnitude higher, respectively. Reciprocal experiments, in which the acid-extractable pool was preloaded with ³H-D-Leu, confirmed this finding. The chirality difference was noted whether pool prelabelling was carried out at 37 or 0°C; but in order to avoid the complications of active transport mechanisms, the competition work reported here was done at 0°C. Similar chirality differences were observed with other hydrophobic amino acids, including His, Ile and Phe, such as, preferential displacement by the L-Leu racemer compared with the D-Leu racemer below mM levels. This was also true for the D and L forms of the non-utilisable isomer of Leu, norleucine (nLeu). We conclude that D-forms of hydrophobic amino acids have lower affinity for similar or the same intracellular binding sites involved in the acid-extractable pool than their L-forms. The significance of these chirality findings to amino acid pools in cells, and to the predominance of L-forms of amino acids in the biosphere is considered.     

Characterization of anther-expressed genes encoding a major class of extracellular oleosin-like proteins in the pollen coat of Brassicaceae†

A large, heterogeneous, highly expressed gene family encoding oleosin-like proteins is described in the Brassicaceae. íeven related cDNA sequences were isolated from Brassica napus anther mRNA using RACE-PCR and compared with other recently described anther-specific oleosin-like genes from B. napus. The expression patterns of four representative members of this diverse gene family were analyzed by Northern blotting and in situ hybridization. In all cases, the genes were expressed specifically in the tapetum of 3–5 mm B. napus buds, which contained microspores at the late-vacuolate and bicellular stages of development. The predicted protein products are ordered into subclasses, each of which has a characteristic C-terminal domain, containing different amino acid motifs or repeated residues. Tryphine (pollen coat) fractions from mature B. napus pollen were found to be particularly enriched in polypeptides of apparent molecular weights 32–38 kDa, plus numerous less abundant polypeptides of less than 15 kDa. The N-terminal 15–20 residues of three of these polypeptides (12, 32 and 38 kDa) were found by microsequencing to be identical to parts of the predicted amino acid sequences of three of the tapetal-expressed oleosin-like genes. This indicates the possibility of post-translational modification of these proteins resulting in a cleavage of the primary translation products in order to generate the mature tryphine polypeptides. These data imply that a large and diverse group of oleosin-like proteins is synthesized in the tapeturn of B. napus anthers and that following tapetal degradation, these proteins, possibly in modified form, then relocate to the developing microspores where they eventually constitute some of the major components of the extracellular tryphine of mature pollen grains. These proteins share a conserved 70 amino acid residue hydrophobic domain and are related structurally to the seed-specific intracellular oleosins, although their biological function may be different.     

Glutamine metabolism in AS-30D hepatoma cells. Evidence for its conversion into lipids via reductive carboxylation

A study was undertaken to assess the role of a physiological concentration of glutamine in AS-30D cell metabolism. Flux of¹⁴C-glutamine to¹⁴CO₂ and of¹⁴C-acetate to glutamate was detected indicating reversible flux between glutamate and TCA cycle -ketoglutarate. These fluxes were transaminase dependent. A flux analysis was compared using data from three tracers that label -ketoglutarate carbon 5, [2-¹⁴C]glucose, [1-¹⁴C]acetate and [5-¹⁴C]glutamine. The analysis indicated that the probability of flux of TCA cycle -ketoglutarate to glutamate was, at minimum, only slightly less than the probability of flux of -ketoglutarate through -ketoglutarate dehydrogenase. The apparent Km for oxidative flux of [¹⁴C]glutamine to¹⁴CO₂, 0.07 mM, indicated that this flux was at a maximal rate at physiological, 0.75 mM, glutamine. Although oxidative flux through -ketoglutarate dehydrogenase was the major fate of glutamine, flux of glutamine to lipid via reductive carboxylation of -ketoglutarate was demonstrated by measuring incorporation of [5-¹⁴C]glutamine into¹⁴C-lipid. In media containing glucose (6 mM), and glutamine (0.75 mM) 47 per cent of the lipid synthesized from substrates in the media was derived from glutamine via reductive carboxylation and 49 per cent from glucose. These findings of nearly equal fluxes suggest that lipogenesis via reductive carboxylation may be an important role of glutamine in hepatoma cells.     

Characteristics of a human epidermal squamous carcinoma cell line at different extracellular calcium concentratrions

A continuous line derived from a human skin squamous cell carcinoma has been grown in media of high, normal and low Ca²⁺ concentrations. The growth rate was unaffected by the Ca²⁺ levels even though morphological changes were observed. Desmosomes were absent at low Ca²⁺ and areas of cell piling were observed at high Ca²⁺. Cell protein staining patterns on polyacrylamide gels were identical for cells grown at the three Ca²⁺ levels. The variations were minor for the glycoproteins reacted with ¹²⁵I-conA. Lactoper-oxidase iodination revealed changes in cell surface proteins, most markedly in the emergence of new proteins at high Ca²⁺.     

The neural basis of a sensory filter in the Jamming Avoidance Response: No grandmother cells in sight

Summary The Jamming Avoidance Response (JAR), during which weakly electric fish modulate their electric organ discharge rate in response to a foreign electric signal of nearly the same frequency is strongest for frequency differences (f s) between 3–8 Hz. We have searched for neural correlates of this behavioral specificity. Single unit recordings in the anterior lateral line ganglion (ALLG), the posterior lateral line lobe (PLLL) and the torus semicircularis (TS) ofEigenmannia virescens were made during electrical stimulation simulating jamming by a nearby conspecific.Contrary to previously published reports (Scheich 1974, 1977) we conclude that f specificity does not lie in a single class of receptors or higher-order units in the PLLL tuned to the most effective f s. No tuning is seen at the receptor level of the PLLL. Specificity seems to be a population effect first visible at the level of the torus semicircularis, with individual units responding most strongly to different f s, but with most units tuned to approximately + and-4 Hz. By having cells tuned to a variety of f s but occurring in proportions corresponding to the observed behavior (and the degree to which f s impair electrolocation), animals would be better equipped to carry out other tasks such as detection of relative motion of objects in space and would also be better able to read complex stimuli corresponding to the more usual case of simultaneous jamming from several conspecifics (Partridge and Heiligenberg 1980).Units in the PLLL show slight differences in the timing of their firing to jamming signals presented at a frequency slightly above (+f) the fish's pacemaker frequency compared to those presented at a frequency slightly below (–f) (Scheich 1977). Firing pattern within the beat cycle produced by interaction of the fish's EOD, or an electrical mimic, S₁, and the foreign signal, S₂, is largely unaffected by the field orientation of the jamming signal. In the torus, by contrast, two classes of units are encountered which completely reverse the pattern of their firing within the beat cycle if the sign of the f is reversed. And, unlike the PLLL cells, those in TS respond differentially to different stimulus field geometries. Units of class 1 appear to compare T-unit input from different sites on the body surface (Heiligenberg and Bastian 1980) whereas those of class 2 additionally appear to receive input from E- and I-units in the PLLL. Abbreviations: see MethodsThis study was supported by grants from the National Science Foundation and the National Institutes of Health.     

The association of the sulphogalactosylglycerolipid of rat brain with myelination

The sulphogalactosylglycerolipid of rat brain is closely associated with the process of myelination, as demonstrated by the following observations. 1. The lipid is barely detectable in rat brain before 10 days of age, accumulates rapidly between age 10 and 25 days, and remains relatively constant in amount (between 0.3 and 0.4mumol per brain) thereafter into adult life. 2. The activity of adenosine 3'-phosphate 5'-sulphatophosphate-galactosyldiacylglycerol sulphotransferase is almost absent before 10 days of age, attains a maximum at age 20 days, and slowly decreases thereafter with increasing age. This developmental pattern correlates well with that of other myelin-specific metabolites. 3. Both the concentration of the sulphogalactosylglycerolipid and the activity of sulphotransferase are greatly decreased in the non-myelinating jimpy mouse. 4. The myelin fraction of rat brain contains most of the sulphogalactosylglycerolipid. The lipid occurs in a diacyl and an alkylacyl form. Determinations of the relative amount of each type in brain showed about a 1:1 mixture in both 21-day-old and adult rats. Rats injected with H(2) (35)SO(4) at 20 days of age lost (35)S from the diacyl form at a higher rate than from the alkylacyl compound over a 21-day period. These data suggest that the diacyl form has a higher turnover than the alkylacyl derivative. The percentage of the total sulpholipid content of brain contributed by the sulphogalactosylglycerolipid is 16% in 21-day-old rats and 8.4% in adult rats.     

Maintenance and induction of morphological differentiation in dissociated mammary epithelium on floating collagen membranes

Summary Dissociated normal mammary epithelial cells from prelactating mice were plated on different substrates in various medium-serum-hormone combinations to find conditions that would permit maintenance of morphological differentiation. Cells cultured on floating collagen membranes in medium containing insulin, hydrocortisone and prolactin maintain differentiation through 1 month in culture. The surface cells form a continuous epithelial pavement. Some epithelial cells below the surface layer rearrange themselves to form alveolus-like structures. Cells at both sites display surface polarization; microvilli and tight junctions are present at their medium-facing or luminal surface and a basal lamina separates the epithelial components from the gel and stromal cells. Occasinal myoepithelial cells, characterized by myofilaments and plasmalemmal vesicles, are identified at the basal surface of the secretory epithelium. In contrast, cells cultured on plastic, glass or collagen gels attached to Petri dishes form a confluent epithelial sheet showing surface polarization, but lose secretory and myoepithelial specializations. If these dedifferentiated cells are subsequently maintained on floating collagen membranes, they redifferentiate. There is little DNA synthesis in cells on collagen gels, in contrast to Petri-dish controls. Protein synthesis in cells on floating collagen membranes increases over T₀ values and remains constant through 7 days in culture whereas it decreases on attached gels; however, if the gels are freed to float, protein synthesis increases sharply and parallels that seen on floating membranes. The work was supported by USPHS Grants CA-05388 and CA-05045 from the National Cancer Institute, DHEW.     

Cytosolic Free Calcium and Gene Expression During Chemical Hypoxia

Understanding the cellular response to hypoxia may help elucidate the role of altered oxidation in neuronal death or abnormal cell function. In PC12 cells, 30 min of chemical hypoxia (i.e., KCN) reduced ATP concentrations by 92%, but diminished viability by only 10%. Ten minutes of hypoxia increased cytosolic free calcium ([Ca2+]i) 2.5-fold above control, but after 30 min of hypoxia, [Ca2+]i was slightly below that of nonhypoxic cells. Short periods of hypoxia also exaggerated the K(+)-induced elevation of [Ca2+]i, but by 30 min these ATP-depleted cells reestablished a calcium gradient that was equal to nonhypoxic, K(+)-depolarized cells. Thus, 30 min of severe ATP depletion left [Ca2+]i and viability relatively unaffected. Nerve growth factor caused slight, but significant, improvements in ATP and viability of hypoxic cells, but had no effect on [Ca2+]i. Although [Ca2+]i was equivalent in control and hypoxic cells after 30 or 60 min, hypoxia abolished the K(+)-stimulated elevation of [Ca2+]i. The nerve growth factor induction of c-fos, an indicator of the genomic response, was diminished by approximately 80%. Thus, hypoxic PC12 cells with greatly reduced ATP stores maintained normal [Ca2+]i, but their ability to respond to external stimulation was impaired. Further, the reduced oxidation that occurs in the brain in a variety of pathological conditions may interfere with the cellular response to stimulation and growth factors.