Some arylamidases from vetch seeds]

Hydrolysis of L-phenylalanyl-p-nitroanilide (PPA) and glycyl-p-nitroanilide by extracts from resting vetch seeds was shown to be the effect of two different arylamidases. One of them, PPAase, was 2000-fold purified on hydroxylapatide, DEAE-cellulose and by gel filtration through Sephadex G-100. The preparation obtained was nearly homogenous chromatographycally. Molecular weight of PPAase, as shown by means of gel filtration, was 66000, K(m) was calculated to be 1.64-10-4 M. PPAase was inhibited by SH-reagents and partially by o-oxyquinoline. Some increase in the enzyme activity was observed in the presence of Ca2+, Mg2+ and Mn2+ in low concentrations. The enzyme hydrolysed amino acid arylamides with hydrophobic side groups and some dipeptides, which had at least one hydrophobic amino acid and did not contain amino acids with polar group.     

Modulation of phospholipase A2 activity associated with human sperm membranes by divalent cations and calcium antagonists

Phospholipase A2 activity in sonicates and acid extracts of ejaculated, washed human sperm was measured using [1-14C] oleate-labeled autoclaved E. coli and 1-[1-14C] stearoyl-2-acyl-3-sn- glycerophosphorylethanolamine as substrates. Phospholipase A was optimally active at pH 7.5, was calcium-dependent, and exclusively catalyzed the release of fatty acid from the 2-position of phospholipids. The activity was membrane-associated, and was solubilized by extraction with 0.18 N H2SO4. Acid extracts of human sperm had the highest specific activity (1709 nmols /h per mg), followed by mouse, rabbit and bull, which were 105, 36 and 1.7 nmols /h per mg, respectively. para-bromophenacyl bromide inhibited human sperm phospholipase A2 activity, but mepacrine was without effect. In the presence of 1.0 mM added CaCl2, phospholipase A2 activity was inhibited by Zn2+ and Mn2+; whereas Cu2+, Cd2+, Mg2+, or Sr2+ had no effect. Zn2+ stimulated activity at low concentrations (10(-6) to 10(-8) M), and inhibited activity in a dose-dependent manner at concentrations of 10(-5) M. The extent of stimulation by low concentrations of Zn2+ was dependent on Ca2+ concentration; at 10(-7) M, Zn2+ activity was stimulated 160% with 0.5 mM CaCl2, and only 120% with 1.0 mM CaCl2. At low concentrations (10(-5) to 10(-7) M), methoxyverapamil (D600) and trifluoperazine stimulated human sperm phospholipase A2 activity, and trifluoperazine but not D600 produced almost complete inhibition between 10(-5) and 10(-4) M of the drug. The significance of human sperm phospholipase A2 activity and its modulation by Ca2+, Zn2+ and Mn2+ in the sperm acrosome reaction is discussed.     

Comet impacts and chemical evolution on the bombarded Earth

Amino acids yields for previously published shock tube experiments are used with minimum Cretaceous-Tertiary (K/T) impactor mass and comet composition to predict AIB amino acid K/T boundary sediment column density. The inferred initial concentration of all amino acids in the K/T sea and in similar primordial seas just after 10 km comet impacts would have been at least 10^–7 M. However, sinks for amino acids must also be considered in calculating amino acid concentrations after comet impacts and in assessing the contribution of comets to the origin of life. The changing concentration of cometary amino acids due to ultraviolet light is compared with the equilibrium concentration of amino acids produced in the sea from corona discharge in the atmosphere, deposition in water, and degradation by ultraviolet light. Comets could have been more important than endogenous agents for initial evolution of amino acids. Sites favorable for chemical evolution of amino acids are examined and it is concluded that chemical evolution could have occurred at or above the surface even during periods of intense bombardment of Earth before 3.8 billion years ago.     

Effect of molybdate on methanogenic and sulfidogenic activity of biomass

The effect of molybdate, a sulfate analog, on the total methanogenic activity (TMA) and total sulfidogenic activity (TSA) of biomass metabolizing synthetic sucrose based substrate containing sulfate was investigated in batch assays. In Phase I of the study, TMA and TSA were assessed twice for four feed changes at a chemical oxygen demand to sulfate (COD/SO(4)(2-)) ratio of 3.5. In Phase II, long-term experiments were conducted for 10-13 feed changes with varying chemical oxygen demand (COD) concentration, sulfate concentration, COD/SO(4)(2-) ratio, molybdate dose and biomass with different growth histories. Assays with 3mM molybdate showed TSA inhibition over 85%. Dose dependency was observed for sulfate concentration, COD/SO(4)(2-) ratio, and biomass history. The minimum concentration that gave over 93% TSA inhibition was 0.25 mM. However, intermediate concentrations of molybdate inhibited methane producing bacteria (MPB) activity. TMA stimulation was observed at 0.75-2.0 mM molybdate.     

Interaction of excitatory and depressant amino acids in the frog retina

(1) Application of excitatory or depressant amino acids (concentrations from 10(-4) to 10(-2) M) could modify response patterns of the retinal ganglion cells to photic stimulus. Excitatory amino acids gave rise to spontaneous discharge while depressant amino acids inhibited spike discharge in response to test flashes. (2) Application of excitatory amino acids of more than 10(-3) M resulted in irreversible termination of spike discharges while recovery was always observed in the case of depressant amino acids even when the concentration of the applied solution was as high as 10(-2) M. No effect was observed when one exciting and one depressant amino acid were properly combined. (3) There is a mixture of four amino acids (two excitatory and two depressant) which could enhance the spike discharge in response to test flashes without giving rise to spontaneous firing. (4) It is implied that proper balance of excitatory and depressant amino acids is important in regulating the excitability of a number of neurons.     

Gustatory responses of eel palatine receptors to amino acids and carboxylic acids

The gustatory receptors of the eel palate were found to be extremely sensitive to amino acids and carboxylic acids. The results obtained are as follows: (a) 11 amino acids which are among naturally occurring amino acids elicited responses in the palatine nerve, but 9 amino acids did not elicit a response even at a high concentration. The effect of D-amino acids was always much less than that of their corresponding L-isomers. There was no appreciable difference in the effectiveness of an alpha-amino acid (alpha-alanine) and beta-amino acid (beta-alanine). (b) The threshold concentrations of the most potent amino acids (arginine, glycine) were between 10(-8) and 10(-9) M. A linear relation between the magnitude of the response and log stimulus concentration held for a wide concentration range for all the amino acids examined. (c) The palatine receptors responded sensitively to various carboxylic acid solutions whose pH was adjusted to neutral. The threshold concentrations varied between 10(-4) and 10(-7) M. The magnitude of the response at 10(-2) M increased with an increase of carbon chain length. (d) The extent of cross-adaptation was examined with various combinations of amino acids. A variety of the response patterns showing complete cross-adaptation, no cross-adaptation, or synergetic interaction was observed. The synergetic interaction was also observed when one amino acid below its threshold concentration was added to the other amino acid below its threshold concentration was added to the other amino acid. No cross-adaptation was observed between amino acids and fatty acids. (e) The treatment of the palate with papain led to loss of the responses to arginine, glycine, and histidine without affecting those to proline and acetic acid. The treatment with pronase E eliminated selectively the response to proline. The possibility that the eel gustatory receptors are responsible for sensing food at a distance was discussed.     

Induction of metabolic shocks in source and sink of two cucurbits by molybdenum stress

Different concentrations of ammonium molybdate (10(-7) to 10(-4)M) affected the levels of metabolites in the source and sink organs of the seedlings of C. melo and C. vulgaris and data were recorded at 7, 14 and 21 days after treatment (DAT) of molybdenum (Mo). Reducing and non reducing sugars declined with an increase in concentration of ammonium molybdate from 10(-7) to 10(-4) M. Soluble protein and dry weight of seedlings increased in source at lower concentration (10(-7) M) and gradually decreased in all other concentrations (10(-6), 10(-5) and 10(-4) M). Starch was slightly accumulated in hypocotyl and fresh weight constantly declined with an increase in ammonium molybdate concentration from 10(-7) to 10(-4) M in all the parts of seedlings viz. cotyledon, hypocotyl and roots. Thus molybdenum at higher concentration induced decline in the metabolite levels in source and sink as well as in transporting organs.     

INHIBITION OF AMINO ACID UPTAKE BY THE ABSENCE OF Na+ IN SLICES OF BRAIN

—The Na+ requirement of amino acid transport was measured in brain slices. The tissue was first washed free of Na+ and then Na+ was replaced by one of the following: choline, Li+, Rb+, or mannose. Amino acid uptake was measured at different times (5–120 min) and at low (10^-7–10^-5m ) and high (10^-3m ) concentrations. Most of the Na+ could be washed out of the tissue; this also decreased K+ levels despite increased K+ in the medium. K+ tissue levels were partially restored when Na+ was added. The absence of Na+ abolished the uptake of Glu, Asp, GABA, Gly, Tau and Pro. Most of the neutral amino acids (Ala, Val, Trp, His) were very strongly inhibited by the absence of Na+ under most experimental conditions. Basic amino acids (Arg, Lys) were not completely inhibited, in that 30 per cent of the equilibrium uptake remained and some of the basic amino acid influx was independent of the Na+ tissue level. The uptake of amines (tyramine, cadaverine, putrescine) did not require Na+, and often was greater in the absence of Na+. We conclude that amino acid uptake in brain slices is Na+ dependent, although the absence of Na+ may affect transport indirectly.     

Phenotypic restoration by molybdate of nitrate reductase activity in chlD mutants of Escherichia coli

ChlD mutants of Escherichia coli are pleiotropic, lacking formate-nitrate reductase activity as well as formate-hydrogenlyase activity. Whole-chain formate-nitrate reductase activity, assayed with formate as the electron donor and measuring the amount of nitrite produced, was restored to wild-type levels in the mutants by addition of 10(-4)m molybdate to the growth medium. Under these conditions, the activity of each of the components of the membrane-bound nitrate reductase chain increased after molybdate supplementation. In the absence of nitrate, the activities of the formate-hydrogenlyase system were also restored by molybdate. Strains deleted for the chlD gene responded in a similar way to molybdate supplementation. The concentration of molybdenum in the chlD mutant cells did not differ significantly from that in the wild-type cells at either low or high concentrations of molybdate in the medium. However, the distribution of molybdenum between the soluble protein and membrane fractions differed significantly from wild type. We conclude that the chlD gene product cannot be a structural component of the formate-hydrogenlyase pathway or the formate-nitrate reductase pathway, but that it must have an indirect role in processing molybdate to a form necessary for both electron transport systems.     

In Vivo Microdialysis Studies on the Effects of Decortication and Excitotoxic Lesions on Kainic Acid-Induced Calcium Fluxes, and Endogenous Amino Acid Release, in the Rat Striatum

The in vivo effects of kainate (1 mM) on fluxes of 45Ca2+, and endogenous amino acids, were examined in the rat striatum using the brain microdialysis technique. Kainate evoked a rapid decrease in dialysate 45Ca2+, and an increase in the concentration of amino acids in dialysates in Ca2+-free dialysates. Taurine was elevated six- to 10-fold, glutamate two- to threefold, and aspartate 1.5- to twofold. There was also a delayed increase in phosphoethanolamine, whereas nonneuroactive amino acids were increased only slightly. The kainic acid-evoked reduction in dialysate 45Ca2+ activity was attenuated in striata lesioned previously with kainate, suggesting the involvement of intrinsic striatal neurons in this response. The increase in taurine concentration induced by kainate was slightly smaller under these conditions. Decortication did not affect the kainate-evoked alterations in either dialysate 45Ca2+ or amino acids. These data suggest that kainate does not release acidic amino acids from their transmitter pools located in corticostriatal terminals.     

Effect of ATP on protein synthesis in cultured nerve tissue of the edible snail]

External ATP in concentrations of 10(-6)--10(-3) M is shown to stimulate the label incorporation from intracellular labeled pool of 14C-leucine into proteins of mollusc nervous tissue. The maximum effect (by 45% higher than in control) is observed at the 10(-5) ATP concentration. In solutions with high concentration of bivalent ions, ATP action increases by 10--15%. Being incubated for an hour in physiological solutions without energic substrates nervous tissue loses 30--50% of labeled amino acids. Outwashing of 14C-leucine depends only a little on the bivalent ion concentration in the external solution and on the presence of helating agents. Addition of 10(-4) M ATP into the solution, completely inhibits the washing of amino acids out of tissue. At low bivalent ion concentrations 14C-leucine incorporation into nervous tissue in the presence of ATP changes inversely to the ATP concentration: low ATP concentrations (10(-5)--10(-6) M) activate label incorporation by 60--40%, whereas high concentrations lead to the corresponding inhibition. This inhibition is due to helating action of ATP.     

The effects of various anions and cations on the regulation of pyruvate dehydrogenase complex activity from pig kidney cortex.

The activity of pyruvate dehydrogenase complex (PDC) purified from pig kidney cortex was found to be affected by various uni- and bi-valent ions. At a constant strength of 0.13 M at pH 7.8, K+, Na+, Cl-, HCO3- and HPO4(2-) had significant effects on the activity of PDC: Na+, K+ and HPO4(2-) stimulated, but HCO3- and Cl- inhibited. The stimulatory effect of Na+ was mediated by a change in the Vmax. of PDC only, whereas K+ produced an increase in Vmax. and a change in the Hill coefficient (h). The extent of stimulation produced by HPO4(2-)4 on the activity of PDC was dependent on the concentrations of K+ and Na+. Both cations at concentrations higher than 40 mM partially prevented the effect of HPO4(2-)4. Cl- and HCO3- anions decreased the Vmax. of the enzyme and increased the S0.5 for pyruvate. The effects of Na+, K+, Cl-, HPO4(2-) and HCO3- on the activity of PDC were additive. In the presence of 80 mM-K+, 20 mM-Na+, 10 mM-HPO4(2-), 20 mM-Cl- and 20 mM-HCO3- the activity of PDC was increased by 30%, the S0.5 for pyruvate was increased from 75 to 158 microM and h was decreased from 1.3 to 1.1. Under these conditions and at 1.0 mM-pyruvate, the activity of PDC was 80% of the maximal activity achieved in the presence of these ions and 4.5 mM-pyruvate. The present study suggests that PDC may operate under non-saturating concentrations for substrate in vivo.     

Responsiveness of RNA degradation to amino acids in cultured rat hepatocytes: comparison with isolated rat hepatocytes.

The role of amino acids in the regulation of RNA degradation was investigated in cultured hepatocytes from fed rats previously labeled in vivo with [6-14C]orotic acid. Rates of RNA degradation were determined between 42 and 48 h of culture from the release of radioactive cytidine in the presence of 0.5 mM unlabeled cytidine. The fractional rate was about 4.4 +/- 0.4%/h in the absence of amino acids (0x). The catabolism of RNA was decreased to basal level (1.5 +/- 0.3%/h) by the addition of amino acids at 10 times normal plasma concentration (10x). The inhibition of RNA degradation, expressed as percentage of maximal deprivation-induced response (0x minus 10x), averaged 60% at normal plasma levels of amino acids. The degree of responsiveness was greatly improved as compared to freshly isolated hepatocytes (20%) and was similar to the sensitivity previously observed with perfused livers. In cultured hepatocytes, the sensitivity of RNA degradation to amino acids was not affected by varying the volume of medium from 1 to 4 ml per dish. In freshly isolated hepatocytes, the inhibitory effect of amino acids was not modified by changing the cell density from 0.5 to 5 x 10(6) cells per ml. In the range of normal plasma concentration of amino acids, the low sensitivity of RNA degradation in isolated hepatocytes persisted with inhibition ranging from 10 to 20%. These findings suggest that the control of RNA degradation in both cultured and isolated hepatocytes is not affected by the total quantity of amino acids available in the medium, but their concentration is crucial. Electron microscopy observations and the inhibitory effect of 3-methyl-adenine in cultured rat hepatocytes partially confirmed the role of the lysosomal system in the increase of RNA degradation and its regulation by amino acids.     

NUTRITIONAL STUDIES OF THE EDIBLE SEAWEED PORPHYRA TENERA. II. NUTRITION OF CONCHOCELIS1

The nutrition of the free-living phase of Conchocelis of P. tenera was studied axenically. Conch-ocelis preferred NO₃, as nitrogen source. Urea and NH₄ in low concentration, asparagine, and lysine were very good N sources. Several other amino acids were also utilized but growth was less abundant. Inorganic and organic phosphates were utilized; they were required at relatively low concentrations. Glycerophosphate gave excellent growth in a comparatively wide range of concentrations (0.1-5 mg P %). The optimal Ca concentration was 10-100 mg %. Needs for boron, manganese, zinc, strontium, rubidium, lithium, and iodine were demonstrated. The iodine effect was remarkable (peak growth with 1μg %); the effective concentration range was very narrow. Iron, cobalt, and bromine seemed to be adequately supplied as impurities of the macro-nutrients. A modified artificial medium (ASP₁₂I) for the Conchocelis phase is presented.     

Axonal surface charges: evidence for phosphate structure.

The effect of different extracellular alkaline-earth cations (Ca2+, Mg2+, Sr2+, Ba2+) upon the threshold membrane potential for spike initiation in crayfish axon has been studied by means of intracellular microelectrodes. This was done at the following extracellular concentrations of the divalent uranyl ion (UO2/2+): 1.0 X 10(-6) M, 3.0 X 10(-6) M, and 9.0 X 10(-6) M. At each concentration employed, extensive neutralization of axonal surface charges by UO2/2+ was evidenced by the fact that equal concentrations (50 mM) of alkaline-earth cations did not have the same effect on the threshold potential. The selectivity sequences observed at the different uranyl-ion concentrations were: 1.0 X 10(-6) M UO2/2+, Ca2+ greater than Mg2+ greater than Sr2+ greater than Ba2+; 3.0 X 10(-6) M UO2/2+, Ca2+ greater than Mg2+ greater than Ba2+ larger than or equal to Sr2+; 9.0 X 10(-6) M UO2/2+, Ca2+ approximately Ba2+ greater than Sr2+ greater than Mg2+. These selectivity sequences are in accord with the equilibrium selectivity theory for alkaline-earth cations. At each of the concentrations used, uranyl ion did not have any detectable effect on the actual shape of the action potential itself. It is concluded that many (if not most) of the surface acidic groups in the region of the sodium gates represent phosphate groups of membrane phospholipids, but that the m gates themselves are probably protein-aceous in structure.     

Effect of polyamines on isoleucyl-tRNA formation by rat-liver isoleucyl-tRNA synthetase.

The effect of polyamines on rat-liver isoelucyl-tRNA formation was studied using isoleucyl-tRNA synthetase purified by column chromatography successively on Sephadex G-200, DEAE-Sephadex A-25, and tRNA-Sepharose 4B. In the presence of 50 mMK+, isoleucyl-tRNA formation was inhibited markedly by 1.5 mM or higher concentrations of Mg2+. However, the addition of spermine to the reaction mixture prevented the inhibitory effect of Mg2+. In the presence of 200 mMK+, the addition of spermine to the reaction mixture stimulated isoleucyl-tRNA formation in the presence of Mg2+ concentrations from 0 to 5 mM. Although the effective concentration was different, spermidine exhibited a similar stimulative effect. The effective concentration of spermine required for stimulation was higher when larger amounts of tRNA were used. The stimulatory effect of isoleucyl-tRNA formation by polyamines was shown to reflect on polypeptide synthesis. When formaldehyde-treated poly(A,U) was used as messenger RNA, polypeptide synthesis from amino acids was stimulated by polyamines, but that from aminoacyl-tRNAs was not stimulated by polyamines.     

Changes in amino acid permeation during sporulation

Changes in amino acid uptake in Bacillus licheniformis and in the amino acid pools of three Bacillus species were investigated, by use of cells from different stages of the life cycle. B. licheniformis contains catalytic uptake systems for all of the 10 amino acids studied. The apparent maximal velocities of uptake decreased during sporulation but did not fall below the range observed for other microorganisms. In sporulating cells, the apparent affinity constants of the uptake systems for individual amino acids remained about the same as in growing cells, i.e., from 2 x 10(-7)m to 7 x 10(-6)m, whereas, in some cases, the apparent maximal velocities decreased significantly. Because the velocity of uptake showed an atypical dependence on substrate concentration, it was postulated that these cells contain two or more uptake systems for each amino acid. Only one of these systems appeared to be operative at a substrate concentration below 10(-6)m. Working at these low substrate concentrations, catalytic activities producing a net efflux of amino acids were demonstrable in vegetative cells in the presence of chloramphenicol, but these exit systems were lost during sporulation. A pool formed by the addition of radioactive algal hydrolysate will exchange with the external medium in vegetative cells but not in sporulating cells. Glutamic acid and alanine comprise at least 60% of the amino acid pool of B. licheniformis A-5, B. subtilis 23, and B. cereus T during all stages of growth and sporulation. The concentrations of the other amino acids in the pool varied extensively, but reflected, in general, the amino acid turnover known to occur during sporulation.     

Effect of spermine on the uptake of amino acids in Micrococcus lysodeikticus

Spermine inhibited the transport of neutral aliphatic amino acids (valine, leucine, isoleucine, alanine, and glycine) into cells of Micrococcus lysodeikticus. On the other hand, spermine did not affect the uptake of basic (arginine and histidine), acidic (glutamic acid), or aromatic (phenylalanine and tyrosine) amino acids. Inhibition of uptake of the neutral amino acids by spermine is apparently of a noncompetitive nature; the V(max) decreased, whereas the apparent K(m) remained unaltered. The inhibition is most likely due to a specific binding of spermine to the carrier(s) of these amino acids. Related polyamines, spermidine and cadaverine, also caused inhibition of valine uptake, though to a lesser extent; spermidine was less active than spermine, and cadaverine showed the weakest effect of all. Valine, leucine, and isoleucine were transported into M. lysodeikticus cells by a common carrier as evidenced from competition experiments. The uptake of these amino acids is an active process; it was temperature-dependent and inhibited by azide (10(-1)m to 2.5 x 10(-2)m) and dinitrophenol (10(-3)m). The intracellular concentration of valine was 100-fold higher than in the medium.     

Effects of amino acids and α-amanitin on bovine embryo development in a simple protein-Free medium

Five experiments, utilizing 3741 embryos produced in vitro, were designed to test the effects of Eagle's nonessential amino acids, and combinations of Eagle's essential amino acids and the RNA polymerase inhibitor α-amanitin on the development of preimplantation bovine embryos in a modified protein-free KSOM medium. Embryos were cultured in 5% O₂:5% CO₂:90% N₂ at 39°C for the first 40–44 hr in modified KSOM, and embryos with ≥4 cells were cultured in modified KSOM-PVA with different amino acids in experiments 1–4, and with the addition of α-amanitin in experiment 5. In experiment 1, addition of 0.5× of the essential amino acids, with different concentrations of nonessential amino acids significantly increased hatching of blastocysts and decreased blastocyst degeneration, but increasing the nonessential amino acids from 1× to 5×, did not stimulate embryo development. In experiments 2–4, increasing only the glycine concentration, or adding each of the 12 essential amino acids singly or several in combination to the medium containing nonessential amino acids, did not significantly improve embryo development. Taurine (0.4 mM) in the modified KSOM medium reduced blastocyst degeneration. In experiment 5, α-amanitin (20 μM) completely inhibited further embryo development when it was added at several stages from 4-cell embryos to morulae. The study with protein-free KSOM plus amino acids provided a completely defined simple medium for culturing bovine embryos, with evidence that continuous mRNA activity and presumed protein synthesis was obligatory to meet the complex and continuous requirements for proteins by the developing blastocyst. Mol. Reprod. Dev. 46:278–285, 1997. © 1997 Wiley-Liss, Inc.     

Transport of methionine in sea-urchin sperm by a neutral amino-acid carrier

A carrier-mediated transport for L-methionine and other neutral amino acids exists in sperm of the sea urchin Lytechinus pictus. The initial rate of L-methionine entry is a Michaelis-Menten function of the methionine concentration in the external medium. The maximum velocity is low [V = 250 pmol h-1 (10(9) sperm)-1 at 22 degrees C] and the affinity is high (Km = 6-10 microM). The initial rate of transport under steady-state exchange conditions is also a Michaelis-Menten function of the external concentration of methionine. The Km determined by this method is about 14 microM. Neutral amino acids compete with L-methionine transport as shown by initial velocity measurements. These results indicate that L-methionine transport is a carrier-mediated process. The temperature dependence of the process is approximately 84 kJ (20 kcal) mol-1 K-1, which is not compatible with a simple diffusion mechanism, but in the range of values usually found for a mediated transport. The transport is largely Na+-independent and does not depend on Ca2+, K+ or H+ gradients. It is only partially sensitive to KCN, showing it is mainly independent of oxidative phosphorylation. The steady-state internal methionine concentration is not a linear function of the external amino acid concentration. This suggests that an exit by diffusion competes with a carrier-mediated concentrative transport in a cellular compartment. This mediated transport is compared to those of higher animal cells.