The influence of intracellular pH of spores on citric acid production by Aspergillus niger

The spores with different intracellular pH values were produced by cultivating Aspergillus niger on potato-dextrose agar at different pH_e values. High citric acid production is obtained using spores with the highest initial pH values (pH_i) around 7.5. After a drop of intracellular pH during germination of spores of about 0.7 units, the conditions for highest rate of metabolic flow through the glycolytic pathway were achieved only by the mycelium grown out of spores with the highest pH_i, since a very narrow pH optimum of 6-phosphofructokinase activity has been found around 7.5.     

Local Ca2 Rise Near Store Operated Ca2 Channels Inhibits Cell Coupling During Capacitative Ca2 Influx

Using a new fluorescence imaging technique, LAMP, we recently reported that Ca²⁺ influx through store operated Ca²⁺ channels (SOCs) strongly inhibits cell coupling in primary human fibroblasts (HF) expressing Cx43. To understand the mechanism of inhibition, we studied the involvement of cytosolic pH (pH_i) and Ca²⁺([Ca²⁺]_i) in the process by using fluorescence imaging and ion clamping techniques. During the capacitative Ca²⁺ influx, there was a modest decline of pH_i measured by BCECF. Decreasing pH_i below neutral using thioacetate had little effect by itself on cell coupling, and concomitant pH_i drop with thioacetate and bulk [Ca²⁺_i rise with ionomycin was much less effective in inhibiting cell coupling than Ca²⁺ influx. Moreover, clamping pH_i with a weak acid and a weak base during Ca²⁺ influx largely suppressed bulk pH_i drop, yet the inhibition of cell coupling was not affected. In contrast, buffering [Ca²⁺_i with BAPTA, but not EGTA, efficiently prevented cell uncoupling by Ca²⁺ influx. We concluded that local Ca²⁺ elevation subjacent to the plasma membrane is the primary cause for closing Cx43 channels during capacitative Ca²⁺ influx. To assess how Ca²⁺ influx affects junctional coupling mediated by other types of connexins, we applied the LAMP assay to Hela cells expressing Cx26. Capacitative Ca²⁺ influx also caused a strong reduction of cell coupling, suggesting that the inhibitory effect by Ca²⁺ influx may be a more general phenomenon.     

Cytochrome b of the Dictyostelium discoideum plasma membrane

Cytochrome b of the plasma membrane of Dictyostelium discoideum was investigated in purified plasma membranes and in solubilized form. The membrane-bound cytochrome b can be reduced by NADH. This reduction is inhibited by p-hydroxymercuribenzoate. The reduced cytochrome b does not react with carbon monoxide. Its apparent molecular weight lies between 13000 and 16000. Tryptic digestion yields a large, heme-containing peptide with an apparent molecular weight between 12000 and 15000. After solubilization with cholate, cytochrome b can be enriched by reversed-phase HPLC, indicating that it contains also a hydrophobic component. With these properties, cytochrome b of the D. discoideum plasma membrane resembles microsomal cytochrome b₅.     

Thyroid hormone stimulates expression of 6-phosphofructo-2-kinase in rat liver

Gel-filtrated human platelets were stimulated with thrombin in the absence and presence of adrenaline. Adrenaline markedly enhanced the thrombin-induced increase in cytoplasmic pH (pH_i) in BCECF-loaded platelets. This rise in pH_i was strongly inhibited by the Na⁺/H⁺ exchange blocker EIPA. The potentiation by adrenaline of thrombin-induced PLC activation measured as [³²P]PA formation and final platelet responses was, however, not blocked by EIPA, even at low concentrations of thrombin. These results indicate that the enhancement by adrenaline of thrombin-induced cytoplasmic alkalinization may be a secondary effect which is not essential for the potentiation by adrenaline of platelet activation by thrombin.     

Spatial aspects of intracellular pH regulation in heart muscle

Intracellular pH (pH_i) is an important modulator of cardiac function. Because it is readily influenced by metabolic processes, pH_i is controlled physiologically. Classical models of intracellular pH regulation comprise acid/base transport proteins expressed in the sarcolemma, acting in concert with intracellular buffers. These two processes are coupled via a diffusive movement of protons. Because intracellular H⁺ buffering is high, H_i⁺-diffusion occurs through a passive shuttling on intrinsic mobile buffers such as acetylated carnosine, anserine and homocarnosine: low molecular weight imidazole compounds. This mechanism is assisted by carbonic buffer, a system regulated biochemically by the enzyme carbonic anhydrase. H_i⁺-mobility via the buffer shuttles is low, and this can result in significant pH_i non-uniformity under conditions of high proton flux across the sarcolemma or within the cell. Spatial regulation of pH_i is complemented by passive H⁺ permeation between cells through gap junctions. This permeation is also mediated via protonated buffers. The control of pH_i is therefore dependent on carrier molecules that spatially shuttle protons within and between cells. In this review, we consider the physiological regulation of H_i⁺-mobility and permeation, and its relevance to pH_i-control in normal and pathophysiological states such as myocardial ischaemia, a clinical condition associated with severe intracellular acidosis.     

Imidazol-1-ylalkanoate esters and their corresponding acids. A novel series of extrinsic H NMR probes for intracellular pH

A novel series of extrinsic probes for intracellular pH (pH_i) determination by ¹H NMR is described. Imidazol-1-ylacetate, malonate, 3-glutarate and 2-succinate esters were synthesized by reaction of imidazole either with -bromoesters or with ,β-unsaturated esters. The corresponding acids were prepared by hydrolysis.     

An autonomously propagating luciferase-encoding vector for Dictyostelium discoideum

We have constructed a luc reporter vector for Dictyostelium discoideum using a 626-bp fragment from the nuclear-associated plasmid Ddp2. The ori from Ddp2 is localized within this fragment and was used to provide an autonomous replication sequence for the reporter vector. This reporter vector was stably retained in D. discoideum AX3K cells without alteration. The vector molecule was also found to exist in relatively low copy number compared to other Dictyostelium vectors in the transformed cells. We demonstrated the utility of this vector as a reporter vector with glycogen synthase promoter/luc fusions of varying sizes.     

A Dictyostelium discoideum DNA fragment complements a Saccharomyces cerevisiae ura3 mutant

Dictyostelium discoideum DNA fragments have been inserted into the chimeric bacterium-yeast plasmid YEp13. Recombinant plasmids were used to transform yeast using a strain of Saccharomyces cerevisiae deficient in OMP decarboxylase activity. Several clones were selected for growth in uracil-free medium. One clone was further analysed and contains a plasmid with a segment of D. discoideum DNA which complements a yeast ura3 mutation.     

From virus to vaccine: developments using the simple eukaryote, Dictyostelium discoideum

Mass vaccination campaigns against viral diseases, both human and animal, depend on the availability of cheap viral antigens. The eukaryote Dictyostelium discoideum has simple growth requirements and rapid growth rates and forms stable cell lines. These features, together with the possibility of secreting recombinant (glyco) proteins into a defined buffer, make the D. discoideum expression system an attractive option for producing economical recombinant subunit vaccines.     

Cocoa shells for heavy metal removal from acidic solutions

The development of economic and efficient processes for the removal of heavy metals present in acidic effluents from industrial sources or decontamination technologies has become a priority. The purpose of this work was to study the efficiency with which cocoa shells remove heavy metals from acidic solutions (pH 2) and to investigate how the composition of these solutions influences heavy metal uptake efficiency. Adsorption tests were conducted in agitated flasks with single-metal solutions (0.25 mM Al, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb and Zn), multi-metal solution (comprised of 0.25 mM of each of the cations above) and an effluent obtained from chemical leaching of metal-contaminated soil, in the presence of different cocoa shell concentrations (5–40 g/l). Results from the single-metal solution assays indicated that the fixation capacity of heavy metals by cocoa shells followed a specific order: Pb > Cr > Cd=Cu=Fe > Zn=Co > Mn=Ni=Al. Cocoa shells are particularly efficient in the removal of lead from very acidic solutions (q_max=6.2 mg Pb/g, pH_i=2.0 and T=22 °C). The presence of other metals and cations in solution did not seem to affect the recovery of lead. It was also observed that the maximum metal uptake was reached in less than 2 h. This research has also demonstrated that the removal of metals caused a decline in solution proton concentration (pH increase) and release of calcium, magnesium, potassium and sodium from the cocoa shells.     

Calcium is required for the increase of dark respiration during diurnal nitrogen fixation by Synechococcus RF-1

Under 12/12 h light/dark cycles, 1 mM ethyleneglycol-bis-(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA, pH 8.0) added at the start of the dark period, inhibited the increase of dark respiration which was associated with nitrogen fixation in Synechococcus RF-1. Twenty-five millimolar NaNO₃ added 30 min before the start of dark period suppressed this respiratory increase. If 1.25 mM CaCl₂ was added to the EGTA-treated sample from 3 to at least 10 h later in the dark period, a quick rise in respiratory rate was observed. This rise was also reduced by 25 mM NaNO₃. Extracellular Ca²⁺ appears to be required for the increase in dark respiration associated with the rhythmic appearance of nitrogenase activity in the dark cycle.     

Highly selective affinity labeling of the primer-binding site of E. coli DNA polymerase I

Membrane-associated phosphoinositidase C activity has been identified in Dictyostelium discoideum using phosphatidylinositol 4,5-bisphosphate as exogenous substrate. Maximal activity was observed with 0.4 mM phosphatidylinositol 4,5-bisphosphate at pH 7.0. The enzyme was stimulated

by micromolar concentrations of free calcium with maximal activity at 100 μM.     

Endogenous ADP-ribosylation of glyceraldehyde-3-phosphate dehydrogenase that is not regulated by nitric oxide in Dictyostelium discoideum

A 41,000 M_r cytosolic protein (p41) in Dictyostelium discoideum was shown to be modified by ADP-ribosylation that was not regulated by nitric oxide (NO). This endogenous ADP-riboxylation was optimal at conditions distinct from those optimal for the NO-stimulated ADP-ribosylation of p41. These two activities were also differentially sensitive to reducing agents and modified different amino acids. The addition of haemoglobin, which sequesters NO, and 3 the NO synthase inhibitors failed to block the endogenous ADP-ribosylation. P41 was purified to homogeneity. The N-terminal sequence of the purified protein was shown to be highly homologous to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Both endogenous and NO-stimulated activities ADP-ribosylated three isoforms of the protein, with pI values of 6.6., 6.8 and 7.0. In each case, the isoform with pI 6.8 was preferentially modified. Experiments using purified GAPDH indicate that both the endogenous and NO-stimulated ADP-ribosylation are self-catalysed modifications.     

Two distinct types of cell surface folic acid-binding proteins in Dictyostelium discoideum

Folic acid is degraded too fast by Dictyostelium discoideum to study binding of this ligand to cell surface binding proteins. Folate deaminase activity was inhibited in the presence of 3.3 × 10⁻⁴ M 8-azaguanine. This inhibitor enabled us to detect two folate binding proteins. One type bound folic acid and deamino-folic acid with the same affinity (K_0.5 = 3–6 × 10⁻⁷ M) and apparently negative cooperativity. Binding to only this type was observed if 8-azaguanine was omitted. The second type bound folic acid noncooperatively with K_d = 7 × 10⁻⁷ M. Deamino-folic acid did not compete even at a 1000-fold excess. This type may correspond to the chemotactic receptor.     

Influence du pH sur la synthèse de l''oxidase (cytochrome a3) chez Bacillus coagulans, microorganisme thermophile, capable d'' “adaptation respiratoire”

In a new series of experiments on Bacillus coagulans (ATCC 11.369), it was demonstrated that this organism possesses a respiratory system with cytochromes b, c₁, c, (a+a₃) and also cytochrome o. A small decrease in the pH of the growth medium from 6.5 to 5.5 increases the respiratory activity by a factor of 4 and induces a variation of the absorption ratio [₆₀₃ (a+a₃)]/[₅₆₀ (b+c)] resulting in a preponderant increase in the ₆₀₃ absorption. The kinetic studies of the respiratory system synthesis during the phenomenon of “respiratory adaptation” have shown that lowering the pH of the adaption medium has the same effect. Spectral studies of membrane fractions (red dithionite) with or without carbon monoxide showed a preferential synthesis of oxidase a₃.     

Activation of the Na-K(NH4)-2Cl-cotransporter from rat submandibular glands in response to VIP

A cellular suspension from rat submandibular glands was prepared with collagenase. The intracellular pH (pH_i) was estimated with 2′,7′-bis-(2-carboxy-ethyl)-5(6)-carboxyfluorescein (BCECF). After exposure to NH₄Cl, the pH_i transiently increased (diffusion of NH₃) and then dropped (influx of NH₄⁺). Isoproterenol increased 2.5-fold the rate of NH₄⁺ influx; bumetanide, an inhibitor of the Na⁺-K⁺-2Cl⁻-cotransporter blocked the response to isoproterenol, confirming that the beta-adrenergic agonist stimulated the cotransporter. Forskolin (1 μmol/L) mimicked the response to isoproterenol. VIP (1 nmol/L-1 μmol/L) also increased the activity of the cotransporter. Cyclic AMP rather than calcium was the mediator of this activation since 1) carbachol which increased the [Ca²⁺]_i fivefold increased the uptake of NH₄⁺ by only 50%; 2) only high concentrations of VIP significantly increased the [Ca²⁺]_i; 3) incubation in the presence of EGTA had no effect on the response to VIP; 4) low concentrations (nmol/L) of the neuropeptide increased the intracellular level of cAMP; and 5) the stimulation of the cotransporter by VIP, forskolin, and isoproterenol was inhibited by H8, an inhibitor of cAMP-dependent protein kinase. It is concluded that the Na⁺-K⁺-2Cl⁻-cotransporter of rat submandibular glands is activated by isoproterenol, forskolin, and neuropeptides of the VIP family by a mechanism involving cAMP-dependent processes. The activation of the cotransporter by VIP could partly explain the potentiating effect of VIP on the response to sialagogues like substance P or muscarinic agonists.¹     

The Xanthomonas type Ⅲ effector Xop AP prevents stomatal closure by interfering with vacuolar acidification

Plant stomata close rapidly in response to a rise in the plant hormone abscisic acid(ABA) or salicylic acid(SA) and after recognition of pathogenassociated molecular patterns(PAMPs). Stomatal closure is the result of vacuolar convolution, ion efflux, and changes in turgor pressure in guard cells. Phytopathogenic bacteria secrete type Ⅲ effectors(T3Es) that interfere with plant defense mechanisms, causing severe plant disease symptoms. Here, we show that the virulence and infection of Xanthomonas...     

An actin-related protein from Dictyostelium discoideum is developmentallyregulated and associated with mitochondria

An actin-related protein (ACLA) has been identified in the cellular slime mould Dictyostelium discoideum. The complete cDNA sequence indicates that within the actin superfamily it belongs to the ARP3 family of actin-related proteins together with Arp66B from Drosophila melanogaster, Actin2 from Bos taurus, act2 from Schizosaccharomyces pombe and possibly act2 from Caenorhabditis elegans. The ACLA mRNA is regulated during development, showing a maximum between 2 and 4 h after starvation. The protein has been expressed in E. coli and antibodies raised against it. Immunofluorescence microscopy shows that ACLA protein co-localises with mitochondria; the protein co-purifies with Dictyostelium mitochondria.     

The physiological ecology of haemocyanin in some selected crabs. II. The characteristics of haemocyanin in relation to terrestrialness

The haemocyanins of five crabs ranging in habit from aquatic to terrestrial have been investigated.

The mean P₅₀ values of the respiratory pigments were determined at 0 mm Hg CO₂ and 28 °C (the average environmental temperature of all the species). Comparison of these data adjusted to the individual mean physiological pH indicate an increase in P₅₀ with terrestrialization, perhaps related to the greater abundance of oxygen in the aerial than in some the aquatic habits, and the progressive elaboration of lung breathing with terrestrialization.

The Bohr shifts (Δ log P₅₀/ΔpH) were determined (using different P_CO2 values to vary pH) and were found to decrease with terrestrialization, perhaps in adaptation to an associated rise in internal P_CO2 (6–8-fold between the aquatic Callinectes sapidus Rathbun and the terrestrial Cardisoma guanhumi Latreille and probably resulting from progressive gill reduction.

The temperature shifts (ΔH cal/mol) of the haemoeyanins were found and it is suggested that they diminish with increasing evironmental temperature and temperature fluctuation accompanying terrestrialization.