Inhibition of Listeria monocytogenes by enterocin 4 during the manufacture andripening of Manchego cheese

The inhibitory effect of enterocin 4, a bacteriocin produced by Enterococcus faecalis INIA 4, on Listeria monocytogenes strains Ohio and Scott A during themanufacture and ripening of Manchego cheese was investigated. Raw ewe's milk wasinoculated with ca 10⁵ cfu ml⁻¹ of L.monocytogenes and with 1% of a commercial lactic starter, 1% of an Ent. faecalis INIA 4 culture, or 1% of each culture. Manchego cheeses were manufactured according tousual procedures. Listeria monocytogenes Ohio counts decreased by 3 log units after8 h and by 6 log units after 7 d in cheese made from milk inoculated with Ent. faecalis INIA 4 or with both cultures, whereas no inhibition was recorded after 60 d in cheese made frommilk inoculated with commercial lactic starter. Listeria monocytogenes Scott A wasnot inhibited by enterocin 4 during cheese manufacture, but decreases of 1 log unit after 7 d andof 2 log units after 60 d were achieved in cheese made from milk inoculated with bothcommercial lactic starter and Ent. faecalis INIA 4.     

Effects of Cd2+ and EDTA on young sugar beets (Beta vulgaris). I. Cd2+ uptake and sugar accumulation

Sugar beets ( Beta vulgaris L. cv. Monohill) grown in a complete nutrient solution, were treated with Cd²⁺ (5 or 50 μ M ) and/or EDTA (10 or 100 μ M ) in different combinations. The Cd contents of five-week-old roots and shoots were determined by atomic absorption spectrophotometry, and the sucrose, glucose and fructose contents were measured enzymatically. The Cd²⁺ uptake in both roots and shoots shows a linear relationship to the concentration of free Cd²⁺ in the nutrient solution. This uptake is diminished in the presence of EDTA, suggesting that the Cd-EDTA complex is unable to penetrate the membranes. The contents of glucose, fructose and sucrose in both roots and shoots decrease with increasing uptake of free Cd²⁺. This may be a secondary effect caused by the inhibition of photosynthesis in the presence of Cd²⁺. EDTA reduces the inhibition of Cd²⁺ on sugar formation and accumulation. In the presence of EDTA alone the sugar content increases somewhat. EDTA slightly influences the dry weights of whole plants. The ratio roots:whole plants increases. Cd²⁺ (≤ 50 μ M ) increases the dry matter portion of roots by ca 30%, but not that of shoots.     

Growth of Bacillus cereus in fermenting tempeh made from various beans and its inhibition by Lactobacillus plantarum

Counts of Bacillus cereus reached ca 10⁸ cfu/g within 40 h in fermenting unacidified horsebean tempeh and resulted in complete spoilage of the product. In fermenting unacidified pea, chickpea and soybean tempeh, B. cereus counts reached 10⁶–10⁷ cfu/g, although the products were not spoiled. Inoculation of these unacidified beans with Lactobacillus plantarum decreased the final count of B. cereus by 2 log units, but had no effect on its growth in unacidified horsebean tempeh and its subsequent spoilage. Acidification of the beans during soaking resulted in a lower rate of B. cereus growth during fermentation. Inoculation of acidified beans with Lact. plantarum resulted in a markedly lower growth rate of B. cereus . In an associative broth culture study, B. cereus was completely inhibited by Lact. plantarum at pH values of about 5·5. Lactobacillus plantarum may be used to control the growth of B. cereus during tempeh production.     

Effects of phloridzin, metavanadate and oligomycin on membrane bound (Na++ K++ Mg2+)ATPase activity in sugar beet roots

To clarify the reaction mechanism of a (Na⁺+ K⁺+ Mg²⁺)ATPase activity in sugar beet roots ( Beta vulgaris L. cv. Monohill) phloridzin, oligomycin (inhibitors of animal ATPases) and metavanadate (NH₄VO₃) have been used. Kinetic studies showed that: 1) Phloridzin inhibition is uncompetitive with respect to MgATP and not influenced by Na⁺ or K⁺. 2) This inhibition is only found in preparations made in the absence of sucrose. 3) Oligomycin and vanadate inhibit the ATPase in different ways. Omission of sucrose from the preparation medium favours vanadate inhibition but suppresses oligomycin inhibition. 4) The kinetic pattern of the Na⁺ activation of the ATPase differs in preparations made in the absence and presence of sucrose, but that of K⁺ activation is the same. – These results indicate that inclusion as against omission of sucrose from the preparation medium causes a conformational change of the membrane fragments/vesicles, which then expose different surfaces to the surrounding medium.     

Integration of an unstable plasmid into the chromosome of Lactobacillus plantarum

Abstract A 2.3 kb Eco RI restriction fragment of Lactobacillus plantarum genomic DNA was cloned into pSA3 to generate pJRI. Both pSA3 and pJR1 were transformed into L. plantarum . Growth of the transformants in the absence of the selection pressure, erythromycin (both plasmids confer Em^r), led to a stable sub-population of EM^r bacteria in which pSA3 and pJR1 had integrated into the L. plantarum genome. Amplification of the chromosomally located plasmids was observed when the organism was grown in the presence of erythromycin. The integrated plasmid sequences were stably maintained in the absence of selection pressure, and did not affect the growth rate of the lactic acid bacterium in rich media.     

Ca2+/Calmodulin-Mediated Regulation of Agonist-Induced Sequestration of Gq Protein-Coupled Histamine H1 Receptors in Human U373 MG Astrocytoma Cells

Abstract: We investigated the regulation by intracellular Ca²⁺ of agonist-induced sequestration of G_q protein-coupled histamine H₁ receptors in human U373 MG astrocytoma cells. Histamine-induced sequestration of H₁ receptors from the cell surface membrane was detected as the loss of [³H]mepyramine binding sites on intact cells accessible to the hydrophilic H₁-receptor antagonist pirdonium. The changes in the pirdonium-sensitive binding of [³H]mepyramine were mirrored by changes in the subcellular distribution of H₁ receptors detected by sucrose density gradient centrifugation. The histamine-induced sequestration of H₁ receptors did not occur in hypertonic medium, in which clathrin-mediated endocytosis is known to be inhibited, but was significantly accelerated in the absence of extracellular Ca²⁺ or in the presence of the calmodulin antagonists W-7 and calmidazolium. Inhibitors of protein kinase C (H-7 and GF109203X), Ca²⁺/calmodulin-dependent protein kinase II (KN-62), or protein phosphatase 2B (FK506) did not alter the time course of H₁-receptor sequestration. These results provide the first evidence that agonist-induced, clathrin-mediated sequestration of G_q protein-coupled receptors is transiently inhibited by Ca²⁺/calmodulin, with the result that receptors remain on the cell surface membrane during the early stage of agonist stimulation.     

Sucrose and ouabain effects on the kinetic properties of a membrane-bound (Na++ K++ Mg2+) ATPase in sugar beet roots

Kinetic studies of a microsomal (Na⁺+ K⁺+ Mg²⁺)ATPase from sugar beet roots ( Beta vulgaris L. cv. Monohill) show that sucrose influences the MgATPase in different ways depending on the presence of K⁺ and/or Na⁺ 1) In the presence of the substrate MgATP and Na⁺ the effect of sucrose follows simple Michaelis-Menten kinetics. 2) In the presence of substrate together with K⁺ or (K⁺+ Na⁺), sucrose has little effect on the ATPase activity. 3) In the presence of Na⁺, onabain acts as an uncompetitive inhibitor with respect to MgATP. 4) In the presence of K⁺ or (K⁺+ Na⁺), the inhibition by ouabain is somewhat depressed and shows non-linearity when 1/v is plotted versus 1/MgATP. 5) Sucrose and Na⁺ activate in a competitive way, so that a successive increase of the Na⁺ level decreases the activation by sucrose. Both K_m and V-values are thereby changed. 6) The sucrose activation in the presence of Na⁺ is also influenced by ouabain. It is, therefore, suggested that Na⁺ may regulate the interference between the Na⁺/K⁺ pump and a sucrose sensitive system.     

Proton-ATPase activity in cells of lactobacilli grown in the presence of propionate

Lactobacillus helveticus ATCC 15009 and CRL 581, and Lact. casei LC3 were grown in a complex medium with and without 15 mmol 1^-1 of neutralized propionic acid and assayed for proton-translocating ATPase activity. The enzyme activity was higher when the medium contained fatty acid than in its absence for all strains studied. Characteristics of this increased ATPase were identical to those of the enzyme located on the membrane of normal cells. The substrate consumption rate of resting cells was increased by propionate. This effect was reverted by the specific H⁺-ATPase inhibitor N,N '-dicyclohexylcarbodiimide indicating that the increment of fermentative activity was related to the H⁺-ATPase activity. These results suggest that the amplification of H⁺-ATPase activity could be involved in the inhibition of lactobacilli growth in cultures where propionic acid is unavoidably present, such as some mixed cultures with propionibacteria.     

Oxidation of Leucine by Leishmania donovani

ABSTRACT. The metabolism of leucine by Leishmania donovani was investigated. Washed promastigotes were incubated with (1-¹⁴C]-or [U-¹⁴C]leucine or [1 -¹⁴C]α-ketoisocaproate (KIC) and ¹⁴C0₂ release was measured. The amount of KIC-derived acetyl-CoA oxidized in the citric acid cycle was computed. Promastigotes from mid-stationary phase cultures oxidized each of these labeled substrates less rapidly than cells from late log phase cultures, and significantly less acetyl-CoA derived from KIC oxidation was oxidized in the citric acid cycle. Glucose was a stronger inhibitor than was acetate of CO₂ formation in the citric acid cycle in log phase promastigotes, but the reverse was observed in cells from mid-stationary phase. Alanine also inhibited leucine catabolism, but glutamate had little effect. Acute hypo-osmotic stress did not affect leucine catabolism, but hyper-osmotic stress caused appreciable inhibition of leucine oxidation. Cells grown under hypo-or hyper-osmotic conditions showed no changes in the effects of hypo-or hyper-osmotic stress on leucine catabolism, i.e. L. donovani is not an osmoconformer with respect to leucine metabolism. Leucine utilization in L. donovani was insensitive to a number of drugs that affect leucine metabolism in mammalian cells, indicating that the leucine pathway in L. donovani is not regulated in the same manner as in mammalian cells.     

EVALUATION OF CULTURE PROTOCOLS AND OXYRASETM SUPPLEMENTATION FOR ARCOBACTER SPP.

Growth of Arcobacter butzleri was evaluated in Brain Heart Infusion broth incubated aerobically, microaerobically, and with Oxyrase^TM supplementation (anaerobically). At initial concentrations of10¹ to 10³ cells/mL, A. butzleri populations reached 7.5 to 8.0 log CFU/ml in 48h at 37C in Oxyrase^TM -supplemented broth. The organism quickly declined in the other two systems to undetectable levels during the initial 24h of incubation. Only moderate population levels (ca. 3 log CFU/ml) could be detected in aerobic and microaerobic systems after 56h incubation. Growth of five Arcobacter spp. strains was evaluated at 30C in Brucella-blood broth, modified Cary and Blair Transport Medium, and a biphase cultural system. Strains were inoculated at a level of ca. 10³ cells/ml and incubated with and without Oxyrase^TM supplementation to the system for 36h. Both the Brucella-blood broth and the biphase system supported good growth of the strains, with counts reaching ca. 8 to 9 log CFU/ml. Modified Cary and Blair Transport Medium was the least effective cultural system. Oxyrase^TM provided slight to moderate stimulation of growth for most strains.     

Changes in Orthophosphate, Pyrophosphate and Long-Chain Polyphosphate Levels in Leishmania major Promastigotes Incubated With and Without Glucose

The intracellular levels of orthophosphate (P₁), pyrophosphate (PP₁) and short- and long-chain polyphosphate (Poly P) were measured in Leishmania major promastigotes incubated in a phosphate-free medium. In the absence of exogenous substrate, the levels of both P₁ and PP₁ increased during a 1 h incubation. The increase in both P₁ and PP₁ was prevented when glucose was present, but glycerol prevented the rise in P₁ only. A rise in P₁ and PP₁ was also seen in cells incubated in the absence of exogenous substrate under anaerobic conditions. This was reversed upon addition of glucose plus oxygen. Polyphosphate, here shown to be present in L. major , was measured by means of a polyphosphate glucokinase assay. Short-chain Poly P content did not differ between cells incubated for 1 h in the absence of exogenous substrate or in the presence of glucose or glycerol. Long-chain Poly P content, however, was lower in cells incubated without glucose than in cells incubated with glucose and was also lower in cells incubated for 1 h with glycerol as compared with freshly washed cells. Up to 61% of the increase in P₁ and PP₁ that occurred in promastigotes incubated in the absence of exogenous substrate could have arisen from the concomitant decrease in long-chain Poly P.     

Lactobacillus amylovorus as a phytase producer in submerged culture

Nineteen strains of lactic acid producing bacteria of the genera Lactobacillus and Streptococcus collected from different culture collections were screened for the production of extracellular phytase. A number of them exhibited the enzyme activity in the fermentation medium but Lactobacillus amylovorus B4552 produced the maximum amounts of phytase ranging from 125 to 146 units ml^-1 extracellularly under the submerged cultivation conditions. The optimum glucose and inorganic phosphate levels for highest phytase production were found to be 1% and 24% mg, respectively. Lactobacillus amylovorus has potential in improving nutritional qualities of cereal and pulse-based food fermentations.     

Tyrosine Hydroxylase Phosphorylation in Bovine Adrenal Chromaffin Cells: The Role of Intracellular Ca2+ in the Histamine H1 Receptor-Stimulated Phosphorylation of Ser8, Ser19, Ser31, and Ser40

Abstract: Tyrosine hydroxylase (TOH), the rate-limiting enzyme in catecholamine biosynthesis, is regulated by phosphorylation. Activation of histaminergic H₁ receptors on cultured bovine adrenal chromaffin cells stimulated a rapid increase in TOH phosphorylation (within 5 s) that was sustained for at least 5 min. The initial increase in TOH phosphorylation (up to 1 min) was essentially unchanged by the removal of extracellular Ca²⁺. In contrast, the H₁-mediated response was abolished by preloading the cells with BAPTA acetoxymethyl ester (50 µ M ) and significantly reduced by prior exposure to caffeine (10 m M for 10 min) to deplete intracellular Ca²⁺. Trypticphosphopeptide analysis by HPLC revealed that the H₁ response in the presence or absence of extracellular Ca²⁺ resulted in a major increase in the phosphorylation of Ser¹⁹ with smaller increases in that of Ser⁴⁰ and Ser³¹. In contrast, although a brief stimulation with nicotine (30 µ M for 60 s) also resulted in a major increase in Ser¹⁹ phosphorylation, this response was abolished in the absence of extracellular Ca²⁺. These data indicate that the mobilization of intracellular Ca²⁺ plays a crucial role in supporting H₁-mediated TOH phosphorylation and may thus have a potentially important role in regulating catecholamine synthesis.     

Sodium ion transport in Neurospora crassa

Na⁺ influx and efflux in Neurospora crassa RL21a can be studied separately to calculate net Na⁺ movements. In the absence of external K⁺, Na⁺ influx was independent of the K⁺ content of the cells, but when K⁺ was present, the inhibition of Na⁺ influx by external K⁺ was higher the higher the K⁺ content. Efflux depended on the K⁺ and Na⁺ content, and on the history of the cells. Efflux was higher the higher the Na⁺ and K⁺ contents, and, in low-K⁺ cells, the efflux was also higher in cells grown in the presence of Na⁺ than when Na⁺ was given to cells grown in the absence of Na⁺. Addition of K⁺ to cells in steady state with external Na⁺ resulted in a net Na⁺-loss. In cells grown without Na⁺ this loss was a consequence of the inhibition of Na⁺ influx. In Na⁺-grown cells, addition of K⁺ inhibited Na⁺ influx and increased Na⁺ efflux.     

Effects of the high pressure of homogenization on some spoiling micro-organisms, representative of fruit juice microflora, inoculated in saline solution

Aims:  This study was aimed to investigate the effects of a high-pressure homogenization (HPH) treatment on some micro-organisms, involved in the spoilage of fruit juices.
Methods and Results:  Lactobacillus plantarum , Lactobacillus brevis , Bacillus coagulans cells, Saccharomyces bayanus , Pichia membranaefaciens and Rhodotorula bacarum were separately inoculated in a saline solution (0·9% NaCl); the initial inoculum was ca. 5 log CFU ml⁻¹. Then, the samples were processed through a homogenizer at 10–150 MPa for 1, 2 or 3 times. Yeasts were completely inactivated at 50–110 MPa with a single pass treatment, while lactic acid bacteria counts were reduced to approximately 1 log CFU ml⁻¹ after a three-steps HPH processing.
Conclusions:  Yeasts were the most sensitive micro-organisms, followed by B. coagulans . On the other hand, lactic acid bacteria appeared resistant to HPH.
Significance and Impact of the Study:  The results of this study provided some useful information on the susceptibility of microflora of juices to homogenization; moreover, they suggested that HPH could be used successfully to inactivate yeasts.     

The effects of growth dynamics upon pH gradient formation within and around subsurface colonies of Salmonella typhimurium

pH measurements made in and around submerged colonies of Salmonella typhimurium grown within a model gelatin gel system using pH-sensitive micro- and macroelectrodes indicated some pH heterogeneity occurring in and around the bacterial colony. Inoculation density, initial pH and glucose concentration were all found to influence colony diameter and metabolism of Salmonella colonies. Colony growth in the presence of glucose, at pH 7.0 with an inoculation density of 1 cell ml^-1 led to a pH fall of 1–2 pH units after 2 d. At pH 5.0, with glucose, colony growth rates were much slower than at pH 7.0, and the pH change varied by less than one pH unit often becoming alkaline. In the absence of glucose, only small pH changes were observed within the medium, although growth rates were similar to those in glucose-containing media. At the higher inoculation density ( ca 1000 cells ml^-1), isolated pH changes were not observed. Morphological changes, such as the production of annular rings, were noted in stationary phase colonies as was alkali production in colonies. These results are discussed in relation to observations with surface colonies.     

Salicylhydroxamic acid-stimulated NADH oxidation by purified plasmalemma vesicles from wheat roots

Purified, right side-out plasmalemma vesicles were isolated from 7-day-old roots of dark-grown wheat ( Triticum aestivum L. cv. Drabant) by aqueous polymer two-phase partitioning. The oxygen consumption by these vesicles at pH 6.5 in the presence of 1 m M NADH [12–29 nmol (mg protein)⁻¹min⁻¹] was 66% inhibited by 1 m M KCN and ca 40% by 1 m M EDTA. It was unaffected by rotenone, antimycin A, carbonyl cyanide trifluoromethoxyphenylhydrazone (FCCP), mersalyl, chlorotetracycline + Ca²⁺, and EGTA. Salicylhydroxamic acid (SHAM) and its analogue, m -chlorobenzhydroxamic acid, stimulated the rate of oxygen consumption 10–20 fold in the presence of 1 m M NAD(P)H with an apparent K_m (SHAM) of ca 40 μ M (with NADH). The dependence of O₂ consumption on NADH concentration in the presence of SHAM (2 m M ) was sigmoidal, possibly due to endogenous catalase activity, and half-maximal rate was obtained at 1.5 m M . In the absence of SHAM the rate increased with increasing acidity and no pH optimum was detectable between pH 4.5 and 8.5. In the presence of SHAM an optimum was observed at pH 6.5 and 0.8 mol of H₂O₂ was produced for every 1 mol O₂ consumed. Endogenous catalase converted this H₂O₂ to O₂ and after complete conversion the stoichiometry was 2 mol NADH consumed for every mol O₃. SHAM was not consumed in the reaction. The possible involvement of a cytochrome P-450/420 system is discussed.     

Does light-promoted export from Commelina benghalensis leaves result from differential light-sensitivity of the cells in the mesophyll-to-sieve tube path?

In contrast to the light-promoted uptake by mesophyll cells, light slightly inhibited sucrose uptake by stripped leaf disks of Commelina benghalensis L. This phenomenon appeared to result from a light-promoted vein-associated release, as light stimulated photosynthate release from stripped disks and inhibited that from mesophyll cells. In the -presence of the resorption-blocker p -chloromercuriphenylsulfonic acid, (PCMBS) the release of preloaded [¹⁴C]-sugars (sucrose, glucose) and [¹⁴C]-amino acids (alanine, asparagine, proline, valine, α-aminoisobutyric acid) from stripped disks was doubled in the light. Illumination enhanced by 20 to 60% the release of endogenous leaf cell compounds (sucrose, H₂PO-₄, K⁺, Mg²⁺, Ca²⁺) from stripped disks in the presence of PCMBS. Light also increased the export of [¹⁴C]-assimilates from intact leaves by 20% after pulse-labelling with ¹⁴CO₂. A model for loading is proposed, based on the differential light sensitivities of the plasma membranes in the mesophyll-to-sieve tube path.     

Salt-induced cooperativity in ATPase activity of plasma membrane-enriched fractions from cultured Citrus cells: kinetic evidence

ATPase activity was studied in plasma membrane-enriched fractions prepared from cultured Citrus sinensis L. cv. Osbeck cells. In general, properties of the plasma membrane ATPase from cultured cells, such as optimal pH and temperature. V_max and K_m were similar to those already observed in higher plants. The effects of high salt concentrations on ATPase activity were studied in membrane fractions derived from salt-sensitive and salt-tolerant cells grown in the presence or absence of salt. NaCl did not have an in vivo effect on V_max and the apparent K_m value for ATP. However, high concentrations of NaCl, or KCl, added in vitro, induced cooperativity in the enzyme and reduced the affinity of the enzyme for its substrate. Isoosmolar concentrations of sucrose or choline chloride failed to do so. Our results suggest that the plasma membrane ATPase of Citrus cells has more than one substrate-binding site on the native form of the enzyme which interact in the presence of salt and act independently in its absence.