Heterologous gene expression in Lactococcus lactis subsp. lactis: synthesis, secretion, and processing of the Bacillus subtilis neutral protease

The Bacillus subtilis nprE gene lacking its own promoter sequence was inserted in the lactococcal expression vector pMG36e. Upon introduction of the recombinant plasmid into Lactococcus lactis subsp. lactis strain MG1363, neutral protease activity could be visualized by the appearance of large clearing zones around colonies grown on milk agar plates. By measuring the activities of the neutral protease and the intracellular enzyme lactate dehydrogenase in culture supernatants and cell fractions, it was demonstrated that the neutral protease was actively secreted into the growth medium. This was corroborated by using the Western blot (immunoblot) technique, which showed the presence of the mature form of the neutral protease in the culture supernatant. On the basis of these results, it is concluded that the B. subtilis neutral protease gene was expressed in L. lactis and that the gene product was secreted into the growth medium and was apparently correctly processed to produced a biologically active protein. The secretion of this particular enzyme may be helpful in achieving accelerated cheese ripening.     

Construction of a lactococcal expression vector: expression of hen egg white lysozyme in Lactococcus lactis subsp. lactis.

A pair of vectors for expression of heterologous genes in Lactococcus lactis was constructed. In addition to an origin of replication that has a broad host range, these vectors contain a multiple cloning site flanked by gene expression signals originating from L. lactis subsp. cremoris Wg2. The two vectors, about 3.7 kilobase pairs in size, differ only in the type of antibiotic resistance they confer to their hosts. pMG36 carries a kanamycin resistance marker, which was replaced by an erythromycin resistance marker in pMG36e. As an example of the use of these vectors, the hen egg white lysozyme-coding sequence was inserted. A fusion protein of the expected size was detected in a transformed L. lactis subsp. lactis strain by using Western blotting (immunoblotting).     

Cloning and expression of a Streptococcus cremoris proteinase in Bacillus subtilis and Streptococcus lactis.

Previously, curing experiments suggested that plasmid pWV05 (17.5 megadaltons [Md]) of Streptococcus cremoris Wg2 specifies proteolytic activity. A restriction enzyme map of pWV05 was constructed, the entire plasmid was subcloned in Escherichia coli with plasmids pBR329 and pACYC184. A 4.3-Md HindIII fragment could not be cloned in an uninterrupted way in E. coli but could be cloned in two parts. Both fragments showed homology with the 9-Md proteinase plasmid of S. cremoris HP. The 4.3-Md HindIII fragment was successfully cloned in Bacillus subtilis on plasmid pGKV2 (3.1 Md). Crossed immunoelectrophoresis of extracts of B. subtilis carrying the recombinant plasmid (pGKV500; 7.4 Md) showed that the fragment specifies two proteins of the proteolytic system of S. cremoris Wg2. PGKV500 was introduced in a proteinase-deficient Streptococcus lactis strain via protoplast transformation. Both proteins were also present in cell-free extracts of S. lactis(pGKV500). In S. lactis, pGKV500 enables the cells to grow normally in milk with rapid acid production, indicating that the 4.3-Md HindIII fragment of plasmid pWV05 specifies the proteolytic activity of S. cremoris Wg2.     

Cloning and expression of a Streptococcus cremoris proteinase in Bacillus subtilis and Streptococcus lactis

Previously, curing experiments suggested that plasmid pWV05 (17.5 megadaltons [Md]) of Streptococcus cremoris Wg2 specifies proteolytic activity. A restriction enzyme map of pWV05 was constructed, the entire plasmid was subcloned in Escherichia coli with plasmids pBR329 and pACYC184. A 4.3-Md HindIII fragment could not be cloned in an uninterrupted way in E. coli but could be cloned in two parts. Both fragments showed homology with the 9-Md proteinase plasmid of S. cremoris HP. The 4.3-Md HindIII fragment was successfully cloned in Bacillus subtilis on plasmid pGKV2 (3.1 Md). Crossed immunoelectrophoresis of extracts of B. subtilis carrying the recombinant plasmid (pGKV500; 7.4 Md) showed that the fragment specifies two proteins of the proteolytic system of S. cremoris Wg2. PGKV500 was introduced in a proteinase-deficient Streptococcus lactis strain via protoplast transformation. Both proteins were also present in cell-free extracts of S. lactis(pGKV500). In S. lactis, pGKV500 enables the cells to grow normally in milk with rapid acid production, indicating that the 4.3-Md HindIII fragment of plasmid pWV05 specifies the proteolytic activity of S. cremoris Wg2.     

Dye permeability at phase transitions in single and binary component phospholipid bilayers

By encapsulating a pH-sensitive dye, phenol red, in multilamellar liposomes of DMPC, DPPC and DMPC/DPPC mixtures, the permeability of these phospholipid bilayers to dye as a function of temperature has been studied. For both DMPC and DPPC liposomes, dye release begins well below the main gel-to-liquid-crystalline phase transition (24°C and 42°C, respectively) at temperatures corresponding to the onset of the pretransition (about 14°C and 36°C, respectively) with DPPC liposomes exhibiting a permeability anomaly at the main phase transition (42°C). The perturbation occurring in the bilayer structure that allows the release of encapsulated phenol red (approx. 5 Å diameter) is not sufficient to permit the release of encapsulated haemoglobin (approx. 20 Å diameter, negatively charged). In liposomes composed of a range of DMPC/DPPC mixtures, dye release commences at the onset of the pretransition range (determined by optical absorbance measurements) and increases with increasing temperature until the first appearance of liquid crystalline phase after which no further dye release occurs. Interestingly, the dye retaining properties of DMPC and DPPC liposomes well below their respective pretransition temperature regions are very different: DMPC liposomes release much encapsulated dye at incubation temperatures of 5°C whilst DPPC liposomes do not.     

RADIOAUTOGRAPHY OF CHOLESTEROL IN LUNG : An Assessment of Different Tissue Processing Techniques

30 Swiss albino mice aged 8 days were injected intraperitoneally with 0.2 ml of a solution of 4% N,N-dimethyl-formamide in 5% dextrose in water containing cholesterol-1,2-³H (~1 mCi/ml). Lung tissue was embedded in an Epon mixture after either acetone and propylene oxide dehydration, partial ethanol and Epon 812 dehydration, or the precipitation of cholesterol by digitonin succeeded by partial dehydration. The distribution of cholesterol-1,2-³H in lung parenchyma in 1µ Epon section radioautograms was compared with that in frozen section radioautograms and was found to be independent of the manner of tissue processing. Grain distribution in the tissue was essentially the same whether 16, 63, 93, or 100% radioactivity was retained in the lung. However, grain distribution in the alveolar spaces differed, presumably due to displacement of pulmonary surfactant, which contains cholesterol. Intracellular distribution of cholesterol, in electron microscope radioautograms, was the same with either 51% or 93% retention of radioactivity in the lung. Loss of radioactivity into the various processing solutions was monitored. The various processing techniques have different drawbacks.     

Flesinoxan Dose-Dependently Reduces Extracellular 5-Hydroxytryptamine (5-HT) in Rat Median Raphe and Dorsal Hippocampus Through Activation of 5-HT1A Receptors

Abstract: The effects of systemic administration of the serotonin (5-hydroxytryptamine) 5-HT_1A receptor agonists flesinoxan and 8-hydroxy-2-(di- n -propylamino)tetralin on extracellular 5-HT were measured using microdialysis probes in both median raphe nucleus and dorsal hippocampus. Both 5-HT_1A agonists dose-dependently decreased dialysate 5-HT levels from both brain regions. The effects of flesinoxan in the median raphe (0.3 mg/kg) and dorsal hippocampus (1.0 mg/kg) could be blocked by the 5-HT_1A receptor antagonist N -[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]- N -(2-pyridyl)cyclohexane carboxamide trihydrochloride (WAY 100,635) at a dose of 0.05 mg/kg s.c. The antagonist itself had no effect at this dosage. Local perfusion of flesinoxan for 30 min through the dialysis probe into the median raphe region at concentrations of 20, 100, and 1,000 n M resulted in a significant decrease in dialysate 5-HT content from both regions. The effect of 100 n M flesinoxan could be blocked by coperfusion of 1,000 n M WAY 100,635. The data indicate that flesinoxan is a potent 5-HT_1A receptor agonist and also support the notion that somatodendritic 5-HT_1A autoreceptors regulate both terminal and somatodendritic 5-HT release.     

Microbes in food processing technology

Abstract: There is an increasing understanding that the microbial quality of a certain food is the result of a chain of events. It is clear that the microbial safety of food can only be guaranteed when the overall processing, including the production of raw materials, distribution and handling by the consumer are taken into consideration. Therefore, the microbiological quality assurance of foods is not only a matter of control, but also of a careful design of the total process chain. Food industry has now generally adapted quality assurance systems and is implementing the Hazard Analysis Critical Control Point (HACCP) concept. Rapid microbiological monitoring systems should be used in these cases. There is a need for rapid and simple microbiological tests which can be adapted to the technology and logistics of specific production processes. Traditional microbiological methods generally do not meet these high requirements. This paper discusses the tests, based on molecular biological principles, to detect and identify microbes in food-processing chains. Tests based on DNA technology are discussed, including in vitro DNA amplification like the polymerase chain reaction (PCR) method and identifications based on RFLP, RAPD and DNA fingerprinting analysis. PCR-haled methodology can be used for the rapid detection of microbes in food manufacturing environments. In addition, DNA fingerprinting methods are suitable for investigating sources and routes of microbial contamination in the food cycle.