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.     

Fluorescence banding in four species of Microtidae: an analysis of the evolutive changes of the constitutive heterochromatin

Three different fluorochrome and specific counterstain combination (DAPI/AMD, DA/DAPI and CMA/DA) treatments were applied to the chromosomes of four Microtidae (Rodentia) species. The results complete the data obtained in our previous paper (Burgos, M., Jiménez, R., & Dìaz de la Guardia, R., Genome 30:540–546, 1988) and prove that the changes in the constitutive heterochromatin in the evolution of the karyotypes of these species are not only due to gain or loss of heterochromatin, but are qualitative with respect to their nucleotide composition, repeated base pair organization or DNA-protein complex modification. These variations lead to the differential response to the fluorescence dye combinations used.     

Effects on efficiency in repetitive lifting of load and frequency combinations at a constant total power output.

Boxes were lifted and lowered repetitively at three different combinations of load and frequency. These combinations were chosen such that the total mechanical power generated was constant. Effects of the varying load or frequency conditions (but constant total mechanical power) on the rate of energy expenditure (M) and on the mechanical efficiency (ME) were measured. Mechanical power was determined from film analysis and separated into external power (generated to lift the load) and internal power (to raise the lifter's body mass). The M was determined from oxygen consumption measurements. The ME was calculated in two ways, depending on the definition of mechanical power, including either the external power only (MEext) or the total power output (MEtot). Despite a constant total mechanical power, M increased at higher loads and lower frequencies. This might be explained by the increasing isometric force required in postural and load control. The M increase resulted in a decrease of MEtot. However, at higher loads and lower frequencies MEext increased, indicating that more external work can be done at the same energy costs at higher loads or lower frequencies, which could be of interest from the point of view of occupational physiology. It would seem that at higher loads or lower frequencies the increased costs for isometric muscle action do not outweigh the benefit of raising the body less frequently. Furthermore, it was found that the MEext in lifting was much lower than the values reported for other kinds of activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prediction of the three-dimensional structure of the rap-1A protein from its homology to theras-gene-encoded p21 protein

rap-1A, an anti-oncogene-encoded protein, is aras-p21-like protein whose sequence is over 80% homologous to p21 and which interacts with the same intracellular target proteins and is activated by the same mechanisms as p21, e.g., by binding GTP in place of GDP. Both interact with effector proteins in the same region, involving residues 32–47. However, activated rap-1A blocks the mitogenic signal transducing effects of p21. Optimal sequence alignment of p21 and rap-1A shows two insertions of rap-1A atras positions 120 and 138. We have constructed the three-dimensional structure of rap-1A bound to GTP by using the energy-minimized three-dimensional structure ofras-p21 as the basis for the modeling using a stepwise procedure in which identical and homologous amino acid residues in rap-1A are assumed to adopt the same conformation as the corresponding residues in p21. Side-chain conformations for homologous and nonhomologous residues are generated in conformations that are as close as possible to those of the corresponding side chains in p21. The entire structure has been subjected to a nested series of energy minimizations. The final predicted structure has an overall backbone deviation of 0.7 å from that ofras-p21. The effector binding domains from residues 32–47 are identical in both proteins (except for different side chains of different residues at position 45). A major difference occurs in the insertion region at residue 120. This region is in the middle of another effector loop of the p21 protein involving residues 115–126. Differences in sequence and structure in this region may contribute to the differences in cellular functions of these two proteins.     

Expression patterns of loricrin in various species and tissues

Abstract. In this study we analyzed the expression patterns of loricrin in various species and tissues using immunohistochemistry, immunoblotting and Northern blots. Loricrin is a glycine-, serine- and cysteine-rich protein expressed very late in epidermal differentiation in the granular layers of normal mouse and human epidermis. Later on in differentiation, loricrin becomes cross-linked as a major component into the cornified cell envelope by the formation of N^ɛ -(γ-glutamyl)lysine isopeptide bonds. This process either occurs directly or by the intermediate accumulation in L-keratohyaline granules of mouse epidermis and human acrosyringia. Loricrin was identified in all mammalian species analyzed by virtue of its highly conserved carboxy-terminal sequences revealing an electric mobility of ∼60 kDa in rodents, rabbit and cow and of ∼35 kDa in lamb and human on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Loricrin is expressed in the granular layer of all mammalian orthokeratinizing epithelia tested including oral, esophageal and fore-stomach mucosa of rodents, tracheal squamous metaplasia of vitamin A deficient hamster and estrogen induced squamous vaginal epithelium of ovary ectomized rats. Loricrin is also expressed in a few parakeratinizing epithelia such as BBN [N-butyl-N-(4–hydroxybutyl)nitrosamine]-induced murine bladder carcinoma and a restricted subset of oral and single vaginal epithelial cells in higher mammals. Our results provide further evidence that the program of squamous differentiation in internal epithelia of the upper alimentary tract in rodents and higher mammals differ remarkably. In addition, we also have noted the distinct distribution patterns of human loricrin and involucrin, another major precursor protein of the cornified cell envelope.     

Behavioral laterality in captive bonobos (Pan paniscus): Replication and extension

We report observational data on behavioral laterality in 10 captive bonobos (Pan paniscus)at the San Diego Zoo. The unimanual measures include carrying, leading limb in locomotion, self-touching, face-touching, reaching, and gestures. We also recorded bimanual feeding in these subjects. A significant population level left-hand bias exists for carrying. Right-hand biases occur for leading limb in locomotion and gestures. During bimanual feeding, the bonobos hold food items with the left hand while feeding with the right hand. Overall, bonobos exhibit behavioral asymmetries that are similar to previous findings in other pongid ape species. The asymmetries in gestures and bimanual feeding represent novel findings with theoretical implications for the origins of tool use and language.     

Antitumour activities of levans produced by Zymomonas mobilis strains

Levans produced by four Zymomonas mobilis strains showed antitumour activity against sarcoma 180 and Ehrlich carcinoma in Swiss albino mice. Levans from two strains (ZAP and CP4) had the highest effects. NMR analysis showed that the polymers were composed only of fructose units. The results suggested that the antineoplasic effect is associated to the polysaccharide molecular weight and that a particular molecular weight range may be responsible for this effect.