Development of immortalized human cerebromicrovascular endothelial cell line as an in vitro model of the human blood-brain barrier

The objective of this study was to generate an immortal cell line representative of specialized human brain microvascular endothelia forming the blood-brain barrier (BBB) in vivo. Human capillary and microvascular endothelial cells (HCEC) were transfected with the plasmid pSV3-neo coding for the SV40 large T antigen and the neomycin gene. The neomycin-resistant transfected cells overcame proliferative senescence, and after a 6-8 wk period of crisis produced immortalization-competent cell colonies. Single-cell clones of near-diploid genotype were isolated from these colonies, propagated, and characterized. Immortalized HCEC (SV-HCEC) exhibited accelerated proliferation rates, but remained serum and anchorage dependent and retained the characteristic cobblestone morphology at confluence. SV-HCEC displayed a stable nuclear expression of SV40 large T antigen, lacked the invasiveness of transformed cells, and maintained major phenotypic properties of early passage control cells including expression of factor VIII-related antigen, uptake of acetylated low-density lipoprotein, binding of fluorescently labeled lectins, expression of transferrin receptor and transferrin receptor-mediated endocytosis, and high activities of the BBB-specific enzymes alkaline phosphatase and gamma-glutamyl transpeptidase. The diffusion of radiolabeled sucrose across SV-HCEC monolayers was fivefold lower than that observed with human lung microvascular endothelial cells. Furthermore, media conditioned by fetal human astrocytes increased the transendothelial electrical resistance of SV-HCEC monolayers by 2.5-fold. Therefore, this newly established human cell line expressing the specialized phenotype of BBB endothelium may serve as a readily available in vitro model for studying the properties of the human BBB.     

A single position in the third transmembrane domains of the human B1 and B2 bradykinin receptors is adjacent to and discriminates between the C-terminal residues of subtype-selective ligands

In order to identify agonist- and antagonist-binding epitopes in the human B1 and B2 bradykinin (BK) receptors, we exploited the ability of these receptors to discriminate between peptide ligands that differ only by the absence (B1) and presence (B2) of a C-terminal Arg. This was done by constructing chimeric proteins in which specific domains were exchanged between these receptors as recently described by us (Leeb, T., Mathis, S. A., and Leeb-Lundberg, L. M. F. (1997) J. Biol. Chem. 272, 311-317). The constructs were then expressed in HEK293 and A10 cells and assayed by radioligand binding and by agonist-stimulated inositol phospholipid hydrolysis and intracellular Ca2+ mobilization. Substitution of the third transmembrane domain (TM-III) of the B1 receptor in the B2 receptor (B2(B1III)) dramatically reduced the affinities of B2-selective peptide ligands including both the agonist BK and the antagonist NPC17731. High affinity binding of both ligands to B2(B1III) was fully regained when one residue, Lys111, in TM-III of this chimera was replaced with the corresponding wild-type (WT) B2 receptor residue, Ser (B2(B1IIIS111)). Replacement of Ser111 with Lys in the WT B2 receptor decreased the affinities of BK and NPC17731 and increased the affinity of the B1-selective des-Arg10 analog of NPC17731, NPC18565. The results show that the C-terminal residue of peptide agonists and antagonists when bound to the B2 receptor is adjacent to Ser111 in the receptor. A Lys at this position, as is the case in the WT B1 receptor, provides a positive charge that repels the C-terminal Arg in B2-selective peptides and attracts the negative charge of the C terminus of B1-selective peptides, which lack the C-terminal Arg. Therefore, the residues at this one single position are crucial in determining the peptide selectivity of B1 and B2 BK receptors.     

Antagonistic activity of Lactobacillus plantarum C11: two new two-peptide bacteriocins, plantaricins EF and JK, and the induction factor plantaricin A

Six bacteriocinlike peptides (plantaricin A [PlnA], PlnE, PlnF, PlnJ, PlnK, and PlnN) produced by Lactobacillus plantarum C11 were detected by amino acid sequencing and mass spectrometry. Since purification to homogeneity was problematic, all six peptides were obtained by solid-phase peptide synthesis and were tested for bacteriocin activity. It was found that L. plantarum C11 produces two two-peptide bacteriocins (PlnEF and PlnJK); a strain-specific antagonistic activity was detected at nanomolar concentrations when PlnE and PlnF were combined and when PlnJ and PlnK were combined. Complementary peptides were at least 10(3) times more active when they were combined than when they were present individually, and optimal activity was obtained when the complementary peptides were present in approximately equal amounts. The interaction between complementary peptides was specific, since neither PlnE nor PlnF could complement PlnJ or PlnK, and none of these peptides could complement the peptides constituting the two-peptide bacteriocin lactococcin G. Interestingly, PlnA, which acts as an extracellular signal (pheromone) that triggers bacteriocin production, also possessed a strain-specific antagonistic activity. No bacteriocin activity could be detected for PlnN.     

World trends in infant feeding

Analysis of the dyadic nature (nutritional, psychological, and biological interaction between mother and infant with each affecting the other) and the economics of infant feeding strongly suggests the superiority of breastfeeding over artificial feeding, regardless of the socioeconomic status of societies. Universally, there are 3 main guidelines for scientifically guided biological infant feeding: 1) to feed the pregnant and lactating mothers with a mixed diet of locally available foods; 2) to breastfeed alone for 4 to 6 months, and 3) to introduce least cost weaning foods based on the concept of multimixes from 4 to 6 months onward, preferably prepared from locally available foods but with continuing lactation into the second year of life, particularly in poorer circumstances. Widespread increases in breastfeeding will protect some 10 million infants annually against marasmus and diarrheal diseases, some 1 million cases against infantile obesity and 100,000 cases against cow's milk allergy. The basic issue involved is the development of practical programs which would improve the pattern of breastfeeding or at least minimize its decline, and to optimize the quantity and quality of milk production, particularly by maternal feeding. This can be done by instituting changes in the emphasis in the education of health professionals, and in procedures at pediatric wards and maternity units in hospitals. The promotion and advertising of infant formulas, especially in poor areas, needs monitoring, and appropriate legislation and enforcement. Lactating mothers who are working should be provided with bonuses, creches, 'nursing pauses' in industry, and other methods which will encourage them to combine the roles of motherhood and salaried worker effectively. Interdisciplinary undertakings between economists, administrators and health professionals are needed in order to improve breastfeeding patterns on a community basis. Reappraisal of modern infant feeding practices should be based on modern scientific knowledge, awareness of successful traditional time-tested adaptation, and on man's ancient biological mammalian heritage.     

Fluorescent brighteners: novel stains for the flow cytometric analysis of microorganisms

Genetic damages are frequently found in both tumor and normal cells at carcinogen exposed areas in the patients with upper aerodigestive tract cancer. These phenomena are explained by the multistage process and/or field cancerization theories. The c-erbB-2 proto-oncogene has been amplified in many human tumors including breast, stomach, kidney and lung cancers. To study the possible evidence of multistage process and/or field cancerization in the development of gastric adenocarcinoma, the amplification statuses of c-erbB-2 proto-oncogene using the Southern hybridization technique were evaluated at the 45 gastric adenocarcinoma specimen sets consisting of tumor tissue, adjacent normal tissue (within 2 cm of the primary tumor), metastatic tissue and normal stomach tissue (at least 5 cm away from primary tumor). As a result, c-erbB-2 proto-oncogene at 2 specimen sets (4.4%) was amplified 2- to 4-fold to normal control status. In these 2 cases, c-erbB-2 proto-oncogene at histologically normal tissue adjacent to tumor tissue was amplified. And, the metastatic tissue of 1 case also exhibited c-erbB-2 proto-oncogene amplification of which the degree was less than that of tumor tissue. From these results, we were able to suspect that c-erbB-2 proto-oncogene amplification in the normal tissue adjacent to tumor tissue could be a biomarker of premalignant changes in a small proportion of gastric adenocarcinoma patients. And, this result might suggest the possible role of multistage process and/or field cancerization in the development of gastric adenocarcinoma.