Trans-Epithelial Transport,Metabolism, and Biological Activity Assessment of the Multi-Target Lupin Peptide LILPKHSDAD (P5) and Its Metabolite LPKHSDAD (P5-Met)

P5 (LILPKHSDAD) is a hypocholesterolemic peptide from lupin protein with a multi-target activity, since it inhibits both 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCoAR) and proprotein convertase subtilisin/kexin type-9 (PCSK9). This work shows that, during epithelial transport experiments, the metabolic transformation mediated by intestinal peptidases produces two main detected peptides, ILPKHSDAD (P5-frag) and LPKHSDAD (P5-met), and that both P5 and P5-met are linearly absorbed by differentiated human intestinal Caco-2 cells. Extensive comparative structural, biochemical, and cellular characterizations of P5-met and the parent peptide P5 demonstrate that both peptides have unique characteristics and share the same mechanisms of action. In fact, they exert an intrinsically multi-target behavior being able to regulate cholesterol metabolism by modulating different pathways. The results of this study also highlight the dynamic nature of bioactive peptides that may be modulated by the biological systems they get in contact with.     

High Frequency Hearing Sensitivity in Adolescent Females of a Lower Socioeconomic Status Over a Period of 24 Years (1985–2008)

PurposeTo examine annually over a period of 24 years, the high frequency hearing sensitivity in different groups of urban female adolescents with a low socioeconomic status (SES) and residential foster care.MethodsHearing screening (15 decibel [dB] hearing level ranging from 1,000 to 8,000 Hertz [Hz]) and threshold (>15 dB hearing level) records were obtained from 8,710 female adolescents (mean age, 15.8 years [range, 12–20 years]), predominantly Hispanic and African American from households with a low SES. Data related to the use of personal listening devices (PLDs), daily hours of usage, occurrence of tinnitus, and hearing thresholds between 1,000 and 8,000 Hz over an 8-year period (2001–2008) were obtained from the adolescents.ResultsHigh frequency hearing loss (HFHL) doubled over the 24-year period from 10.1% in 1985 to 19.2% in 2008. In comparison with the general adolescent population, this group of female adolescents presented with a higher percentage of bilateral mild or greater degrees of HFHL at two or more frequencies including 3,000, 4,000, and 6,000 Hz. Use of PLDs increased four-fold, from 18.3% (n = 68) in 2001 to 76.4% (n = 227) in 2008. Of the total number reporting tinnitus (n = 286), 99.7% (n = 285) also reported regular PLD use. A significant relationship was found between PLD use and reported tinnitus and HFHL irrespective of time of use of PLD.ConclusionsIncreased incidence of HFHL, reported tinnitus, PLD use, and hours of daily use in at-risk female adolescents of a low SES was found. A frequency interval of 3,000–6,000 Hz should be included in hearing screening protocols to identify potentially disabling hearing loss. Hearing conservation strategies need to be developed and/or modified that target and reach at-risk children and adolescents.     

Intracellular zinc stores protect the intestinal epithelium from Ochratoxin A toxicity

Ochratoxin A (OTA) is a harmful mycotoxin frequently contaminating foods, feeds and beverages. OTA was reported to be nephrotoxic, immunotoxic, hepatotoxic and a potential carcinogen, with yet poorly characterized mechanisms. Although intestinal cells are relatively resistant to high concentrations of OTA, interaction with other dietary factors or specific nutritional conditions may increase OTA toxicity to the intestinal mucosa. The role of intracellular zinc stores in protecting the integrity of intestinal mucosa has been investigated in human Caco-2/TC7 cells challenged with OTA. Zinc depletion of cells incubated with TPEN, a specific zinc chelator, caused an increase of tight junction permeability in OTA treated cells, accompanied by increased apoptosis. These effects were fully reverted by zinc supplementation during TPEN treatment, showing a specific role for this micronutrient in enterocyte defence mechanisms from OTA toxicity. A complex perturbation of zinc homeostasis was also demonstrated by analyzing the expression of genes coding for proteins involved in cellular zinc. In particular, zinc-dependent up-regulation of the metallothionein gene MT2A upon OTA treatment may indicate that the mycotoxin acts through generation of redox imbalance and that zinc deprivation reduces the intracellular defence mechanisms against noxious insults.     

Epithelial cells in culture as a model for the intestinal transport of antimicrobial agents.

The bioavailabilities of orally administered drugs depend to a great extent on their capability of being transported across the intestinal mucosa. In an attempt to develop an in vitro model for studying the intestinal transport of drugs, we used an intestinal epithelial cell line (Caco 2) derived from a human colon adenocarcinoma. A renal epithelial cell line (MDCK) was also used to determine the tissue specificity of drug transport. These cell lines, which were grown on filters, form a monolayer of well-polarized cells coupled by tight junctions and can be used for transcellular transport experiments. We studied the transport of nine antimicrobial agents with different physicochemical and pharmacokinetic characteristics using these epithelial cell monolayers to determine whether this model could be predictive of oral bioavailability. The transepithelial passage was assayed from the apical (AP) to the basolateral (BL) side and in the opposite direction (BL to AP) in both cell lines. Radioactively labeled mannitol was used to monitor the intactness of the cell monolayer during drug passage. The results indicated that all antimicrobial agents tested tended to behave in vitro generally according to their known in vivo absorptive characteristics. In addition, the use of epithelia from different tissues enabled us to divide the drugs into four groups according to their behaviors and suggested the existence of different transport mechanisms. In particular, two antibiotics, gentamicin and teicoplanin, showed no passage in either direction or cell line, in accordance with their very poor in vivo absorbances after oral administration. In contrast, rifapentine, rifampin, and nalidixic acid passed very efficiently at similar rates in both directions and cell lines in a concentration-dependent, nonsaturable manner, which is suggestive of passive diffusion down a concentration gradient. Of the remaining drugs, isoniazid and novobiocin sodium showed some differences in passage between the two cell lines and, given their ionized state at the pH that was used, may use the paracellular route. Finally, trimethoprim and D-cycloserine exhibited differences in passage both with respect to polarity and cell line; in particular, trimethoprim had a faster rate of passage only in Caco 2 cells and in the BL to AP direction, while D-cycloserine was exclusively transported by Caco 2 cells in the AP to BL direction. In both cases it is possible that active transport mechanisms are involved.     

Characterization of the mucins produced by normal human colonocytes in primary culture

Mature goblet cells filled with mucin ready for secretion represent about one third of the cells in primary cultures of human colonocytes. In the present study characterization of the mucins produced by cultured human colonocytes was made by histochemical methods by lectin and monoclonal antibody binding. Two monoclonal antibodies and three lectinsDolichos biflorus (DBA),Helix pomatia (HPA) andArachis hypogea (PNA) recognizing epitopes or sugar haptens characteristic of different stages of mucin glycoprotein maturation, were employed. The reacivity to these probes was tested both on cultured colonocytes and on tissue sections of the normal colon mucosa. The results show that the mucins produced in culture are glycosylated to the mature form, as they show the same reactivity to lectins and antibodies of the mucins expressed in tissue sections of the normal colon mucosa. In addition, it is demonstrated that cultured human colonocytes do not express mucins reactive to PNA, which are characteristic of tumors. Since the cultured colonocytes maintain the expression of differentiated functions for at least three days, they may offer a useful model to study metabolism, function and regulation of colon mucins in health and disease.

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Résumé Des cellules caliciformes matures remplies de mucine prêtes à la sécrétion constituent environ 1/3 de toutes les cellules dans des cultures primaires de colonocytes humains. Au cours de l'étude présente, les mucines produites par des cultures de colonocytes humains ont été typisées par des méthodes histochimiques, par liaison à la lectin et à des anti-corps monoclonaux. Deux anti-corps monoclonaux et trois types de lectin Dolichos biflorus (DBA), Helix pomatio (HPA) et Arachis hypogea (PNA) susceptibles de reconnaïtre les épitopes ou les haptènes d'hydrate de carbone caractéristiques de différentes phases de la maturation des glycoprotéines des mucines ont été emplyés. Les réactivités à ces substances ont été testéees à la fois sur des cultures de colonocytes et sur des segments de tissu muqueux coliques normaux. Les résultats montrent que les mucines produites en culture sont glycosylées dans les formes matures car elles présentent la même réactivité aux lectins et aux anti-corps des mucines tels que mesurés dans des segments de tissue de muqueuse colique normale. De plus, on a démontré que les colonocytes humains cultivés ne produisent pas de mucine réactive au PNA qui sont caractéristiques des tumeurs. Etant donné que les colonocytes humains cultivés conservent l'expression d'une différenciation des fonctions pour au moins trois jours, ils peuvent constituer un modèle utile pour étudier le métabolisme de la fonction et de la régulation des mucines coliques chez le sujet sain et pathologique.

     

Ultrastructural studies of spontaneous in vitro transformation of cultured marrow monocyte-macrophage cells from a patient with congenital hypoplastic anemia

The CM-S cell line was established from the bone marrow of a patient suffering from congenital hypoplastic anemia (syndrome of Diamond-Blackfan). The cells grew in suspension in liquid culture and were dependent for their continuous replication in vitro on growth factors produced by the same cells seeded at high density. Initially, undifferentiated blasts, immature myeloid, megakaryocytic and, rarely, erythroid cells were observed. Eventually, a population of cells with characteristics of monocyte-macrophage precursors predominated. These cells could be induced to terminal macrophage differentiation by incubation with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate. During this period (over 150 continuous passages), the cells failed to form colonies in agar and to give rise to tumors when inoculated into athymic mice. On prolonged passages, however, the cells gradually increased their growth capacity in liquid culture and became capable of forming colonies in agar and tumors in animals. Ultrastructural studies revealed that the expression of differentiated traits markedly changed as a function of time: after 277 passages, the transformed cells, although displaying characteristics of monocyte precursors, appeared blocked at this stage and no longer responded to 12-O-tetradecanoylphorbol-13-acetate.     

The effects of in vitro ageing on the composition of the intracellular free amino acid pool of human diploid fibroblasts

The composition of the free amino acid pool of the human diploid fibroblast cell line, BCL-D1, was analysed by automated ion-exchange chromatography throughout the life-span of the culture. Significant age-related changes, independent of growth effects, were found with six essential amino acids: a decrease in tyrosine, phenylalanine, leucine, isoleucine and valine, and an increase in methionine. The nature of the changes were suggestive of a decline in the operation of the L-system of amino acid transport with in vitro age. However, it is also possible that age-related changes in the proportions of tRNA iso-acceptors and/or the availability and accuracy of tRNA synthetases were involved.     

Placental expression of ceruloplasmin in pregnancies complicated by severe preeclampsia

There is consensus that ischemia/reperfusion injury associated with preeclampsia (PE) promotes both placental damage and the release of factors leading to maternal endothelium dysfunction, a hallmark of this potentially life-threatening syndrome. These factors include plasminogen activator inhibitor-1 (PAI-1) and soluble fms-like tyrosine kinase-1 (sFlt-1). The goal of this study was to further characterize placental factors involved in the pathophysiology of PE. Thus, DNA microarray gene profiling was utilized to identify mRNA differentially regulated in placentas from women with severe PE compared to both preterm (PC) and term control (TC) groups. Microarray studies detected an upregulation of mRNA for ceruloplasmin, a copper-containing iron transport protein with antioxidant ferroxidase properties, in PE compared to PC and TC placentas, respectively. Quantitative real-time PCR confirmed these results by demonstrating significant increases in ceruloplasmin mRNA in PE vs PC and TC placentas. Supporting previous reports, the expression of sFlt-1 and PAI-1 were also upregulated in PE placentas. Immunohistochemistry localized ceruloplasmin to the intervillous space in PE and PC placentas, whereas stronger syncytial staining was noted in PE. Western blotting confirmed a significant increase in ceruloplasmin levels in placental tissue in PE compared to PC groups. PCR identified the presence of mRNA for ceruloplasmin in primary cultures of syncytiotrophoblasts, but not villous-derived fibroblasts, suggesting that syncytium is the site of ceruloplasmin synthesis in placenta. Hypoxic treatment (1% O(2)) of syncytiotrophoblasts enhanced levels of ceruloplasmin mRNA approximately 25-fold, a significantly greater upregulation than that noted for PAI-1 and sFlt-1, suggesting that enhanced ceruloplasmin expression is a sensitive marker of syncytial hypoxia. We suggest that syncytial ceruloplasmin and its associated ferroxidase activity, induced by the hypoxia accompanying severe PE, is important in an endogenous cellular program to mitigate the damaging effects of subsequent reperfusion injury at this site.     

Cytotoxic activity of human lymphocytes against differentiated intestinal tumour cell lines.

The natural killer (NK) and lymphokine-activated killer (LAK) cytotoxic activity of human peripheral blood lymphocytes (PBL) against various human tumour cell lines from intestinal origin (WIDR, HT29, Caco-2) has been investigated. The differentiated Caco-2 cells were then used as a model to investigate the cytotoxic activity against enterocyte-like target cells. Caco-2 were seeded on polycarbonate filters and maintained in culture for at least 15 days to allow the differentiation and formation of tight junctions. The integrity of tight junctions was assayed by measuring [3H]mannitol flux from apical to basolateral compartment. Cytotoxic analysis showed that both differentiated and undifferentiated Caco-2 cells were similarly susceptible to NK and LAK activity. The capacity of cytotoxic lymphocytes to kill enterocyte-like cells with intact junctional complex may suggest a direct role of cytotoxic lymphocytes in causing intestinal lesions under inflammatory conditions.