A new principle for tight junction modulation based on occludin peptides

The aim of this study was to investigate whether peptides from the extracellular loops of the tight junction protein occludin could be used as a new principle for tight junction modulation. Peptides of 4 to 47 amino acids in length and covering the two extracellular loops of the tight junction protein occludin were synthesized, and their effect on the tight junction permeability in Caco-2 cells was investigated using [14C]mannitol as a para-cellular marker. Lipopeptide derivatives of one of the active occludin peptides (OPs), synthesized by adding a lipoamino acid containing 14 carbon atoms (C14-) to the N terminus of the peptide, were also investigated. Peptides corresponding to the N terminus of the first extracellular loop of occludin increased the permeability of the tight junctions without causing short-term toxicity. However, the peptides had an effect only when added to the basolateral side of the cells, which could be partly explained by degradation by apical peptidases and aggregate formation. By contrast, the lipopeptide C14-OP90-103, which protects the peptide from degradation and aggregation, displayed a rapid apical effect. The l- and d-diastereomers of C14-OP90-103 had distinctly different effects. The d-isomer, which releases intact OP90-103 from the lipoamino acid, displayed a rapid and transient increase in tight junction permeability. The l-isomer, which releases OP90-103 more rapidly, gave a more sustained increase in tight junction permeability. In conclusion, C14-OP90-103 represents a prototype of a new class of tight junction modulators that act on the extracellular domains of tight junction proteins.     

Defective gap junctional intercellular communication in lung cancer: loss of an important mediator of tissue homeostasis and phenotypic regulation

Gap junctions provide direct pathways for the exchange of molecules and ions between neighboring cells, a process known as gap junctional intercellular communication (GJIC). This GJIC is important for homeostasis and regulation of mitosis, differentiation, and apoptosis. Gap junctions are present in lung airway and alveolar epithelial cells and, in addition to the above roles, might coordinate ciliary beating and surfactant secretion. GJIC is decreased in human and mouse lung carcinoma cells because of reduced expression of the gap junction protein, connexin43 (Cx43), and defects in signal transduction pathways that mediate Cx43 function. This reduced GJIC is important in the behavior of lung carcinoma cells because forced expression of Cx43 in lung carcinoma cells inhibits their growth and tumorigenicity. In this report, we summarize our studies on the role of GJIC in lung neoplasia.     

NAD(P)H:quinone oxidoreductase (DT-diaphorase) activity and mRNA content in normal and neoplastic mouse lung epithelia

DT-diaphorase (DTD) is a flavoprotein which catalyzes obligate two-electron reduction of a diverse group of substrates. We have reported previously that non-tumorigenic mouse lung alveolar type-II pneumocytes have high DTD activity, while cell lines derived from lung tumors do not. In contrast, other investigators, using human lung tissue, reported increased DTD activity in tumors compared with normal tissue. We therefore investigated DTD associated with mouse lung neoplasia in vivo as well as in vitro. Pulmonary tumors had far less DTD activity compared with normal mouse lung. Correspondingly, a tumorigenic mouse lung cell line which arose as a spontaneous transformant of a normal cell line had very low DTD activity compared with non-tumorigenic lung cells. DTD-specific mRNA levels were also much higher in normal cell lines than in neoplastic ones. DTD was localized histochemically in type-II pneumocytes in situ, but was not observed by this technique in normal bronchiolar epithelia or in tumor cells. These data show that, unlike what has been observed in human lung cancer, a marked decrease in DTD content and activity accompanied mouse lung tumorigenesis in vivo and neoplastic transformation in vitro.     

Effects of adrenalectomy and corticosterone administration on mouse lung tumor susceptibility and histogenesis

The effects of adrenalectomy (Ax) on urethan-induced lung tumors were determined in strains of mice that vary in their respective tumor susceptibilities: A/J (sensitive), BALB/cByJ (intermediate), and C57BL/6J (B6, resistant). Ax increased tumor number in both A/J (by 25%) and B6 mice (by 400%), but not in BALB/cByJ mice. The relative proportions of adenomas exhibiting the alveolar or papillary histological growth patterns were unchanged. Implantation of corticosterone-containing pellets into adrenalectomized B6 mice restored tumor multiplicity to that of sham-operated mice and into adrenalectomized A/J mice reduced multiplicity below that of sham-operated mice. Corticosterone, therefore, regulates neoplastic development of mouse lung epithelial cells.     

Role of inflammation in mouse lung tumorigenesis: a review

Peritumoral and intratumoral macrophages are associated with human and mouse lung cancer The mouse model allows manipulation of the macrophage population to experimentally evaluate its contribution to tumor growth. Genetic and pharmacologic strategies also permit testing the invol vement of specific inflammatory mediators in tumor progression. Among those endogenous mediators thus identified are interleukin (IL)-10, glucocorticoids, prostacyclin, nitric oxide, and surfactant apoprotein D (SP-D); serum SP-D levels are a useful biomarker to monitor tumor growth rate. The importance of understanding the mutually antagonistic roles of individual prostaglandins downstream from cycloxygenase (COX) and how this affects the efficacy of COX-inhibitory drugs is discussed. Promising drug candidates include synthetic glucocorticoids such as budesonide and the sulfone derivative of sulindac, apotosyn.     

Effects of neutron radiation on mouse hair matrix cell populations.

We have investigated the effects of a single low dose of 42 rads neutron radiation on hair matrix cell kinetics. Anagen hair skin sites in 3-mo-old Carworth Farms no. 1 female mice were exposed to a fast neutron beam yielding a modal energy of 3.6 MeV. In contrast to low LET (linear energy transfer) radiation at a 2 1/2 fold higher dose, neutrons induced a markedly prolonged depression (greater than 60%) in the mitotic index for more than 18 hr postradiation. In addition, from 10--18 hr after neutron irradiation sharp elevations (greater than 80%) in the labeling indices apparently reflected significant impairment in cell maturation rates and/or slowed exit rates from the proliferative compartments. Postradiation reductions of up to 20% in the size of proliferating matrix cell populations were found. At the cellular level, this study confirms previous findings of a disproportionately high relative biological effectiveness for neutron radiation following low dose level exposure.     

Functional changes in the regulatory subunit of the type II cyclic adenosine 3':5'-monophosphate-dependent protein kinase isozyme during normal and neoplastic lung development

The abilities of cyclic adenosine 3':5'-monophosphate (cAMP) and cyclic 8-azidoadenosine 3':5'-[32P]monophosphate (8-N3-[32P]cAMP) to bind to the regulatory subunit (RII) of the type II cAMP-dependent protein kinase isozyme and to cause subsequent dissociation of the holoenzyme were compared in extracts from adult and neonatal mouse lung and lung adenoma. RII in extracts from adult lung exhibits equal numbers of high- (Kd 15 nM) and low- (Kd 230 nM) affinity 8-N3-[32P]cAMP binding sites. In the neonate, the proportion of high-affinity sites is reduced to 20% while, in lung adenoma, only low-affinity RII binding is observed. Low-affinity RII binding is correlated with an inability of cAMP to dissociate the type II holoenzyme completely. Sucrose gradient sedimentation of adult lung cytosol in the presence of cAMP shows complete dissociation of the type I isozyme, while only some of the type II holoenzyme is dissociated. This is in contrast to the case with lung tumor cytosol, in which only low-affinity binding is observed and no apparent dissociation of the type II isozyme occurs. cAMP does promote RII dephosphorylation within the holoenzyme, however, suggesting that cAMP can bind to RII without dissociating the tetramer. Consistent with this interpretation, photoincorporation of 8-N3-[32P]cAMP prior to sucrose gradient sedimentation results in the formation of a photolabeled RII complex which sediments at the same rate as does the holoenzyme. Two-dimensional gel electrophoresis of RII photolabeled at low and high concentrations of 8-N3-[32P]cAMP suggests that these altered binding and dissociation characteristics of the type II isozyme are not due to the presence of a structurally altered RII molecule. After DEAE-cellulose chromatography of lung cytosol, only high-affinity RII binding is observed, and all of the RII can now be dissociated with cAMP. Low-affinity binding may thus reflect either an altered conformational state of RII or the interaction of the type II kinase with other cytosolic molecules which can affect RII binding and dissociation without altering the functional properties of the type I isozyme.     

A simple multi-purpose cannula system for access to the brain and/or systemic vascular system of unanesthetized animals

By utilizing a pedestal system mounted on the skull containing a cerebral guide cannula with hub, and a vascular catheter which is exteriorized within the pedestal, an easily made, multi-purpose system has been developed using materials which are available from any scientific or medical supplier. This technique is adaptable for use in a wide variety of animal species and can be used in unanesthetized or anesthetized preparations.