Short communication: Lysine retained among 2 lipid-coated lysine products after exposure to alfalfa or corn silage with different amounts of acidity.

We conducted 2 experiments to determine lysine loss from 2 lipid-coated lysine products after mixing with silage. In our first experiment, we mixed 2 lipid-coated lysine products, crystalline lysine or crystalline lysine and amounts of lipid identical to amounts included in lipid-coated lysine products, with alfalfa or corn silage that had 2 different amounts of acidity. Lysine appeared to disassociate from lipid-coated lysine products in a nonlinear manner after mixing with either alfalfa or corn silage at different amounts of acidity. Additionally, silage source and acidity affected amounts of lysine released from lipid-coated lysine products after mixing. In a corresponding experiment, in vitro estimates of lysine available to ruminal microbiota after mixing with alfalfa or corn silage at different amounts of acidity were measured by ammonia release. In vitro measures were conducted with or without monensin to allow estimates of effects of monensin on amounts of lysine released from the 2 lipid-coated lysine products. It is unclear whether in vitro estimates of lysine fermentation from lipid-coated lysine are truly reflective of ruminal degradation of lysine from lipid-coated lysine because amounts of time needed to measure differences between different lysine sources were greater than typical estimates of mean ruminal particulate retention time. Nonetheless, monensin apparently reduced ammonia release from lysine, but ammonia release from lipid-coated lysine did not differ from crystalline lysine. Clearly, methods of manufacture together with physical and chemical characteristics of diet can affect amounts of lysine provided from lipid-coated lysine products to ruminants.

Development and clinical evaluation of a new sensor design for buccal pulse oximetry in horses.

BACKGROUND: The use of pulse oximetry in horses is limited due to inadequate readings with conventional transmission sensor probes. OBJECTIVES: The objectives of this study were to 1) develop an improved sensor design for horses to be used at an appropriate anatomical site, and 2) evaluate this design in an experimental study. STUDY DESIGN: In vivo experiment. METHODS: A new sensor design for reflectance pulse oximetry at the buccal mucosa was developed. A conventional Nonin 2000SL sensor for transmission pulse oximetry was included into this design. Three different prototypes (N1, N2a, N2b) were constructed and used with the Nonin 2500A Vet pulse oximetry monitor. Thirteen anaesthetised warmblood horses were included into a desaturation protocol (100-70% SaO2 ). SpO2 and pulse frequency values were recorded, using SaO2 calculated from blood gas analysis and invasive pulse frequency measurements as reference methods. Bias and precision were evaluated by calculations of the root mean square deviation (Arms ). The agreement of the methods was tested with Bland-Altman analysis. RESULTS: The quality of the pulse frequency readings determined the quality of the SpO2 -readings. Good pulse signal strength resulted in a SpO2 -accuracy comparable to that of the original sensor (Nonin 2000SL: Arms = 3%; N1: Arms = 3.60%; N2b: Arms = 3.46%). Especially at heart rates ≤30 bpm, pulse rate readings that were about twice as high as the reference value occurred. Their exclusion from the dataset resulted in a pulse rate accuracy similar to that of the original sensor. Bland-Altman plots showed limits of agreement typical of pulse oximeters. MAIN LIMITATIONS: The pulse frequency accuracy requires further improvement. The usability in clinical cases needs to be tested. CONCLUSIONS: The new sensor design has been shown to be suitable for buccal pulse oximetry in horses.

Lysine bioavailability among 2 lipid-coated lysine products after exposure to silage.

We conducted 2 experiments to determine lysine bioavailability from 2 lipid-coated lysine products. In an in vitro experiment we mixed each lipid-coated lysine product with either alfalfa- or corn-silage at different amounts of acidity. Scanning electron micrographs indicated that surface structure of each lipid-coated lysine particle was eroded after mixing with silage. Additionally, visual evaluation of scanning electron micrographs suggested that peripheral surface abrasion of lipid-coated lysine may be greater when lipid-coated lysine was mixed with alfalfa silage in comparison to corn silage. In a corresponding experiment, in vivo measures of lysine bioavailability to sheep from 2 lipid-coated lysine products and lysine-HCl were determined after mixing in corn silage. Plasma lysine concentrations increased linearly (P < 0.01) in response to abomasal lysine infusion indicating that our model was sensitive to increases in metabolizable lysine flow. Bioavailability of each lipid-coated lysine source and dietary lysine-HCl were calculated to be 23, 15, and 18%, respectively. Even though each dietary source of lysine increased plasma lysine, rates of increases in plasma lysine from one lipid-coated lysine source (linear; P = 0.20) and lysine-HCl (linear; P = 0.11) were not different from plasma lysine levels supported by diet alone. However, the rate of plasma lysine increase in response to lysine from the other lipid-coated lysine source was greater (P = 0.04) than plasma lysine from feed alone. Nonetheless, the rate of plasma lysine increase in response to lipid-coated lysine did not differ (P ≥ 0.70) from the rate of plasma lysine increase from lysine-HCl. Clearly, methods of manufacture, together with physical and chemical characteristics of diet, can impact amounts of metabolizable lysine provided from lipid-coated lysine products. Direct measures of lysine bioavailability from lipid-coated lysine products after mixing with diets should be based on measurements with the products treated similarly to the method of feeding.

Ruthenium red-mediated suppression of Bcl-2 loss and Ca(2+) release initiated by photodamage to the endoplasmic reticulum: scavenging of reactive oxygen species.

The photosensitizer 9-capronyloxytetrakis (methoxyethyl) porphycene localizes predominantly in the endoplasmic reticulum (ER) and, to a lesser extent, in mitochondria of murine leukemia L1210 cells. Subsequent irradiation results in the loss of ER > mitochondrial Bcl-2 and an apoptotic response. Although an increase in cytosolic Ca(2+) was observed after irradiation, apoptosis was not inhibited by either the presence of the calcium chelator BAPTA or by the mitochondrial uniporter inhibitor ruthenium amino binuclear complex (Ru360). Moreover, neither reagent prevented the loss of Bcl-2. Ruthenium red (RR) devoid of Ru360 prevented Bcl-2 loss, release of Ca(2+) from the ER and the initiation of apoptosis. Since RR was significantly more sensitive than Ru360 to oxidation by singlet oxygen, we attribute the protective effect of RR to the quenching of reactive oxygen species. Although cytosolic and (to a lesser extent) mitochondrial Ca(2+) levels were elevated after photodynamic therapy, these changes were apparently insufficient to contribute to the development of apoptosis.

Kinetics of depression and recovery of murine hepatic cytochrome P-450 levels after treatment with the interferon inducer polyriboinosinic-polyribocytidylic acid.

In vivo treatment of randombred Swiss Webster mice with polyriboinosinic-polyribocytidylic acid (poly l X poly C) inhibited the induction of cytochrome P-450's by both 3-methylcholanthrene [(MCA) CAS: 56-49-5] and phenobarbitol [(PB) CAS: 50-06-6]. Concomitant treatments with poly l X poly C and a single dose of MCA inhibited the induction of P-450's for 24 hours and delayed the obtainment of the MCA-induced P-450 levels for approximately 48-72 hours. When cytochrome P-450 levels were induced by four successive daily treatments of MCA or PB and when poly l X poly C was given on only the 1st day, induction of P-450's was completely suppressed for 24 hours and obtainment of the maximal P-450 level was delayed by 72-96 hours. Treatment with poly l X poly C of animals preinduced for P-450's by four successive daily treatments with either PB or MCA decreased the P-450 content to the noninduced basal level within 24 hours. The effect was temporary in the MCA-treated mice since P-450 content recovered to the MCA-preinduced levels within 72 hours. PB-dependent P-450 induction was short lived, and no recovery occurred after poly l X poly C treatment of PB-preinduced mice. Reduced hepatic cytochrome P-450 contents correlated with decreased abilities of liver homogenates to metabolically activate benzo [a]pyrene (CAS: 50-32-8) and N-acetyl-2-aminofluorene (CAS: 53-96-3), as scored in an Ames Salmonella typhimurium revertant assay.

Murine epidermal xanthine oxidase activity: correlation with degree of hyperplasia induced by tumor promoters.

Topical application of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) to SENCAR mouse skin results within 48 h in a 3-fold elevation of xanthine oxidase (XO) activity, an enzyme capable of generating the reactive oxygen species superoxide and hydrogen peroxide. The antiinflammatory steroid fluocinolone acetonide, an inhibitor of TPA-induced hyperplasia, as well as the multiple stages of tumor promotion as defined in SENCAR mice (Stages I and II), inhibited the TPA-dependent elevation of epidermal XO activity. Neither tosylphenylalanyl chloromethyl ketone nor retinoic acid, inhibitors of promotion Stages I and II, respectively, had significant effects on TPA-induced hyperplasia or elevated XO activity. The nonpromoting but hyperplasiogenic agents ethyl phenylpropiolate and acetic acid significantly elevated XO activity within 48 h of topical application. The non-phorbol ester tumor promoter benzoyl peroxide also elevated XO activity consistent with the degree of induced hyperplasia. Multiple treatments with TPA or ethyl phenylpropiolate resulted in a sustained elevation of XO activity which peaked at five treatments and then declined. Sustained inhibition of XO activity by p.o. administration of allopurinol did not inhibit the TPA-induced hyperplasia as determined histologically. These results suggest that the TPA-dependent elevation of epidermal XO activity is associated with the hyperplasia induced by the agent, and is a consequence of the hyperplasia rather than the cause of it.

Potentiation of photodynamic therapy by ursodeoxycholic acid.

Ursodeoxycholic acid (UDCA) protects cells from the apoptotic effects of hydrophobic bile acids and some other cytotoxic agents. We observed the opposite result when assessing the effects of UDCA on the apoptotic response to mitochondrial photodamage induced by photodynamic therapy (PDT). Two photosensitizers with predominantly mitochondrial specificity were used: a porphycene we have designated CPO; and the tin etiopurpurin SnET2. UDCA potentiated the loss of mitochondrial potential, release of cytochrome c into the cytosol, activation of caspase-3, and apoptotic cell death after irradiation of photosensitized murine leukemia L1210 or hepatoma 1c1c7 cells. These effects were not observed when UDCA was added after irradiation. Glyco-UDCA and tauro-UDCA, conjugated forms of UDCA that are formed in vivo, were as effective as UDCA in promoting PDT phototoxicity. Because UDCA does not act by enhancing intracellular accumulation of the photosensitizing agents used in this study, we propose that the mode of action of UDCA involves the sensitization of mitochondrial membranes to photodamage. UDCA is used currently in gastroenterology for several indications. The drug may offer a means for promoting the efficacy of PDT with minimal adverse effects.

Tumor copromoting activity of gamma-interferon in the murine skin multistage carcinogenesis model.

Recombinant DNA-derived murine gamma-interferon (rMuIFN-gamma) was tested in the murine skin multistage carcinogenesis model as a modulator of 12-O-tetradecanoylphorbol-13-acetate (TPA) promotion. Female SENCAR mice were topically initiated with 7,12-dimethylbenz(a)anthracene and promoted twice weekly with TPA for 20 weeks. Intraperitoneal administration of rMuIFN-gamma 1 day prior to TPA treatment affected neither the kinetics of papilloma development nor the percentage of mice that developed tumors. However, papilloma multiplicities could be either inhibited or increased depending upon the dose of rMuIFN-gamma. Papilloma multiplicities for mice receiving 100, 500, 1000, and 5000 units of rMuIFN-gamma were 184, 122, 105, and 84% of TPA control values, respectively. In contrast, twice weekly i.p. treatments of 7,12-dimethylbenz(a)anthracene initiated mice with only rMuIFN-gamma for 20 weeks did not promote the development of any tumors. Consequently, TPA functioned as a copromoter in those situations in which combined TPA and IFN-gamma treatments elevated papilloma multiplicities. Collectively, the current study demonstrates that rMuIFN-gamma can systemically modulate TPA-dependent promotion in mouse skin.

12-O-tetradecanoylphorbol-13-acetate-dependent induction of xanthine dehydrogenase and conversion to xanthine oxidase in murine epidermis.

Both xanthine dehydrogenase (XD) and xanthine oxidase (XO) catalyze the conversion of hypoxanthine to xanthine, and xanthine to uric acid. Topical application of a promoting dose of 12-O-tetradecanoylphorbol-13-acetate (TPA) to the dorsal skin of female SENCAR mice resulted in a 3.0-3.5-fold elevation of epidermal XO specific activity. Epidermal XO specific activity was maximally elevated 48-96 h after TPA treatment, and required 11 days to return to control levels. Although TPA increased the XO/(XD + XO) ratio from 0.45 to 0.7, the conversion of preexisting XD to XO could not solely account for the TPA-dependent elevation in XO specific activity since control XD plus XO activity was less than just the XO activity in TPA-treated epidermis. Topical application of cycloheximide simultaneously with, or 12 h after, TPA treatment inhibited the TPA-dependent increases in the XO/(XD + XO) ratio and XO specific activities. Collectively, these results suggest that the increased XO activity detected following TPA treatment is the consequence of TPA-induced XD synthesis, and a conversion of existing and newly synthesized XD to XO. In addition, the in vivo promoting activities of analogues of TPA could be correlated with their abilities to elevate XO activity (TPA greater than phorbol-12,13-dibenzoate much greater than 4-O-methyl-TPA = phorbol).

Effect of extracellular calcium concentration on the metabolism of polycyclic aromatic hydrocarbons by cultured mouse keratinocytes.

Cultures of adult mouse epidermal keratinocytes (MEKs) were utilized to determine whether the metabolism and metabolic activation of polycyclic aromatic hydrocarbons varied as a function of extracellular calcium (Ca2+) concentration. MEKs grown in low Ca2+-containing medium (0.05-0.10 mM) maintain basal cell morphology and proliferate while increasing the Ca2+ concentration in the medium to 1.2-1.4 mM signals the cells to undergo terminal differentiation. Relative to cultures of undifferentiated MEKs (low Ca2+), cultures of differentiated MEKs that had been switched to high Ca2+ medium 48 h prior to treatment with benzo(a)-pyrene [B(a)P] and 7,12-dimethylbenz(a)anthracene (DMBA) exhibited more rapid overall metabolism of both hydrocarbons. The greatest differences in the metabolism of B(a)P and DMBA between the two types of cultures occurred after a 3-6-h lag period. In addition, the levels of DNA-adducts formed from B(a)P and DMBA after a 24-h exposure to the hydrocarbon were 4- and 3-fold higher respectively, in cultures of differentiated MEKs (high Ca2+). Higher levels of mutagenesis and cytotoxicity were also observed in cocultures of Chinese hamster lung V-79 cells and MEKs that had been switched to high Ca2+-containing medium. In cocultures treated with the hydrocarbons at the time of Ca2+ shift, several hours elapsed before differences in mutagenesis were apparent between high and low Ca2+-containing cultures. This lag period was eliminated if the MEKs were switched to high Ca2+ medium 24 h prior to exposure to DMBA. Based on the present data, we propose that the expression and inducibility of certain enzyme activities involved in the metabolism of B(a)P and DMBA by cultured MEKs is regulated by the extracellular Ca2+ concentration and possibly the Ca2+-induced differentiation of MEKs.

Alteration in the distribution of the epidermal protein filaggrin during two-stage chemical carcinogenesis in the SENCAR mouse skin.

The histidine-rich epidermal protein filaggrin was purified from urea extracts of newborn SENCAR mouse epidermis. The protein had a molecular weight of 28,000 and an amino acid composition distinctive for this class of proteins. The purified protein was a phosphate acceptor in an in vitro protein kinase assay. Rabbit antibodies raised against filaggrin were used in an indirect immunofluorescent survey of the distribution of filaggrin in the epidermis after single and multiple 12-O-tetradecanoylphorbol-13-acetate treatments as well as in papillomas and carcinomas. The immunofluorescent pattern of acetone-treated adult SENCAR mouse epidermis showed primarily granular layer fluorescence. A single topical 12-O-tetradecanoylphorbol-13-acetate treatment increased immunofluorescence in basal and suprabasal cells. Large papillomas produced by a dimethylbenz[a]anthracene initiation-12-O-tetradecanoylphorbol-13-acetate promotion protocol showed increased fluorescence in all layers. Exuberant papillomas showed a pleomorphic distribution of filaggrin with alternating positive and negative areas of immunofluorescence. Filaggrin immunofluorescence in invasive carcinomas was negative or only slightly positive. The distribution of filaggrin as detected by indirect immunofluorescence is a good indicator of maturation and differentiation in experimental tumors, and its presence correlates with the absence of aggressive or invasive growth.

Inhibition by 2,6-dithiopurine and thiopurinol of binding of a benzo(a)pyrene diol epoxide to DNA in mouse epidermis and of the initiation phase of two-stage tumorigenesis.

The chemotherapeutic agent 6-mercaptopurine was previously shown to inhibit the binding of 7r,8t-dihydroxy-9,10t-oxy-7,8,9,10-tetrahydro-benzo(a) pyrene (BPDE-I) to DNA in Chinese hamster ovary cells. Two compounds related to 6-mercaptopurine, 2,6-dithiopurine (DTP) and thiopurinol (TP), have been tested for inhibition of the binding of BPDE-I to epidermal DNA in mouse skin. Doses of test compound (0.2-20 mumol) or solvent control were applied to the shaved backs of female SENCAR mice. Fifteen min later, 200 nmol [3H]BPDE-I were applied to the same area and 3 h later the mice were sacrificed and epidermal DNA was purified and adduct formation was quantitated radiometrically. At the highest doses studied, DTP and TP inhibited DNA binding by 90 and greater than 80%, respectively. The dose necessary to inhibit DNA binding by 50% was about 0.8 mumol for DTP and about 2 mumol for TP. To test whether this protective effect was long-lasting, the time between application of purinethiol and [3H]BPDE-I was systematically increased. Although the level of protection was decreased by increasing the time between applications, both compounds inhibited binding 50-60% even after 24-48 h. A radioactive compound tentatively identified as a TP-BPDE-I adduct could be recovered from epidermal homogenates following topical application of TP and BPDE-I. We used a standard two-stage initiation-promotion protocol to test the effects of these compounds on mouse skin carcinogenesis. Mice were treated with 0, 1, or 10 mumol of either TP or DTP, and 15 min later were treated with an initiating dose of BPDE-I (200 nmol). Twice weekly promotion with 12-O-tetradecanoylphorbol-13-acetate was begun 2 weeks later and continued for 23 weeks. A dose-dependent inhibition of tumor incidence and multiplicity was noted with both compounds. Treatment of skin with 10 mumol of DTP prior to initiation lowered the number of papillomas per mouse by greater than 90% compared to solvent controls; a 10-fold lower dose resulted in about 50% inhibition. The 10-mumol dose of TP resulted in about 50% inhibition. Mice were examined for 50 weeks for the presence of squamous cell carcinomas. Compared to the positive control group, 10 mumol DTP inhibited carcinoma incidence and lowered the total number of carcinomas by 90-95%. Treatment with 10 mumol TP had no significant effect on carcinoma incidence, and only slightly lowered the total number of carcinomas.

Dissociation of sensitivities to tumor promotion and progression in outbred and inbred SENCAR mice.

The sensitivity of outbred SENCAR mice and inbred SENCAR (SSIN) mice to multistage carcinogenesis was studied. Tumors were induced using either 7,12-dimethylbenz[a]anthracene or N-methyl-N'-nitro-N-nitrosoguanidine as initiators and 12-O-tetradecanoylphorbol-13-acetate or benzoyl peroxide as promoting agents. Although the number of papillomas per mouse was higher in SSIN than in outbred SENCAR mice, the number of carcinomas observed in the SSIN strain was significantly lower regardless of the initiator or promoter used. It was also observed that the expression of markers of premalignant progression (i.e., dysplasia, expression of keratin K13, and loss of keratin K1 expression) was markedly suppressed in SSIN papillomas. After 50 wk of promotion with 12-O-tetradecanoylphorbol-13-acetate, the pattern of expression of K13 and K1 in SSIN mice was comparable to the pattern observed in outbred SENCAR mice after 10 to 20 wk of promotion with 12-O-tetradecanoylphorbol-13-acetate. It was also observed that 67% of the tumors induced in SSIN mice by initiation with 7,12-dimethylbenz[a]anthracene exhibited a mutation in codon 61 of the Ha-ras-1 gene. This latter finding suggests that the differences observed in tumor progression between the inbred strain and the outbred stock are not related to a genetic alteration in the Ha-ras-1 gene but rather to an independent event that we have postulated to involve a putative suppressor gene. The data reported here suggest that the putative gene(s) that confers susceptibility to tumor promotion was segregated from the gene(s) involved in tumor progression during selection and inbreeding of the SENCAR mouse stock.

Production of hydrogen peroxide by murine epidermal keratinocytes following treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate.

The ability of murine epidermal cells to produce intracellular hydrogen peroxide was analyzed by flow cytometry and the measurement of 2',7'- dichlorofluorescin (DCFH) oxidation. Epidermal cells isolated from acetone-treated CD-1 mice for 24 h were relatively homogeneous in cell size and density and oxidized low levels of DCFH. However, following 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment of mice (10 micrograms; 24 h), two cytokeratin-positive populations of cells were identified that were heterogeneous with respect to size and density. These two TPA-derived cell populations oxidized levels of DCFH that were time and dose dependent and were between 2- and 10-fold higher than levels of DCFH oxidized by cells isolated from acetone-treated mice. The ability of catalase, the enzyme that detoxifies hydrogen peroxide, to suppress DCFH oxidation to control levels suggested that intracellular hydrogen peroxide was responsible for the enhanced rate of DCFH oxidation in epidermal cells isolated from TPA-treated mice. The ability of mouse epidermal keratinocytes to oxidize DCFH in response to TPA treatment was confirmed using a cloned keratinocyte cell line. These results suggest that specific subpopulations of keratinocytes produce elevated levels of intracellular peroxides following treatment with TPA either in vivo or in culture.

A mechanism for the proapoptotic activity of ursodeoxycholic acid: effects on Bcl-2 conformation.

Ursodeoxycholic acid (UDCA), a relatively nontoxic bile acid, enhanced the apoptotic response of tumor cells to both photosensitizers that cause photodamage to Bcl-2 and to the nonpeptidic Bcl-2/Bcl-x(L) antagonist HA14-1. The latter agent binds to the surface pocket formed by the BH1, BH2 and BH3 domains of Bcl-2 and Bcl-x(L). Fluorescence polarization studies indicated that affinity of HA14-1 for Bcl-2 was enhanced in the presence of UDCA. Moreover, Bcl-2 photodamage was promoted by UDCA using a photosensitizing agent with affinity for the endoplasmic reticulum, a site of Bcl-2 localization. Fluorescence resonance energy transfer (FRET) studies revealed that the proximity of Bcl-2 to a hydrophobic photosensitizing agent embedded in liposomes was enhanced by UDCA. Since photodamage will occur only if a protein is in close contact with a photosensitizing agent, we propose that these findings support the hypothesis that UDCA causes a conformational change in Bcl-2, promoting HA14-1 binding and enhancing affinity for certain membrane-bound photosensitizers.

Release of cytochrome c and activation of pro-caspase-9 following lysosomal photodamage involves Bid cleavage.

Photodynamic therapy (PDT) protocols employing lysosomal sensitizers induce apoptosis via a mechanism that causes cytochrome c release prior to loss of mitochondrial membrane potential (DeltaPsi(m)). The current study was designed to determine how lysosomal photodamage initiates mitochondrial-mediated apoptosis in murine hepatoma 1c1c7 cells. Fluorescence microscopy demonstrated that the photosensitizer N-aspartyl chlorin e6 (NPe6) localized to the lysosomes. Irradiation of cultures preloaded with NPe6 induced the rapid destruction of lysosomes, and subsequent cleavage/activation of Bid, pro-caspases-9 and -3. Pro-caspase-8 was not activated. Release of cytochrome c occurred at about the time of Bid cleavage and preceded the loss of DeltaPsi(m). Extracts of purified lysosomes catalyzed the in vitro cleavage of cytosolic Bid, but not pro-caspase-3 activation. Pharmacological inhibition of cathepsin B, L and D activities did not suppress Bid cleavage or pro-caspases-9 and -3 activation. These studies demonstrate that photodamaged lysosomes trigger the mitochondrial apoptotic pathway by releasing proteases that activate Bid.

On the nature of superconductivity in the anisotropic dichalcogenide NbSe2{CoCp2}x.

We present a detailed study of the superconducting properties of the weakly pinned, quasi-two-dimensional superconductor 2H-NbSe2, and its intercalated variant NbSe2{CoCp2}0.26. The intercalation of 2H-NbSe2 with the organometallic donor molecule cobaltocene (CoCp2) hardly affects the superconducting properties within the layers. However, the properties perpendicular to the layers change significantly due to the large expansion of the layer spacings of the host lattice in the c-direction by a factor of about two. In particular, the superconducting anisotropy factor Γ increases from 3.3 in the parent compound 2H-NbSe2 up to 4.4 in the intercalated species. Therefore, NbSe2{CoCp2}0.26 is an excellent candidate to analyze how the anisotropy effects the superconducting mechanism in layered dichalcogenides, and to evaluate the various models proposed in the literature to account for the anisotropy in 2H-NbSe2. While a two-gap model and an anisotropic single-gap model are competing concepts to describe the almost linear T(2)-dependence of ΔC/T in low-dimensional dichalcogenides, our comparative study suggests that a single-gap model with an anisotropic Fermi-surface is sufficient to capture the ΔC/T(T) behavior in our samples qualitatively.

Conversion of xanthine dehydrogenase to xanthine oxidase occurs during keratinocyte differentiation: modulation by 12-O-tetradecanoylphorbol-13-acetate.

The distributions of xanthine dehydrogenase (XD) and xanthine oxidase (XO) in subpopulations of murine keratinocytes differing in their stages of terminal differentiation were determined by enzymatic analyses. Keratinocytes were isolated from the skins of female SENCAR mice that had been treated 72 h earlier with either acetone or 12-O-tetradecanoylphorbol-13-acetate (TPA). The ratio of XO/(XD + XO) specific activities was used as an index of the XD to XO conversion. The XO/(XD + XO) ratios for basal cell, suprabasal cell, granular cell plus squamae, and horny sheet preparations isolated from acetone- or TPA-treated mice were 0.35, 0.35, 0.45, 0.75 and 0.28, 0.29, 0.58, and 1.0, respectively. Total XD + XO and XO specific activities in each subpopulation derived from TPA-treated mice were approximately twice the values measured in their control counterparts. Suspension culturing of basal cell keratinocytes in methylcellulose induced terminal differentiation and a conversion of XD to XO. The kinetics of keratin disulfide crosslinking and the XD to XO conversion were similar and preceded cornification. Collectively, these studies demonstrate that the conversion of XD to XO occurs primarily during the later stages of keratinocyte terminal differentiation. Furthermore, the increases in XO activity measured in epidermal homogenates after TPA treatment are due to TPA-dependent increases in 1) the relative proportions of keratinocytes undergoing differentiation, 2) tissue XD content, and 3) increased conversion of XD to XO.

Induction of suppressor T cells and inhibition of contact hypersensitivity in mice by 12-O-tetradecanoylphorbol-13-acetate and its analogs.

12-O-tetradecanoylphorbol-13-acetate (TPA) and its analogs were surveyed for their abilities to modify contact hypersensitivity (CHS) responses in SENCAR mice. Sensitization of dorsal skin with 2,4-dinitrofluorobenzene (DNFB) and subsequent challenge of the ear 5 d later resulted within 24 h in ear swelling and increased vascular permeability (as measured by the extravasation of Evans Blue dye). Treatment of dorsal or ventral skin with TPA 4 times (application made every 3 or 4 d) prior to sensitization on the dorsum inhibited subsequent induction of CHS by DNFB challenge. Maximum suppression of CHS required sensitization at the site of TPA treatment. Suppression occurred over a narrow dose range of TPA (0.1-1.0 micrograms), and qualitatively correlated with the tumor incidences scored in an initiation-promotion multistage skin carcinogenesis experiment. Multiple applications (4x) of the promoters phorbol-12,13-dibenzoate (10 micrograms) and mezerein (2 micrograms) also suppressed CHS, whereas the non-promoter phorbol (20 micrograms) and the first stage tumor promoter 4-O-methyl TPA (20 micrograms) had no effect. Adoptive transfer of splenocytes isolated from mice pre-treated with TPA prior to DNFB sensitization inhibited the development of CHS in recipient mice that were sensitized and challenged with DNFB, but not oxazolone. Splenocyte preparations depleted of T lymphocytes prior to transfer could not suppress CHS in recipient mice. Conversely, suppressive activity was concentrated in splenocyte preparations depleted of adherent cells/monocytes. Collectively, these studies demonstrate that TPA treatment of murine epidermis prior to sensitization with hapten can inhibit subsequent hapten-dependent elicitation of CHS. This suppression is mediated in part by antigen-specific suppressor T cells. Furthermore, there is a qualitative correlation between the complete and second stage in vivo tumor-promoting activities of TPA and its analogs, and their abilities to inhibit CHS.

Quantitative RT-PCR on CYP1A1 heterogeneous nuclear RNA: a surrogate for the in vitro transcription run-on assay.

A quantitative reverse transcription polymerase chain reaction (RT-PCR) assay was developed to amplify a region of the CYP1A1 heterogeneous nuclear RNA (hnRNA) transcript encompassing the first intron-exon boundary. The RT-PCR protocol uses a CYP1A1 recombinant RNA internal standard identical to the target hnRNA except for an engineered unique internal restriction site. Its inclusion enables normalization between reactions and a measurement of the absolute number of target hnRNA transcripts. Specificity for the hnRNA was achieved by using intron-directed primers in both the RT and the PCR. Nuclear run-on assays and the hnRNA RT-PCR assay detected an equivalent increase in transcription of Cyp1a-1 in cultured murine Hepa 1c1c7 cells following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The RT-PCR assay also revealed TCDD-dependent transcriptional activation of the Cyp1a-1 gene in murine skin, a tissue unsuited to the nuclear run-on assay because of inherent difficulties associated with the isolation of nuclei. These examples demonstrate that the hnRNA RT-PCR assay is a facile surrogate for the nuclear run-on assay. Moreover, the sensitivity and design characteristics of the RT-PCR assay suggest the potential for its broad application in general transcriptional research.