Type 3 protein kinase C localization to the nuclear envelope of phorbol ester-treated NIH 3T3 cells.

We have examined the immunocytochemical localization of protein kinase C (PKC) in NIH 3T3 cells using mAbs that recognize Type 3 PKC. In control cells, the immunofluorescent staining was similar with mAbs directed to either the catalytic or the regulatory domain of PKC. Type 3 PKC localized in a diffuse cytoplasmic pattern, while the nuclei were apparently unstained. Cytoskeletal components also were Treatment of the cells with phorbol 12-myristate 13-acetate (PMA) resulted in a redistribution of PKC with a specific increase in nuclear PKC. Compared to control cells, the staining with the anticatalytic domain mAbs changed markedly, covering the entire cell surface. In contrast, the staining by the antiregulatory domain mAb did not cover the cell surface and the nuclei remained unstained; these results suggest that PKC activation leads to a conformational change of the regulatory domain such that the epitope recognized by the antiregulatory domain mAb is not readily accessible. We have demonstrated by three criteria that PMA treatment specifically increased PKC in the nucleus: (a) immunofluorescent staining in isolated nuclei increased; (b) Western blots showed that our mAbs detected only one protein, the 82-kD PKC, whose level increased in nuclear lysates from PMA-treated cells; and (c) PKC activity increased in nuclear lysates. In fractionation studies we demonstrated that PKC specifically localized to the nuclear envelope fraction. These results demonstrate that PMA activation leads to a rapid redistribution of Type 3 PKC to the nuclear envelope, and suggests that this isozyme may play a role in mediating PKC-induced changes in gene expression.

Solution structures of stromelysin complexed to thiadiazole inhibitors.

Unregulated or overexpressed matrix metalloproteinases (MMPs), including stromelysin, collagenase, and gelatinase. have been implicated in several pathological conditions including arthritis and cancer. Small-molecule MMP inhibitors may have therapeutic value in the treatment of these diseases. In this regard, the solution structures of two stromelysin/ inhibitor complexes have been investigated using 1H, 13C, and 15N NMR spectroscopy. Both-inhibitors are members of a novel class of matrix metalloproteinase inhibitor that contain a thiadiazole group and that interact with stromelysin in a manner distinct from other classes of inhibitors. The inhibitors coordinate the catalytic zinc atom through their exocyclic sulfur atom, with the remainder of the ligand extending into the S1-S3 side of the active site. The binding of inhibitor containing a protonated or fluorinated aromatic ring was investigated using 1H and 19F NMR spectroscopy. The fluorinated ring was found to have a reduced ring-flip rate compared to the protonated version. A strong, coplanar interaction between the fluorinated ring of the inhibitor and the aromatic ring of Tyr155 is proposed to account for the reduced ring-flip rate and for the increase in binding affinity observed for the fluorinated inhibitor compared to the protonated inhibitor. Binding interactions observed for the thiadiazole class of ligands have implications for the design of matrix metalloproteinase inhibitors.

The tetracycline analogs minocycline and doxycycline inhibit angiogenesis in vitro by a non-metalloproteinase-dependent mechanism.

The tetracycline analogs minocycline and doxycycline are inhibitors of metalloproteinases (MMPs) and have been shown to inhibit angiogenesis in vivo. To further study the mechanism of action of these compounds we tested them in an in vitro model of angiogenesis: aortic sprouting in fibrin gels. Angiogenesis was quantitated in this system by a unique application of planar morphometry. Both compounds were found to potently inhibit angiogenesis in this model. To further characterize the activity of these compounds against MMPs, we determined the IC50S of both compounds against representatives of three classes of metalloproteinases: fibroblast collagenase, stromelysin, and gelatinase A. Doxycycline was found to inhibit collagenase, gelatinase A and stromelysin with IC50S of 452 microM, 56 microM and 32 microM, respectively. Minocycline was found to inhibit only stromelysin in the micromolar range with an IC50 of 290 microM. Since these results suggest that these compounds may not have been inhibiting in vitro angiogenesis by an MMP-dependent mechanism, we decided to test the effects of the potent MMP inhibitor BB-94. This compound failed to inhibit aortic sprouting in fibrin gels, thus strongly suggesting that both doxycycline and minocycline act by an MMP-independent mechanism. These results have implications for the mechanism of action of tetracycline analogs, particularly where they are being considered for the treatment of disorders of extracellular matrix degradation including periodontal disease, arthritis, and tumor angiogenesis.

The design and synthesis of purine inhibitors of CDK2. III.

Cyclin-dependent kinases (CDKs) belong to a class of enzymes that control the ability of a cell to enter into and proceed through the cell division cycle. Using purine as a scaffold, we have synthesized a number of nanomolar inhibitors of CDK-2/cyclin E. In this report, the synthesis of a series of piperidine-substituted purine analogs will be presented, as well as some of their in vitro and in vivo biological effects.

Mitotic and pigment-translocating activities of cultured chromatophores of the guppy, Lebistes reticulatus.

Using Ham's F-12 medium, an in vitro culture system permitting cellular survival for over 6 months has been developed for the chromatophores of the guppy. In this culture system, the various types of chromatophores (melanophores, erythrophores and xanthophores) migrated out of the explanted tail fin tissue, retained their pigmentation, and displayed both mitotic and pigment-translocating activities. The mitotic activity was evident during the first 3 or 4 weeks in culture, whereas the pigment-translocating ability persisted for 16 weeks. The cultured chromatophores of male fish displayed pigment aggregation in response to adrenergic agents (epinephrine and norepinephrine) and pigment dispersion in response to alpha-melanocyte stimulating hormone (alpha-MSH), cyclic AMP and dibutyryl cyclic AMP. Cyclic GMP did not elicit pigment-translocating responses in any of the chromatophores.

Denial of aging: age identification and reference group orientations.

A 10-year longitudinal study of the age-identities of persons 70 and older revealed that many rejected the possibility that they were, in fact, "old." Although there was increased acknowledgment in the restudy of being old, a majority of respondents continued to define themselves in other ways (i.e., as middle-aged or elderly). The importance of comparative reference groups for aging denial was tested. As hypothesized, favorable self-evaluations versus age peers were positively correlated with younger self-images. These comparative evaluations were shown to be as useful as "positional" variables in explaining age-identity. The findings demonstrate the utility of a reference-group perspective in explaining diverse psychological adaptations to late-life role changes.

Structural effects and life satisfaction among the aged.

This study investigates whether adjustment, or life satisfaction, is a function of the structural effect of community age density, the behavioral varaibles of total social interaction and age-graded interaction, or both. The sample contains one hundred eight-five persons seventy years of age and older in a midwestern state who have resided in their community at least five years. Thus, the study examines the influence of community structure on life satisfaction, age-graded interaction, and total interaction in a "real world" setting by using a relatively non-mobile sample. The findings suggest that no significant relationship exists between community structure and either total or age-graded interaction. Age-graded interaction, however, is significantly correlated with life satisfaction in large communities.

The stereotype of "old." A review and alternative approach.

Within the literature, a negative stereotype of "old" has been emphasized which, it has been argued, is important for self-concept in late life. This paper questions the validity of this argument and presents an alternative model more congruent with extant data. Forty-seven reports of research on stereotypes of old age were analyzed. It was found that 21 studies utilized older persons in the sample, and half of these were based on institutionalized or indigent aged. A positive stereotype of old age was reported in several studies. Thus, the assumption that the aged accept a negative stereotype of old age may not be valid. An alternative theoretical model is presented. From the framework of cognitive dissonance theory, it is argued that the acceptance of a negative or positive stereotype by the aged is related to objective indicators of old age, the subjective definition of self, and self-concept.

Social interaction and life satisfaction: an empirical assessment of late-life patterns.

Problems of social and psychological adjustment in later life have been examined by numerous investigators. Some have found positive relationships between social interaction and personal adjustment, while others have found interaction and adjustment to be unrelated. The purpose of the research reported here was to examine how different ways of measuring interaction may affect its relationship with personal adjustment. Data were obtained in interviews with 218 noninstitutionalized persons aged 70 and older. Findings indicate that both the number of persons interacted with, and the frequency of this interaction, are of little importance for the adjustment of older people. We suggest that the quality, rather than quantity, of social interaction is crucial to understanding adaptations to old age.

Work and retirement: a test of attitudinal relationships.

Recent studies report contradictory findings on the hypothesis of an inverse relationship between work satisfaction and retirement attitude. In an effort to clarify the situation, it has been suggested that only in instances where work acts as a key organizing factor in the workers' lives should the inverse relationship be observed. Data testing these hypotheses were analyzed from a study of employed males aged 50 years of age and older (N=1,922) residing in a midwestern state. Results of this study offer only marginal support for the hypothesis. A work satisfaction-retirement attitude typology based on combinations of the two attitudinal areas is discussed.

The work-satisfaction, retirement-attitude typology: profile examination.

After a typology is developed based on work satisfaction and attitude toward retirement, profiles are examined of respondents appearing in four typological categories. Data at three points in time (1964, 1966, 1974) from 1,922 older (50+) employed males suggest that membranes of some types will be more susceptible to negative consequences of life-cycle change (work to retirement) than others. Findings from socioeconomic status, age, social participation, health, community, housing, family, work, morale, and longevity variables indicate pre-retirement planning approaches should be developed for those having different work-retirement attitudes.

Patterns of epithelial expression of Fos protein suggest important role in the transition from viable to cornified cell during keratinization.

An antibody directed against the DNA-binding region of c-fos was used to localize the distribution of cells positive for Fos protein in epithelial tissues. The antibody consistently bound to the nuclei of epithelial cells in the late stages of differentiation, just prior to cornification. The epidermis, palate, buccal mucosa, gingiva, tongue, forestomach and vagina in estrus all produced this type of labelling, suggesting a burst of expression immediately before cell death and cornification. The differentiating cells of the hair follicle, including the hair and inner root sheath, were also labelled. Non-keratinized tissues including junctional epithelium, embryonic epidermis and diestrus vaginal epithelium showed little or no Fos labelling. With the onset of keratinization at 18 days gestation or with induction of estrus in ovariectomized mice with estradiol benzoate, the epidermis and vagina expressed Fos protein in the manner typical for keratinized tissues. The Er/Er mutant epidermis, a tissue that is blocked in its ability to keratinize, overexpresses Fos with Fos-positive cells appearing in virtually every cell layer. Gel shift analysis demonstrates the presence of a functional AP-1 complex in epidermal extracts that is recognized by our antibody. Our data suggest that the expression of Fos is intricately related to epithelial cell differentiation, specifically in relation to the process of cornification and cell death.

Changes in attitudes toward retirement: evidence from a panel study of older males.

Although societal evaluations of retirement may be changing, we hypothesized that attitudes toward retirement change little as persons pass through the years normally associated with retirement. To test this and other hypotheses, data were analyzed from a panel study of 1,152 older Iowa males. Results suggest that changes in retirement attitudes tend to be relatively insignificant. Selected occupational categories differ, however, as do employed and retired persons at the end of the 10-year study. Also, results vary somewhat by the item used to measure attitudes toward retirement, with more negative attitudes toward personal dimensions of retirement and greater acceptance of the societal dimensions.

Anomia, self-esteem, and life satisfaction: interrelationships among three scales of well-being.

The extent to which anomia, self-esteem, and life satisfaction are conceptually distinct was studied by using factor analysis of the items from Srole's anomia scale, Rosenberg's scale of self-esteem, and the LSI-Z. The data came from interviews held with 1,332 older men living in nonmetropolitan areas of Iowa. The analysis demonstrated that, while the concepts of anomia and self-esteem are distinct, the domain of life satisfaction overlaps those of anomia and self-esteem. The representation of the five components of life satisfaction by the items of the LSI-Z is questioned, and caution in using the six-items version of Rosenberg's measure of self-esteem with older adults is suggested.

Adhesion of HT-29 colon carcinoma cells to E-selectin results in increased tyrosine phosphorylation and decreased activity of c-src.

Adhesion of metastatic cancer cells at secondary sites is known to be regulated by several families of adhesion proteins, including selectins and integrins. Colon carcinoma cells have been shown to tether to and roll on both stimulated endothelial cells and purified E-selectin. We have demonstrated that HT-29 human colon carcinoma cells adhere specifically to an E-selectin-IgG chimera. Upon adhesion to E-selectin, the amount of tyrosine phosphorylation of several proteins in HT-29 cell lysates increases compared with cells in bovine serum albumin-coated wells on phosphotyrosine Western blots; this increase is statistically significant. This effect is specific for adhesion to E-selectin, since addition of an E-selectin blocking monoclonal antibody (MAb), E3, to the wells causes a statistically significant decrease in tyrosine phosphorylation relative to E-selectin alone on phosphotyrosine Western blots. One protein that is affected this way has been identified as c-src. Kinase assays show a dose-dependent and statistically significant decrease in c-src activity upon adhesion to E-selectin, which correlates with an increase in phosphorylation of Tyr 527, the negative regulatory tyrosine. CnBr digestion of 32P-labeled c-src shows an increase in phosphorylation of tyrosine 527 after adhesion to E-selectin. Our results may identify a signaling pathway involving the E-selectin ligand on HT-29 cells and c-src.

Increased PKA and PKC activities accompany neuronal differentiation of NT2/D1 cells.

After retinoic acid treatment, a large percentage of cells of the human embryonal carcinoma cell line NT2/D1 differentiate into neuronal cells. We demonstrate here that the differentiated cells, but not the undifferentiated cells, contain high levels of neurofilament mRNA. We have also measured mRNA, protein, and activity levels of two kinases, cAMP-dependent protein kinase (PKA) and protein kinase C (PKC), in order to explore the role of protein kinases in the establishment of the differentiated state. RNA levels for the catalytic (C alpha and C beta) subunits of PKA increased after differentiation. Total PKA activity levels increased 7-fold in the differentiated cells. Parallel with this, a rise in the level of catalytic subunit protein occurred. A 12-fold induction of Type 2 (beta) PKC mRNA levels was observed after neuronal differentiation. Increases in PKC activity and in Type 2 (beta) and Type 3 (alpha) PKC protein levels also accompanied differentiation. These changes in PKA- and PKC-specific RNA levels and enzyme activity may be necessary for production and maintenance of the differentiated state in these cells.

Interstitial collagenase is required for angiogenesis in vitro.

Human umbilical vein endothelial cells (HUVECs) invade collagen gels and establish vascular-like structures within the gel following stimulation with phorbol esters. This process was quantitated by measuring release of radioactivity from gels composed of [3H]collagen. Collagen was steadily degraded over the period of several weeks by phorbol ester-treated cells while little collagenolysis by cells not receiving phorbol ester was noted. Examination of matrix metalloproteinases (MMPs) secreted by HUVECs revealed a prominent induction of interstitial collagenase. Production of the mature forms of gelatinase A was also stimulated, as was the secretion of gelatinase B. Stromelysin was not detected. Two inhibitors of MMPs, the naturally occurring tissue inhibitor of metalloproteinases (TIMP; 10 micrograms/ml) and the synthetic, peptide inhibitor BB-94 (1 microM) were both effective at blocking HUVEC-mediated collagen degradation. Morphological examination of control, PMA-treated HUVECs, as well as PMA-treated HUVECs receiving TIMP or BB-94, revealed that MMP inhibition resulted in a block to invasion and tubule formation within the collagen gels. Similar results for MMP expression and inhibition of tubule formation in vitro were obtained with human dermal microvascular endothelial cells. Examination of collagen proteolytic fragments revealed that both BB-94 and TIMP blocked cleavage of the alpha 1 and alpha 2 chains of type I collagen and the appearance of tropocollagen fragments A and B, demonstrating that the inhibitors were acting directly upon interstitial collagenase. Our results demonstrate that interstitial collagenase is required for angiogenesis in vitro.

Phorbol myristate acetate inhibits growth in S49 cells: isolation of resistant variants.

We have used S49 mouse lymphoma cells to study phorbol ester effects on growth. Treatment of wild-type (wt) cells with phorbol 12-myristate 13-acetate (PMA) results in growth arrest within 72 hr. We have selected variants that are resistant to PMA-induced growth arrest, based on a selection in the presence of 10 nM PMA. We have characterized one of these variants, termed 21.1, in detail. The 21.1 and wt cells contain similar levels of protein kinase C (PKC) as determined by [3H]phorbol 12,13-dibutyrate ([3H]PDBu) binding. Treatment of both wt and 21.1 cells with PMA results in translocation of PKC to the membrane, suggesting that the coupling between PKC and an immediate biological response is intact. PMA treatment leads to the phosphorylation of many similar proteins in wild-type and 21.1 cells. However, in the 21.1 cells there is a prominent substrate of approximately 70 kilodaltons (kD) which is no longer phosphorylated after PMA treatment. In wild-type cells ornithine decarboxylase (ODC) activity and mRNA levels are decreased within 1 hr of PMA treatment. Likewise, ODC levels are decreased in the 21.1 cells after exposure to PMA even though PMA only slightly modulates the growth of these cells. The 21.1 cells represent a unique line with a dominant phenotype in which ODC expression is uncoupled from the growth state of the cell. These cells may represent a good model system in which to examine the steps involved in phorbol ester growth regulation in S49 cells.