Dephosphorylation or antibody binding to the carboxy terminus stimulates pp60c-src.

Phosphorylation of pp60c-src at Tyr-527, six residues from the carboxy terminus, has been implicated in regulation of the protein-tyrosine kinase activity of pp60c-src. Here we show that dephosphorylation of pp60c-src by phosphatase treatment in vitro caused a 10- to 20-fold increase in pp60c-src protein-tyrosine kinase activity. Binding of specific antibody to the region of pp60c-src which contains phosphotyrosine-527 also increased kinase activity. Each treatment increased phosphorylation of added substrates and of Tyr-416 within pp60c-src by a similar mechanism that involved altered interactions with ATP and increased catalytic rate. We suggest that the phosphorylated carboxy terminus acts as an inhibitor of the protein kinase domain of pp60c-src, unless its conformation is altered by either dephosphorylation or antibody binding. The antibody additionally stimulated the phosphorylation of forms of pp60c-src that had reduced gel mobility, much like those phosphorylated in kinase reactions containing pp60c-src activated by polyomavirus medium tumor antigen. These in vitro experiments provide models for the activation of pp60c-src in cells transformed by polyomavirus. We also show that autophosphorylation of pp60c-src at Tyr-527 occurs only to a very limited extent in vitro, even when Tyr-527 is made available for phosphorylation by treatment with phosphatase. This suggests that other protein-tyrosine kinases may normally phosphorylate Tyr-527 and regulate pp60c-src in the cell.

Vaccinia virus growth factor stimulates tyrosine protein kinase activity of A431 cell epidermal growth factor receptors.

Infection of A431 cells with vaccinia virus, or exposure to a mitogenic polypeptide secreted by vaccinia virus-infected cells, induces tyrosine phosphorylation of epidermal growth factor receptors.

Neoplastic transformation induced by an activated lymphocyte-specific protein tyrosine kinase (pp56lck).

The lck proto-oncogene encodes a lymphocyte-specific member of the src family of protein tyrosine kinases. Here we demonstrate that pp56lck is phosphorylated in vivo at a carboxy-terminal tyrosine residue (Tyr-505) analogous to Tyr-527 of pp60c-src. Substitution of phenylalanine for tyrosine at this position resulted in increased phosphorylation of a second tyrosine residue (Tyr-394) and was associated with an increase in apparent kinase activity. In addition, this single point mutation unmasked the oncogenic potential of pp56lck in NIH 3T3 cell transformation assays. Viewed in the context of similar results obtained with pp60c-src, it is likely that the enzymatic activity and transforming ability of all src-family protein tyrosine kinases can be regulated by carboxy-terminal tyrosine phosphorylation. We further demonstrate that overexpression of pp56lck in the murine T-cell lymphoma LSTRA as a result of a retroviral insertion event produces a kinase protein that despite wild-type primary structure is nevertheless hypophosphorylated at Tyr-505. Thus, control of normal growth in this lymphoid cell line may have been abrogated through acquisition of a posttranslationally activated version of pp56lck.

Preparation and characterization of the nonionic detergent-soluble proteins of human stratum corneum.

Nonionic detergent-soluble (NIDS) proteins from human stratum corneum have been prepared by the combined action of detergent and mechanical stimulation of normal human skin. SDS-polyacrylamide gel electrophoresis studies revealed approximately 15 components falling into the molecular weight ranges of 10,000-15,000, 24,000-37,000, and 44,000-68,000. Immunization of rabbits with this material gave antisera which demonstrated 3 or 4 antigenic components using 2-dimensional immunoelectrophoresis. Studies with 125I-labeled NIDS protein indicated that the main precipitate on 2-dimensional immunoelectrophoresis was associated with SDS-polyacrylamide gel components of molecular weight 15,000 and 30,000. Indirect immunofluorescence studies on human skin sections revealed localization of NIDS protein antigens throughout the suprabasal epidermis but concentrated in the stratum granulosum and stratum corneum. Localization studies using strips of stratum corneum obtained by the skin surface biopsy technique revealed a pericellular type of distribution of the NIDS protein antigens.

Regulation of parathyroid hormone-related protein gene expression in murine keratinocytes by E1A isoforms: a role for basal promoter and Ets-1 site.

PTHrP gene expression was evaluated in a murine keratinocyte line, Pam 212K, transformed with E1A and ras. We found that the 12S-E1A oncogene, with or without ras transformation, markedly reduced PTHrP mRNA expression. Using transient transfection assays, we found that the 12S isoform repressed activity from a 5'PTHrP-driven reporter gene. E1A-induced repression of PTHrP reporter constructs appears to be mediated by sequences within minimal promoter region. The 13S-E1A isoform did not repress PTHrP reporter gene activity, and a 13S-deletion mutant that lacked the repressor domains activated a subset of reporter constructs. Mutation of an Ets-1 binding site upstream of the basal promoter substantially decreased activation of reporter constructs by this 13S-deletion mutant. These findings suggest that the E1A oncoprotein may serve as a model for both activation and repression of PTHrP gene expression.

Activation of PTHrP gene expression in squamous carcinoma cell lines by mutant isoforms of the tumor suppressor p53.

We have evaluated the status of p53 expression in three squamous carcinoma cell lines that express high levels of PTHrP mRNA and protein and also cause hypercalcemia when grown in nude mice. All three of these lines possess a single p53 allele, each of which harbors a missense point mutation that gives rise to it mutant p53 protein with a denatured conformation. Using site-directed mutagenesis, we created a p53 expression construct bearing a missense mutation at codon 158, identical to that expressed by one of the cell lines. This construct and p53 constructs expressing representative denatured conformation mutants were then used to develop stably transfected lines, which expressed increased levels of PTHrP mRNA. Promoter-specific RNase protection indicated that this increase was due primarily to transcripts originating from the two TATA promoters, and not the GC-rich initiator element within the PTHrP gene. Cotransfection of mutant p53 expression vectors with a series of reporter constructs under the control of the human PTHrP promoter region showed that mutant p53 isoforms activated constructs containing multiple promoter elements and flanking sequences, but failed to activate constructs with individual promoters in isolation. These findings suggest that the activation of PTHrP gene expression by mutant p53 isoforms displaying a denatured conformation is dependent on interactions with sequences in the PTHrP gene regulatory region beyond the basal TATA promoters.

Preliminary studies into percorneal penetration and elemental content of the stratum corneum using X-ray microanalysis.

A technique is described which measures the penetration of substances through the stratum corneum (SC) and the distribution of elements in this structure employing scanning electron microscopy and energy dispersive analysis. Preliminary observations show that the normal SC shows a concentration gradient of potassium, high in the surface layers and lowest deeper down whereas the reverse is true for phosphorus. It has been shown that sulphur rapidly tranverses the SC and seems to penetrate through all parts of the horny layer whereas lead and zinc do not easily enter this structure.

Is the stratum corneum of uninvolved psoriatic skin abnormal?

A variety of abnormalities of the uninvolved skin have been reported in psoriasis, but there are few studies in which abnormalities of the stratum corneum (SC) have been investigated. In this study we have examined the intracorneal cohesion and structural detail of corneocytes of the SC from involved and uninvolved sites in 24 patients with psoriasis and 10 controls. We have found that intracorneal cohesion is increased in the involved and uninvolved skin of psoriatic patients compared to controls and that there are abnormalities of stratum corneum and corneocyte structure as determined by scanning electron microscopy. The changes in the uninvolved sites may well be due to the increased rate of epidermal cell production in these areas.

Formation of the horny layer in psoriasis--primarily defective or secondarily affected.

It is our belief that any abnormality in keratinization is secondary to an increased rate of epidermal cell production and decreased transit time and research into the underlying nature of psoriasis should concentrate on the issue of whether the stimulus to increased epidermopoietic activity is dermally or epidermally derived.

In vivo and in vitro inhibition of human chorionic gonadotropin-induced testosterone production in rat testis by bursa of fabricius extract.

A bursa of Fabricius homogenate extract (BHE) was used to investigate the endocrine regulation function of this avian organ. In vivo and in vitro results indicated that BHE inhibited human chorionic gonadotropin (HCG)-induced testosterone production by rat testes. Leydig cells from collagenase-dispersed rat testes, when treated with BHE, showed a dose-response related depression of testosterone production under HCG stimulation. Young male rats, injected simultaneously with BHE and HCG, failed to show the marked testosterone production peak observed in rats injected with HCG only. However, in vivo treatments with BHE, in the absence of HCG stimulation, did not inhibit basal testosterone production. These results indicate that the bursa of Fabricius produces an endocrine regulation factor that inhibits HCG-induced testosterone production both in vivo and in vitro. Further investigation is necessary to isolate the active factor and determine its mechanism of action.

Carcinoma of the cervix and cervical cytology--short epidemiological review.

Carcinoma of the cervix is an important disease of well-documented epidemiology but uncertain cause. It causes appreciable morbidity and mortality in all countries, including Zimbabwe, with a significant load on curative services. Epidemiology and the role of cytology are reviewed. Cytology screening programmes have suffered from an inability to cover whole populations, particularly less affluent and/or socially disadvantaged groups, which are most at risk. Despite this difficulty, the magnitude of the problem makes it necessary to continue its study. In particular, efforts are needed in developing countries to study incidence, to better define high risk groups and to devise economical ways of detecting more cases in the earlier stages.

Choriocarcinoma presenting as an intussusception--a case report.

A case of choriocarcinoma presenting as an ileo-ileal intussusception in a sixteen year old girl is presented. Review of the literature reveals this to be the second case ever reported with such an unusual presentation of choriocarcinoma.

Anisotropic magnetoresistance components in (Ga,Mn)As.

We explore the basic physical origins of the noncrystalline and crystalline components of the anisotropic magnetoresistance (AMR) in (Ga,Mn)As. The sign of the noncrystalline AMR is found to be determined by the form of spin-orbit coupling in the host band and by the relative strengths of the nonmagnetic and magnetic contributions to the Mn impurity potential. We develop experimental methods yielding directly the noncrystalline and crystalline AMR components which are then analyzed independently. We report the observation of an AMR dominated by a large uniaxial crystalline component and show that AMR can be modified by local strain relaxation. Generic implications of our findings for other dilute moment systems are discussed.

Choose a path.

Managed care, organizational downsizing, and reengineering create two different paths for nurses to choose: one of opportunity and one of victimization. APNQ interviews author Jane Schweitzer and features excerpts from her book Tears and Rage: The Nursing Crisis in America.

Collaboration and the practice of nurse-midwifery.

In the United States there are growing examples of physician and certified nurse-midwife collaborations capitalizing on the strengths of both disciplines working in the best interest of the patient and her family. The philosophy and practice of certified nurse-midwives (CNMs), however, are not embraced universally by all physicians, and childbirth continues to be treated and reimbursed as a disease process. As Americans seek to establish a partnership with those providing their care, they will naturally embrace advanced practice nurses and reach a mutually beneficial health outcome.

Healing in the midst of chaos.

All aboard for an imaginary journey to the village of Health Care Chaos where the different actors and issues involved in the very real state of health care come alive. The author incorporates an interview with Virginia Trotter Betts, President of the American Nurses Association, to highlight the controversy surrounding the involvement and role of advanced practice nurses.

Second licensure.

"Question the Answers" is a column in Advanced Practice Nursing Quarterly featuring a lively discussion of contemporary, and at times, controversial issues in advanced practice nursing. The column will incorporate written testimony from clinical and educational experts from the editorial board as well as from the field. Contact column editor: Cheryl Slagle King, MPM, RN, CNAA, to contribute to this feature.

Prescriptive authority.

"Question the Answers" is a column in Advanced Practice Nursing Quarterly featuring a lively discussion of contemporary, and at times, controversial issues in advanced practice nursing. The column will incorporate written testimony from clinical and educational experts from the editorial board as well as from the field. Contact column editor: Cheryl Slagle King, MPM. RN, CNAA, to contribute to this feature.

Ending the silent conspiracy: sexual harassment in nursing.

Sexual harassment in health care institutions presents a formidable challenge for nurse leaders as solutions and accountabilities get mired in power, politics, and a historical tradition of secrecy and silence. Harassment is devastating to all individuals, but especially to women because it blatantly violates the victim's personal autonomy and individual sense of equality. Sexual harassment lingers in the corridors of all types of organizations, preying on women as they struggle to overcome cultural and socially imposed barriers. Nurse executives must learn the subtle complexities surrounding harassment, voice strong opposition against harassment, and take swift and comprehensive action to prevent sexual harassment.

Effects of protein kinase C activation after epidermal growth factor binding on epidermal growth factor receptor phosphorylation.

The possible role of epidermal growth factor (EGF) receptor phosphorylation at threonine 654 in modulating the protein-tyrosine kinase activity of EGF-treated A431 cells has been studied. It has been suggested that EGF could indirectly activate a protein-serine/threonine kinase, protein kinase C, that can phosphorylate the EGF receptor at threonine 654. Protein kinase C is known to be activated, and threonine 654 is phosphorylated, when A431 cells are exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA). The protein-tyrosine kinase activity of EGF receptors is normally evidenced in EGF-treated cells by phosphorylation of the receptor at tyrosine. This is inhibited when TPA-treated cells are exposed to EGF. We now show that receptor phosphorylation at threonine 654 can also be detected in EGF-treated A431 cells, presumably due to indirect stimulation of protein kinase C or a similar kinase. Some receptor molecules are phosphorylated both at threonine 654 and at tyrosine. Since prior phosphorylation at threonine 654 inhibits autophosphorylation, we propose that protein kinase C can phosphorylate the threonine 654 of autophosphorylated receptors. This provides evidence for models in which protein kinase C activation, consequent upon EGF binding, could reduce the protein-tyrosine kinase activity of the EGF receptor. Indeed, we find that 12-O-tetradecanoylphorbol-13-acetate, added 10 min after EGF, further increases threonine 654 phosphorylation and induces the loss of tyrosine phosphate from A431 cell EGF receptors.