Survey of pharmacists' subtherapeutic INR management and anticoagulation bridging practices.

WHAT IS KNOWN AND OBJECTIVE: Despite extensive warfarin use, optimal management of subtherapeutic international normalized ratios (INRs) remains unclear. This study assessed the differences in bridging practices among pharmacists with varying levels of experience, residency training and prescribing privileges. METHODS: An electronic survey was distributed to two ambulatory care pharmacist e-mail lists. Respondents indicated if they would utilize parenteral anticoagulation bridging in 16 clinical scenarios at three therapeutic time points. The scenarios included patients with atrial fibrillation (AFib) (CHADS2 score of 3-4), AFib (CHADS2 score of 5-6) and venous thromboembolism (VTE). The AFib time points were as follows: anticoagulation initiation, early phase (<1 month) and maintenance phase (>1 month). VTE time points included early phase (<1 month), months 2-3 and maintenance phase (>3 months). RESULTS AND DISCUSSION: The survey was completed by 143 respondents. In only three of the scenarios did >50% of respondents indicate they would utilize parenteral anticoagulation bridging. No statistically significant differences in bridging practices were identified between pharmacists providing anticoagulation services in different clinic settings. However, there were significant differences in bridging practices between pharmacists with varying levels of experience, residency training and prescribing privileges in some, but not all of the scenarios. WHAT IS NEW AND CONCLUSION: The standards of care for subtherapeutic INRs warrant further definition.

Effect of cooking on banana and plantain texture.

The effect of temperature and duration of cooking on plantain and banana fruit texture and cytpoplasmic and cell wall components was investigated. The firmness of both banana and plantain pulp tissues decreased rapidly during the first 10 min of cooking in water above 70 degrees C, although plantain was much firmer than banana. Cooking resulted in pectin solubilzation and middle lamella dissolution leading to cell wall separation (as observed by SEM). Dessert banana showed more advanced and extensive breakdown than plantain. Although dessert banana had a higher total pectin content than plantain, the former had smaller-sized carboxyethylenediaminetetraacetic acid (CDTA) soluble pectic polymers which are associated with plant tissues that have a propensity to soften. Plantain had higher levels of starch and amylose than banana but this was associated with a firmer fruit texture rather than a softening due to cell swelling during starch gelatinization. Different cooking treatments showed that cooking in 0.5% of CaCl(2) solution and temperatures below 70 degrees C had significant effects on maintenance of pulp firmness.

Cloning and characterization of Lnk, a signal transduction protein that links T-cell receptor activation signal to phospholipase C gamma 1, Grb2, and phosphatidylinositol 3-kinase.

A cDNA encoding a signal transduction protein with a Src homology 2 (SH2) domain and a tyrosine phosphorylation site was cloned from a rat lymph node cDNA library. This protein, which we designate Lnk, has a calculated molecular weight of 33,988. When T lymphocytes were activated by antibody-mediated crosslinking of the T-cell receptor and CD4, Lnk became tyrosine phosphorylated. In activated T lymphocytes, phospholipase C gamma 1, phosphatidylinositol 3-kinase, and Grb-2 coimmunoprecipitated with Lnk. Our results suggest that Lnk becomes tyrosine phosphorylated and links the immediate tyrosine phosphorylation signals of the TCR to the distal phosphatidylinositol 3-kinase, phospholipase C gamma 1 and Ras signaling pathways through its multifunctional tyrosine phosphorylation site.

Determination of type I transglutaminase in differentiating normal and neoplastic human keratinocytes by an in situ radioimmunoassay.

The presence of type I transglutaminase was determined in the neoplastic human keratinocyte line SqCC/Y1 and in normal human epidermal keratinocytes (NHEK) by an in situ radioimmunoassay which corresponded directly with the measurement of type I transglutaminase enzymatic activity. Dexamethasone induced differentiation of SqCC/Y1 cells caused a marked increase in transglutaminase immunoreactivity and enzymatic activity over non-steroid treated cells in a concentration-related and a time-related fashion. Retinoic acid suppressed the dexamethasone induced increase in type I transglutaminase immunoreactivity in differentiating SqCC/Y1 cells. The type I transglutaminase radioimmunoassay should be useful in studies focusing on the regulation of transglutaminase activity in normal and neoplastic keratinocytes, and for rapidly screening agents for their effects on squamous cell differentiation.

Immediate and delayed (late-phase) dermal contact sensitivity reactions in guinea pigs. Passive transfer by IgG1 antibodies, initiation by mast cell degranulation, and suppression by soybean proteinase inhibitor.

Specific immediate and delayed (24-hour) local dermal reactivity to oxazolone and dinitrochlorobenzene (DNCB) was passively transferred to naive guinea pigs by the intradermal injection of immune guinea pig serum and its IgG1 fraction. In both actively sensitized and passively sensitized animals, neutrophils were the major cells present in the immediate reaction to the specific contactant. Basophils and mononuclear cells were the major cells present in the delayed reaction to the contactant. This late-phase reaction is, therefore, a cutaneous basophil hypersensitivity (CBH) response. The serum factor that passively transferred this CBH response was stable when heated at 56 degrees C for 1-4 h. Since transfer factor, cutaneous basophil hypersensitivity factor and guinea pig IgE are heat-sensitive, these factors probably made little or no contribution to this response. Because proteinase inhibitors are known to inhibit mast-cell degranulation in vitro, we tested the effect of soybean proteinase inhibitor in vivo. This inhibitor suppressed both the immediate and the delayed skin reactivities mediated by intradermal contactant-specific IgG1. These studies support the following concept: IgG1 in guinea pigs (and IgE in human beings) sensitize mast cells to specific antigens. Such antigens degranulate mast cells, releasing histamine and other mediators for the immediate hypersensitivity reaction, and cause mast cells to produce cytokines that recruit basophils, eosinophils and mononuclear cells for the late-phase (CBH) reaction.

Glycosylation of annexin I and annexin II.

Human placental annexin I and annexin II were shown to be glycosylated by one-dimensional affinity blotting with the lectin concanavalin A, which recognizes D-mannose and D-glucose residues. Further evidence that annexin I and annexin II are glycosylated was provided by the finding that these proteins incorporated D-[2,6-3H]mannose and D-[6-3H]glucose when they were biosynthesized by the human squamous carcinoma cell line SqCC/Y1. Annexin I and annexin II could be rapidly purified from a human placental membrane extract by concanavalin A-Sepharose, which indicated that these proteins contain two biantennary mannosyl residues.

Antigen-specific IgG1-mediated epidermal cell injury: a component of contact hypersensitivity reactions in guinea pigs, measurable in vitro in full-thickness skin explants.

Guinea pigs were sensitized by the topical application of either dinitrochlorobenzene (DNCB) or oxazolone on days 1, 2, 3, and 10. Seventeen days after the first treatment with the sensitizer, full-thickness 1.0-cm2 explants of untreated areas of skin were topically exposed in vitro to these contactants. Compared to the response of skin from control guinea pigs, skin from specifically sensitized animals showed a dose-related increase in the number of epidermal cells containing vacuoles. A specific increase in epidermal microblistering paralleled the increase in epidermal vacuolization. In addition, skin explants from sensitized animals (exposed to the contactant) showed a specific decrease in the incorporation of [14C]leucine. Full-thickness skin explants from unsensitized guinea pigs were sensitized in vitro by the intradermal injection of serum IgG1 fraction from oxazolone-sensitized guinea pigs. In such passively sensitized explants, the specific contactant produced an increase in the number of epidermal vacuoles, an increase in the amount of microblistering, and a decrease in the number of mast cells detectable by Giemsa staining. To elicit this specific response, the concentration of the specific contactant had to be mildly injurious, as well as antigenic. This requirement for nonspecific injury could be met by topically exposing skin explants to a nonspecific irritant followed by a sub-threshold concentration of the specific contactant. In contrast to vacuole formation and blistering, contactant-specific degranulation of mast cells (measured by the decrease in their number) did not require irritant levels of the contactant. These studies show that several components of contact sensitivity reactions can be reproduced in vitro by the passive transfer of sera containing antigen-specific immunoglobulins. Banks of such sera might, therefore, be useful in identifying (in human populations) many pre-existing sensitivities to chemical compounds.

Annexin I and involucrin are cross-linked by particulate transglutaminase into the cornified cell envelope of squamous cell carcinoma Y1.

The squamous cell carcinoma line, SqCC/Y1, like natural squamous epithelia, forms a cornified cell envelope during differentiation which can be directly correlated with an increase in particulate transglutaminase activity. When transglutaminase is activated in these cells by calcium ionophore X-537A, annexin I and involucrin become incorporated into the cornified cell envelope and cannot be extracted with solutions containing sodium dodecyl sulfate (SDS) and beta-mercaptoethanol. This effect is specific for annexin I; thus, the amounts of annexins II and IV that were extractable from cells by SDS and beta-mercaptoethanol did not change following treatment with ionophore X-537A. Annexin I could be cross-linked in vitro to itself and to other endogenous proteins by transglutaminase extracted from the particulate fraction of SqCC/Y1 cells. Immunofluorescence studies showed that cross-linked annexin I and involucrin form an envelope-like structure in SqCC/Y1 cells during differentiation that cannot be extracted by EGTA and Triton X-100. The amount of staining of this envelope structure corresponded directly to the particulate transglutaminase activity of these cells. Annexin I monoclonal and polyclonal antibodies were shown to bind to purified cornified cell envelopes from SqCC/Y1. These studies suggest that particulate transglutaminase regulates a function of annexin I during the differentiation of SqCC/Y1 cells by covalently cross-linking this protein into the cornified cell envelope.

Purification of annexin I and annexin II from human placental membranes by high-performance liquid chromatography.

Annexin I and annexin II were extracted from human placental membranes with ethylene glycol bis(beta-amino-ethyl ether)-N,N'-tetraacetic acid (EGTA) and purified by high-performance liquid chromatography by measuring their ability to inhibit phospholipase A2 activity in vitro. Neither protein was capable of binding to a DEAE-5PW HPLC column at neutral pH; however, they were resolved through binding to a Mono S column and passage through size-exclusion HPLC columns. Annexin I and its covalently linked dimer (36 and 66 kDa, respectively, by sodium dodecyl sulfate (SDS)-gel electrophoresis) reacted in one-dimensional immunoblots with monoclonal antibodies to annexin I and calpactin II, and with monoclonal and polyclonal antibodies to lipocortin I, confirming that annexin I, calpactin II, and lipocortin I are the same or closely related proteins. Milligram amounts of monomeric annexin I containing negligible amounts of the cross-linked dimeric annexin I were selectively isolated from placental membranes by using buffers containing the sulfhydryl reagent iodoacetic acid. Milligram amounts of cross-linked annexin I were selectively isolated when placental membranes were initially treated with buffers that did not contain iodoacetic acid and then extracted with Triton X-100, suggesting that sulfhydryl-dependent transglutaminase activity contributes to the selective isolation of this protein. A third phospholipase A2-inhibitory protein (35 kDa by SDS-gel electrophoresis) that reacted in immunoblots with monoclonal antibodies to calpactin I and annexin II, indicating their similar identity, was isolated. The procedure employed allows the rapid purification of annexins I and II in milligram amounts from placental membranes within 2 days.

A survey of state Medicaid policies for coverage of screening mammography and Pap smear services.

In the winter of 1990, we surveyed all states and the District of Columbia to ascertain Medicaid policies on screening mammography and Pap smear--two critical preventive cancer screens for women. Forty-four state Medicaid programs cover screening mammography and all 51 jurisdictions cover Pap smear services. However, the extent of coverage and reimbursement rates vary widely across states. Only a small minority of states reported age or frequency limits for screening; five of these are in conflict with nationally recommended guidelines.

Full-thickness human skin explants for testing the toxicity of topically applied chemicals.

This report describes a model organ-culture system for testing the toxicity of chemical substances that are topically applied to human skin. In this system, the viable keratinocytes in the full-thickness skin explants are protected by the same keratinized layer as skin remaining on the donor, and toxicity can be assessed microscopically and/or biochemically. The human skin specimens were discards from a variety of surgical procedures. They were cut into full-thickness 1.0-cm2 explants, and briefly exposed to the military vesicant sulfur mustard (SM), which was used as a model toxicant. The explants were then organ cultured in small Petri dishes for 24 h at 36 degrees C. In the 0.03-1.0% dosage range, a straight-line dose-response relationship occurred between the concentration of SM applied and the number of paranuclear vacuoles seen histologically in the epidermis. Within the same SM dosage range, there was also a proportional decrease in 14C-leucine incorporation by the explants. Thus, the number of paranuclear vacuoles reflected decreases in protein synthesis by the injured epidermal cells. The epidermis of full-thickness untreated (control) human skin explants usually remained viable for 7 d when stored at 4 degrees C in culture medium. During storage, a relatively small number of paranuclear vacuoles developed within the epidermis, but the explants were still quite satisfactory for testing SM toxicity. Incubation (for 4 or 24 h at 36 degrees C) of such control skin explants reduced (often by 50%) the small number of paranuclear vacuoles produced during 4-7 d of storage. This reduction was probably caused by autolysis of many of the vacuolated cells. Two types of paranuclear vacuoles could be identified by both light and electron microscopy: a storage type and a toxicant type. The storage type seemed to be caused by autolysis of cell components. The toxicant type seemed to be caused by an invagination of the plasma membrane. Only toxicant-type vacuoles increased appreciably in number when skin explants were exposed to mustard, and to other toxicants.

Role of inflammatory cells in the metabolic activation of polycyclic aromatic hydrocarbons in mouse skin.

Oxidants, such as those generated by activated polymorphonuclear leukocytes (PMNs) during inflammation, have been implicated in the metabolic activation of procarcinogens to their ultimate carcinogenic form. In this study we examined the effect of inflammation on the metabolic activation of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BP 7,8-dihydrodiol) to a covalent binding species in mouse epidermis. Interaction of BP 7,8-dihydrodiol with 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated murine leukocytes resulted in the generation of both a chemiluminescent intermediate and one that covalently bound to the DNA of cocultured epidermal keratinocytes. Topical treatment of mouse skin with TPA led to an influx of PMNs into the skin beginning several hours after application. Myeloperoxidase activity, a marker for neutrophils, increased 15-fold in the skin by 16 hr after TPA treatment. Dual applications of TPA at both 16 hr before and concurrently with administration of [3H]BP 7,8-dihydrodiol led to a 50% enhancement of the level of carcinogen that was covalently bound to epidermal DNA. However, a single application of TPA, either 16 hr before or concurrently with BP 7,8-dihydrodiol administration, had no enhancing effect, suggesting that both initial recruitment of PMNs into the skin and subsequent stimulation of oxidant production by the PMNs were required to enhance carcinogen binding. By contrast, no enhancement of benzo[a]pyrene binding was observed by TPA treatments in vivo. However, TPA-stimulated neutrophils did not activate this procarcinogen to a chemiluminescent metabolite in vitro. These results suggest that oxidants generated by metabolically stimulated PMNs can activate penultimate polycyclic aromatic hydrocarbons, such as BP 7,8-dihydrodiol, to potentially genotoxic metabolites in vivo and further define a role for inflammation in carcinogenesis.

Studies on flower longevity in Digitalis : The role of ethylene in corolla abscission.

The flowers of Digitalis purpurea respond to pollination by rapid corolla abscission without any loss of corolla turgor, nor any significant loss of corolla constituents, relative to the corollas of unpollinated flowers of a similar age. The corollas of unpollinated flowers too eventually abscise, 6 d after the stigma opens, however, they do so with only a minimal loss of fresh weight or corolla constituents. Pollination causes an increase in ethylene production detectable within 1 h. Increased ethylene production occurs initially only from the upper portion of the style, later from the lower portion, and lastly, between 23 and 48 h after pollination, from the ovary plus calyx. The pollination response can be induced by exogenous ethylene, the degree of weakening of the corolla abscission zone being dependent upon the concentration and duration of the exposure period and on the stage of flower development. The regulation of ethylene biosynthesis and its involvement in the control of pollination-induced corolla abscission are discussed.

Studies on flower longevity in Digitalis : Pollination induced corolla abscission in Digitalis flowers.

Flower lifespan was terminated by corolla abscission 5-6 days after stigma opening in the unpollinated flower. Increased pollen loads produced increased seed set and reduced flower longevity progressively to a minimum of one day after pollination with pure pollen. Weakening of the abscission zone was detectable 8 h after pollination, whilst the pollen tubes were still within the stigmatic zone, suggesting that a stimulus, moving at 4 mm h(-1) minimum, was transmitted through the style and ovary. Soon after pollination removal of the stigma prevented the pollination-induced corolla abscission. Later it was necessary to remove the stigma and upper style, and later still the whole style to delay abscission. The progressive induction of the stigma and style took place at a rate of 1.5 mm h(-1), in advance of the pollen tubes which grew at 0.75 mm h(-1). It was not possible to reproduce the pollination effects by application of indoleacetic acid (IAA) to the stigma or the style.

Purification and properties of a protease from Lupinus angustifolius during germination.

A protease, present in Lupinus angustifolius cotyledons on the fifth day after germination and assayable by following the release of amino groups from gliadin has been purified to the degree that the associated protein of the extract is undetectable. The enzyme is capable, under varying conditions, of releasing amino groups from lupin alpha, beta and gamma conglutins and possesses a mean molecular weight, by dodecylsulphate/polyacrylamide gel electrophoresis and Sephadex G-75 gel filtration of 27500 +/- 450. The isoelectric point is 9.0 +/- 0.848 with a pH optimum of pH 4.0 using gliadin as substrate.

Concanavalin A-mediated agglutination of plant plastids.

Cucumber (Cucumis sativus L.) and pear (Pyrus domestica Medik.) fruit proplastids, and pea (Pisum sativum L., cv. Meteor) leaf chloroplasts, extracted by osmotic rupture of protoplasts isolated after degradation of the cell walls by cellulase and pectinase, agglutinated in the presence of Con A. Agglutination of cucumber proplastids was inhibited by anti-Con A and by methyl α D-gluco/manno pyranosides but not by methyl α D-galactopyranoside. Fluorescein isothiocyanate-conjugated Con A (FITC-Con A) rendered agglutinated clumps fluorescent. If cellulase was omitted from the macerating medium, Con A-mediated agglutination did not occur even if proplatids were subsequently incubated with cellulase. Proplastids and chloroplasts extracted by conventional mechanical disruption methods were not agglutinated by Con A and did not acquire fluorescence with FITC-Con A. However, cucumber proplastids so extracted could be agglutinated by Con A if incubated with cellulase after preparation.