Pharmacist compensation for cognitive services: focus on the physician office and community pharmacy.

We provide a stepwise approach for the clinical pharmacy practitioner in the physician clinic or community pharmacy setting to secure compensation for cognitive services. How to establish compensation for pharmacist services is explored, including evaluating the payer mix, developing a relationship with the first- or third-party payer, becoming credentialed with a third-party payer, and creating a fee structure. We detail the physical process of billing, which involves completing appropriate billing forms, appropriately using billing codes, documenting cognitive services in the patient record, and obtaining the proper waivers and/or approvals to provide specific services such as laboratory services and immunizations.

Mechanism, specificity and general properties of the yeast enzyme catalysing the formation of inosine 34 in the anticodon of transfer RNA.

In yeast, inosine is found at the first position of the anticodon (position 34) of seven different isoacceptor tRNA species, while in Escherichia coli it is present only in tRNAArg. The corresponding tRNA genes all have adenosine at position 34. Using as substrates in vitro T7-runoff transcripts of 31 plasmids carrying each natural of synthetic tRNA gene harbouring an anticodon with adenosine 34, we have characterised a yeast enzyme that catalyses the conversion of adenosine 34 to inosine 34.

Defective transfer RNA-queuine modification in C3H10T1/2 murine fibroblasts transfected with oncogenic ras.

tRNA isoacceptors for aspartic acid, asparagine, histidine, and tyrosine are modified in the anticodon wobble position with the deazaguanine analogue queuine. Queuine modification is defective in many tumors and transformed cell lines, and the extent of hypomodification correlates with staging and outcome in numerous human tumors. The molecular role of queuine modification in normal cells and the mechanisms of queuine hypomodification in tumors are unknown.

6-Thioguanine-induced growth arrest in 6-mercaptopurine-resistant human leukemia cells.

The thiopurines 6-thioguanine (6TG) and 6-mercaptopurine (6MP) are cytotoxic to proliferating cells by a mechanism involving incorporation into DNA via the purine salvage pathway, and resistance to these agents can be conferred by lack of the salvage pathway enzyme hypoxanthine-guanine phosphoribosyltransferase. However, human and murine hypoxanthine-guanine phosphoribosyltransferase-deficient leukemia cell lines have been shown to respond to 6TG by growth arrest and differentiation by a mechanism apparently not involving incorporation of 6TG into DNA. If so, leukemia cells resistant to 6MP should still respond to 6TG by growth arrest via an undescribed epigenetic mechanism.

Detection of human tRNAs with antisense oligonucleotides.

Regulated expression and modification of tRNA isoacceptors may play an important role in the control of gene expression during such processes as differentiation and immune activation. However, the development of techniques for the identification and quantitation of multiple tRNA isoaccepting species has been hindered by the relative physicochemical similarity among individual isoacceptors and their high degree of post-transcriptional modification. We have used antisense DNA oligonucleotides derived from the T stem to acceptor stem region of six human tRNAs and one murine tRNA to detect individual tRNA isoacceptors in slot blots, Northern blots, and dot blots of human tRNA.

Absence of tRNA-guanine transglycosylase in a human colon adenocarcinoma cell line.

Queuosine (Q), found exclusively in the first position of the anticodons of tRNA(Asp), tRNA(Asn), tRNA(His) and tRNA(Tyr), is synthesized in eucaryotes by a base-for-base exchange of queuine, the base of Q, for guanine at tRNA position 34. This reaction is catalyzed by the enzyme tRNA-guanine transglycosylase (EC 2.4.

An enzyme-linked immunosorbent assay (ELISA) for the detection and quantitation of the tumor marker 1-methylinosine in human urine.

A highly sensitive enzyme-linked immunoassay (ELISA) was developed to detect and quantify the tumor marker, 1-methylinosine (m1I), in human urine. The rabbit antisera was highly specific for m1I with negligible or no inhibition by other nucleosides excreted into urine. Using the competitive ELISA, nanogram amounts of m1I were easily measured directly in urine.

Queuine, a tRNA anticodon wobble base, maintains the proliferative and pluripotent potential of HL-60 cells in the presence of the differentiating agent 6-thioguanine.

6-Thioguanine (6-TG)-induced differentiation of hypoxanthine phosphoribosyltransferase (IMP: pyrophosphate phosphoribosyltransferase, EC 2.4.2.

Modification of the anticodon wobble position of tRNA(Ala) in vitro does not require 5' or 3' processing.

Maturation of eukaryotic tRNA molecules requires nuclear processing as well as nuclear and cytoplasmic modification of specific nucleotides. Nucleotide modifications within the anticodon are found in the majority of all tRNAs and are among the last maturation events to occur in vivo. We show that 5' and 3' processing of SP6 polymerase-generated transcripts are not necessary for the in vitro modification of A----I in the anticodon of tRNA(Ala).

Nontumorigenic squamous cell carcinoma line converted to tumorigenicity with methyl methanesulfonate without activation of HRAS or MYC.

Plasticity of human tumor populations could account for the reason why many tumorigenic human cell lines lose this feature when grown in culture. Methyl methanesulfonate (MMS) was used to convert premalignant squamous cell carcinoma (SCC) cell line SCC-83-01-82 to a malignant phenotype. The MMS-treated SCC-83-01-82 cells (MMS-SCC-83-01-82) produced progressively growing tumors in 5 of 11 splenectomized BALB/c nude mice within 3-5 months.

Differential effect of 6-ethylmercaptopurine on c-myc expression in wild-type and HGPRT-deficient HL-60 cells.

A variety of compounds inhibit the growth and induce differentiation of human promyelocytic leukemia (HL-60) cells. HL-60 subclones that lack the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) can also be induced to differentiate with purine analogs. Mechanisms by which purine analogs induce differentiation offer unique possibilities for cancer chemotherapy.

Noncorrelative c-myc and ras oncogene expression in squamous cell carcinoma cells with tumorigenic potential.

The distribution of heterogeneous cell types within human tumors was examined, and the biological behavior of tumors and different tumor cell lines was evaluated following implantation into surrogate hosts. In situ hybridization and immunohistochemistry were used to examine the expression of oncogenes and localization of the squamous cell carcinoma cell surface-associated antigens. Increased levels of H-ras mRNA and p21 protein were present in six tumors, but enhanced c-myc mRNA expression was observed in just two tumors.

Altered queuine modification of transfer RNA involved in the in vitro transformation of Chinese hamster embryo cells.

Altered queuine modification of tRNA has been correlated to neoplastic transformation, but no direct cause and effect relationship has been defined. In the present study, a potential role for this alteration has been assigned. The tRNA in normal Chinese hamster embryo cells is significantly more queuine modified than the tRNA in their transformed Chinese hamster embryo counterparts, even though the specific activity of the queuine modification enzyme is much lower in Chinese hamster embryo cells than in transformed Chinese hamster embryo cells.

6-ethylmercaptopurine-mediated growth inhibition of HL-60 cells in vitro irrespective of purine salvage.

A variety of purine analogs inhibit the growth and induce the differentiation of human promyelocytic leukemia (HL-60) cells that lack the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT). Mechanisms by which purine analogs induce differentiation offer unique potential for cancer chemotherapy. The guanine analogs, 6-thioguanine and 8-azaguanine, induce granulocytic differentiation of HGPRT-deficient HL-60 promyelocytes.

Identifying inhibitors of queuine modification of tRNA in cultured cells.

Altered queuine modification of tRNA has been associated with cellular development, differentiation, and neoplastic transformation. Present methods of evaluating agents for their ability to induce queuine hypomodification of tRNA are tedious, time-consuming, and not readily amenable to examining cell-type or tissue specificity. Therefore, a rapid, small-scale assay was developed to identify agents that alter queuine modification of tRNA in cultured cells.

Inhibition of phorbol ester-mediated phenotypic changes in cultured cells by hypoxanthine.

Hypoxanthine induces the differentiation of certain transformed cells in vitro, so analyses were undertaken to determine whether this purine metabolite might influence the expression of transformed phenotypes induced in normal cells by chemical agents. Chinese hamster embryo cells and human skin fibroblasts in culture were treated with the promoting agent phorbol-12,13-didecanoate (PDD) with or without prior treatment with 3-methylcholanthrene (MCA), and various phenotypic effects were monitored. Hypoxanthine was found to inhibit significantly the formation of type III foci and the increase in saturation density observed for Chinese hamster cells treated with MCA plus the phorbol ester.

Guanine analog-induced differentiation of human promyelocytic leukemia cells and changes in queuine modification of tRNA.

Treatment of hypoxanthine-guanine phosphoribosyltransferase (HGPRT)-deficient human promyelocytic leukemia (HL-60) cells with 6-thioguanine results in growth inhibition and cell differentiation. 6-Thioguanine is a substrate for the tRNA modification enzyme tRNA-guanine ribosyltransferase, which normally catalyzes the exchange of queuine for guanine in position 1 of the anticodon of tRNAs for asparagine, aspartic acid, histidine, and tyrosine. During the early stages of HGPRT-deficient HL-60 cell differentiation induced by 6-thioguanine, there was a transient decrease in the queuine content of tRNA, and changes in the isoacceptor profiles of tRNA(His) indicate that 6-thioguanine was incorporated into the tRNA in place of queuine.

Enhancement of the chemical transformation of Chinese hamster embryo cells in vitro by 7-methylguanine.

The RNA catabolite 7-methylguanine has been shown to inhibit queuine modification of tRNA in Chinese hamster embryo cells under conditions leading to in vitro transformation. Phorbol ester tumor promoters also induce queuine hypomodification of tRNA in normal human cells, and this effect was reported to be correlated directly to the appearance of an altered (transformed) cell phenotype. Based on this common macromolecular alteration, 7-methylguanine was evaluated for its ability to enhance the chemically induced transformation of cultured cells.

Urinary nucleosides in leukemia: laboratory and clinical applications.

Urinary nucleosides offer a number of useful laboratory and clinical applications in the study and analysis of leukemia. There are significant differences in the excretion of modified nucleosides between normal individuals and individuals with various forms of leukemia, as well as between leukemia patients at opposite ends of the clinical spectrum, i.e.

Hematopoiesis and the inosine modification in transfer RNA.

Human promyelocytic leukemia (HL-60) cells were used to begin to evaluate the role in hematopoiesis of inosine biosynthesis in the tRNA anticodon wobble position; a reaction involving the enzymatic insertion of performed hypoxanthine. Dimethyl sulfoxide (DMSO) and hypoxanthine were found to induce the differentiation of HL-60 cells in a synergistic manner, and the induced differentiation was independent of changes in the purine catabolic enzymes adenosine deaminase and purine nucleoside phosphorylase. The short-term exposure of HL-60 cells to DMSO plus hypoxanthine resulted in enhanced leucine incorporation, and a model is presented showing how the inosine modification reaction in tRNA may be involved.

Inhibition of queuine uptake in cultured human fibroblasts by phorbol-12,13-didecanoate.

The modified base queuine is inserted posttranscriptionally into the first position of the anticodon of tyrosine tRNA, histidine tRNA, asparginine tRNA, and aspartic acid tRNA. Phorbol-12,13-didecanoate (PDD) effects a decrease in the queuine content of tRNA in cultured human foreskin fibroblasts. The present data suggest that this results from a PDD-mediated inhibition of queuine uptake.

Relationship between a tumor promoter-induced decrease in queuine modification of transfer RNA in normal human cells and the expression of an altered cell phenotype.

With normal human skin fibroblasts in culture, a transient decrease in queuine modification of tRNA precedes a phorbol ester tumor promoter-induced 5- to 10-fold increase in saturation density. Subsequently, an increase in the queuine content of cellular tRNA (to levels comparable to those in untreated cultures) precedes a decrease in saturation density. This reversal of the phorbol ester-induced alteration in tRNA modification occurs in the continued presence of the tumor promoter, and it parallels an increased ability of the cells to salvage queuine from catabolized endogenous tRNA.

Altered growth properties of normal human cells induced by phorbol 12,13-didecanoate.

The tumor promoter phorbol 12,13-didecanoate (PDD) significantly altered the growth properties of early passage normal human skin cells in vitro in culture medium supplemented with elevated concentrations of selected amino acids. Continuous treatment of cells with 10(-7) or 10(-8) M PDD resulted in a 5 to 10-fold increase in saturation density at early passages followed by a long-term two- to fourfold increase. The PDD-treated cultures remained in exponential growth at cell densities greater than 10-fold higher than the control cultures.

Adenine nucleotide metabolism and compartmentalization in isolated adult rat heart cells.

The metabolism and intracellular compartmentalization of adenine nucleotides in a preparation of adult rat heart myocytes showing good morphology, viability, and tolerance to calcium ion has been examined by high performance liquid chromatography. These myocytes contain an average of 23 nmol adenine nucleotide per milligram protein which is about 60% of the adenine nucleotide content of intact rat heart tissue. The loss of adenine nucleotide occurs during the incubation and washing steps that increase the yield of viable cells, rather than during the collagenase perfusion.

Inosine biosynthesis in transfer RNA: a postulated role in immune regulation.

A hypothesis is put forth describing a role in immune regulation for inosine biosynthesis in the anticodon of tRNA. The enzymatic insertion of hypoxanthine into the tRNA wobble base position is predicted to be a control point for the translation of proteins and peptides required for normal immune function. The substrate for inosine biosynthesis in tRNA, hypoxanthine, is an intermediate in the purine catabolic pathway, and defects in this pathway are associated with inherited immunodeficiency diseases.