Adenine nucleotide-dependent regulation of assembly of bacterial tubulin-like FtsZ by a hypermorph of bacterial actin-like FtsA.

Cytokinesis in bacteria depends upon the contractile Z ring, which is composed of dynamic polymers of the tubulin homolog FtsZ as well as other membrane-associated proteins such as FtsA, a homolog of actin that is required for membrane attachment of the Z ring and its subsequent constriction. Here we show that a previously characterized hypermorphic mutant FtsA (FtsA*) partially disassembled FtsZ polymers in vitro. This effect was strictly dependent on ATP or ADP binding to FtsA* and occurred at substoichiometric levels relative to FtsZ, similar to cellular levels. Nucleotide-bound FtsA* did not affect FtsZ GTPase activity or the critical concentration for FtsZ assembly but was able to disassemble preformed FtsZ polymers, suggesting that FtsA* acts on FtsZ polymers. Microscopic examination of the inhibited FtsZ polymers revealed a transition from long, straight polymers and polymer bundles to mainly short, curved protofilaments. These results indicate that a bacterial actin, when activated by adenine nucleotides, can modify the length distribution of bacterial tubulin polymers, analogous to the effects of actin-depolymerizing factor/cofilin on F-actin.

Expression and Regulation of Human Cytochrome P450 4F Isoforms in Tissue Samples and Under TNF-Alpha Challenges

CYP4F (Cytochrome P4504F) enzymes metabolize endogenous molecules including leukotrienes, prostaglandins and arachidonic acid. The involvement of these endogenous compounds in inflammation has led to the hypothesis that changes in the inflamed tissue environment may affect the expression of CYP4Fs during the pro-inflammatory state, which in turn may modulate inflammatory conditions during the anti-inflammatory state. We demonstrated that inflamed tissues have different levels of CYP4F isoform expression profiles in a number of human samples when compared to the average population. The CYP4F isoform expression levels change with the degree of inflammation present in tissue. Further investigation in cell culture studies revealed that inflammatory cytokines, in particular TNF-α, play a role in regulating the expression of the CYP4F family. One of the isoforms, CYP4F11, had different characteristics than that of the other five CYP4F family members. CYP4F11 metabolizes xenobiotics while the other isoforms metabolize endogenous compounds with higher affinity. CYP4F11 also was expressed at high quantities in the brain, and was up-regulated by TNF-α, while the other isoforms were not expressed at high quantities in the brain and were down-regulated by TNF-α. We identified the AP-1 protein of the JNK pathway as the signaling protein that causes significant increase in CYP4F11 expression. Since TNF-α stimulation causes a simultaneous activation of both JNK pathway and NF-κB signaling, we investigated further the role that NF-κB plays on expression of the CYP4F11 gene. We concluded that although there is a significant increase in CYP4F11 expression in the presence of TNF-α, the activation of NF-κB signaling inhibits CYP4F11 expression in a time dependent manner. The expression of CYP4F11 is only significantly increased after 24 hours of treatment with TNF-α; at shorter time points NF-κB signaling overpowers the JNK pathway activation. We believe that these findings may in the future lead to improved drug design for modulating inflammation.

Multiple site phosphorylation of tyrosine hydroxylase: A potential role for protein kinase C in the regulation of catecholamine synthesis

Tyrosine hydroxylase (TH), the initial and rate limiting enzyme in the catecholaminergic biosynthetic pathway, is phosphorylated on multiple serine residues by multiple protein kinases. Although it has been demonstrated that many protein kinases are capable of phosphorylating and activating TH in vitro, it is less clear which protein kinases participate in the physiological regulation of catecholamine synthesis in situ. These studies were designed to determine if protein kinase C (PK-C) plays such a regulatory role.^ Stimulation of intact bovine adrenal chromaffin cells with phorbol esters results in stimulation of catecholamine synthesis, tyrosine hydroxylase phosphorylation and activation. These responses are both time and concentration dependent, and are specific for those phorbol ester analogues which activate PK-C. RP-HPLC analysis of TH tryptic phosphopeptides indicate that PK-C phosphorylates TH on three putative sites. One of these (pepetide 6) is the same as that phosphorylated by both cAMP-dependent protein kinase (PK-A) and calcium/calmodulin-dependent protein kinase (CaM-K). However, two of these sites (peptides 4 and 7) are unique, and, to date, have not been shown to be phosphorylated by any other protein kinase. These peptides correspond to those which are phosphorylated with a slow time course in response to stimulation of chromaffin cells with the natural agonist acetylcholine. The activation of TH produced by PK-C is most closely correlated with the phosphorylation of peptide 6. But, as evident from pH profiles of tyrosine hydroxylase activity, phosphorylation of peptides 4 and 7 affect the expression of the activation produced by phosphorylation of peptide 6.^ These data support a role for PK-C in the control of TH activity, and suggest a two stage model for the physiological regulation of catecholamine synthesis by phosphorylation in response to cholinergic stimulation. An initial fast response, which appears to be mediated by CaM-K, and a slower, sustained response which appears to be mediated by PK-C. In addition, the multiple site phosphorylation of TH provides a mechanism whereby the regulation of catecholamine synthesis appears to be under the control of multiple protein kinases, and allows for the convergence of multiple, diverse physiological and biochemical signals. ^

Studies on regulation of skeletal muscle differentiation by growth factors

Skeletal muscle differentiation involves sequential events in which proliferating undifferentiated myoblasts withdraw from the cell cycle and fuse to form multinucleated myotubes. The process of fusion is accompanied by the disappearance of proteins associated with cell proliferation and the coordinate induction of a battery of muscle-specific gene products, which includes the muscle isoenzyme of creatine kinase, nicotinic acetylcholine receptor, and contractile proteins such as alpha-actin. The molecular events associated with myogenesis are particularly amenable to experimental analysis because the events which occur in vivo can be recapitulated in vitro using established muscle cell lines. Initiation of myogenic differentiation in vitro can be achieved by removing serum from the culture medium. Myogenesis, therefore, can be considered to be regulated through a repression-type of mechanism by components in serum. The objectives of this project were to identify the components involved in regulation of myogenesis and approach the mechanism(s) whereby these components achieve their regulatory function. Initially, the effects of a series of polypeptide growth factors on myogenesis were examined. Among them TGF$\beta$ and FGF were found to be potent inhibitors of myogenic differentiation which did not affect cell proliferation. The inhibitory effects of these growth factors on differentiation requires their persistent presence in the culture medium. After myoblasts have undergone fusion, they become refractory to the inhibitory effects of TGF$\beta$, FGF, and serum. When fusion is inhibited by the presence of EGTA, a Ca$\sp{2+}$ chelator, muscle-specific genes are expressed reversibly upon removal of inhibitory growth factors. Subsequent exposure of biochemically differentiated cells to serum or TGF$\beta$ leads to down-regulation of muscle-specific genes. Stimulation with serum also leads to reentry of myocytes into the cell cycle, whereas fused myotubes are irreversibly and terminally differentiated. Measurement of levels of TGF$\beta$ receptors reveals that under non-fusing conditions, TGF$\beta$ receptor levels in biochemically differentiated myocytes remained as high as in undifferentiated myoblasts, while during terminal differentiation, TGF$\beta$ receptors decreased at least five-fold. Thus, down-regulation of TGF$\beta$ receptors is coupled to irreversible differentiation, but not reversible differentiation in the absence of fusion. The possible involvement of second messenger systems, such as cAMP and protein kinase C, in the pathway(s) by which TGF$\beta$, FGF, or serum factors transduce their signals from the cell surface to the nucleus was also examined. The results showed that myogenic differentiation is subject to negative regulation through cAMP elevation-dependent and cAMP elevation-independent pathways and that serum mitogens, TGF$\beta$ and FGF inhibit differentiation through a mechanism independent of cAMP-elevation or protein kinase C activation. ^

B cell memory and idiotype network regulation

This investigation examined the clonal dynamics of B-cell expression and evaluated the role of idiotype network interactions in shaping the expressed secondary B-cell repertoire. Three interrelated experimental approaches were applied. The first approach was designed to distinguish between regulatory influences controlled by the major histocompatibility complex (MHC) and regulatory influences controlled by non-MHC factors including the idiotype network. This approach consisted of studies on the clonal dynamics and heterogeneity of the expressed IgG antibody repertoire of BALB/c mice. The second approach involved the analysis of the clonal dynamics of antibody responses of outbred rabbits. This analysis was coupled with studies to detect the occurrence and activity of constituents of the idiotype network. In the third approach the transfer of rabbit lymphocytes from immunized donors to MHC matched naive recipients was used to examine the effects of recipient non-MHC immunoregulatory influences on the expression of donor memory B-cells. Although many memory B cells were unaffected by non-MHC influences, these data show that non-MHC immunoregulatory influences can affect the expression of B-cells in the secondary response of inbred mice and outbred rabbits. The results also indicate that most IgG antibody responses are heterogeneous and are characterized by a stable group of dominant clonotypes. Clonal dominance and B-cell memory were found to be established early in an immune response. The expression of B memory clones appeared to be favored over the expression of virgin B cells. The injection of anti-tetanus antibody induced the antigen independent production of anti-tetanus antibody, probably through idiotypic mechanisms. These results demonstrate that both antibody and antigen can affect the expressed B-ceIl repertoire. Thus, idiotypic interactions are capable of influencing the expression of B-cells and these findings support the existence and function of an idiotype network with strong immunoregulatory potential. ^

Regulation of adenylyl cyclase by protein kinase C and P$\sb2$ purinergic agonists

Activation of protein kinase C (PKC) causes multiple effects on adenylyl cyclase (AC), (i) an inhibition of (hormone) receptor/G$\sb{\rm s}$ coupling, consistent with PKC modification of the receptor and (ii) a postreceptor sensitization consistent with a PKC-mediated modification of the stimulatory (G$\sb{\rm s}$) or inhibitory (G$\sb{\rm i}$) G-proteins or the catalyst (C) of AC. In L cells expressing the wild-type beta-adrenergic receptor ($\beta$AR) 4-$\beta$ phorbol 12-myristate-13-acetate (PMA) caused 2-3-fold increases in the K$\sb{\rm act}$ and V$\sb{\rm max}$ for epinephrine-stimulated AC activity and an attenuation of GTP-mediated inhibition of AC. Deletion of a concensus site for PKC phosphorylation (amino acids 259-262) from the $\beta$AR eliminated the PMA-induced increase in the K$\sb{\rm act}$, but had no effect on the other actions of PMA. PMA also increased the K$\sb{\rm act}$ and V$\sb{\rm max}$ for prostaglandin E$\sb1$ (PGE$\sb1$)-stimulated AC and the V$\sb{\rm max}$ for forskolin-stimulated AC. Maximal PMA-induced sensitizations were observed when AC was assayed in the presence of 10 $\mu$M GTP and 0.3 mM (Mg$\sp{++}$).^ Liao et al. (J. Biol. Chem. 265:11273-11284 (1990)) have shown that the P$\sb2$ purinergic receptor agonist ATP stimulates hydrolysis of 4,5 inositol bisphosphate (PIP$\sb2$) by phospholipase C (PLC) in L cells. To determine if agonists that stimulate PLC and PMA had similar effects on AC function we compared the effects of ATP and PMA. ATP caused a rapid 50-150% sensitization of PGE$\sb1$-, epinephrine-, and forskolin-stimulated AC activity with an EC$\sb{50}$ of 3 $\mu$M ATP. The sensitization was similar (i.e. Mg$\sp{++}$ and GTP sensitivity) to that caused by 10 nM PMA. However, unlike PMA ATP did not affect the K$\sb{\rm act}$ for hormone-stimulated AC and its effects were unaltered by down-regulation of PKCs following long term PMA treatment. Our results demonstrate that a PKC concensus site in the $\beta$AR, is required for the PMA-induced decrease in receptor/G$\sb{\rm s}$ coupling. Our data also indicate that activation of P$\sb2$ purinergic receptors by ATP may be important in the sensitization of AC in L cells. The mechanism behind this effect remains to be determined. ^

Intron involvement in the regulation of MuSVts110 RNA splicing

The murine sarcoma virus MuSVts110 exhibits an alternative RNA splicing pattern. Like other simple retroviruses, MuSVts110 pre-mRNA splicing is balanced to allow the production of both spliced and unspliced RNA during the replicative cycle. In addition to balance, MuSVts110 RNA splicing exhibits a unique growth-temperature restriction to splicing; temperatures below 33$\sp\circ$C are permissive for splicing while temperatures of 37$\sp\circ$C or above are non-permissive. Previous work has established that this thermosensitive splicing phenotype is mediated in cis by viral transcript features. Here we show that at least three sequence elements regulate the MuSVts110 splicing phenotype. First, the MuSVts110 branchpoint (BP) and poly-pyrimidine tract (PPT) were found to be determinants of overall splicing efficiency. Wild-type MuSVts110 possesses a weak BP and PPT adjacent to the 3$\sp\prime$ splice site. Introduction of a strong BP caused MuSVts110 splicing to proceed to virtual completion in vivo, thus losing any vestige of balance or thermosensitivity. In in vitro splicing extracts, the strong BP overcame a blockade to wt MuSVts110 splicing at both the first and second catalytic steps. Weakening the consensus nature of the strong BP allowed the recovery of thermosensitive splicing in vivo, and reinstated the blockades to splicing in vitro, arguing that a suboptimal BP is an unusual manifestation of the proportional splicing pattern of retroviruses. The PPT is essential for accurate recognition of the BP sequence by the splicing machinery. Lengthening the PPT of MuSVts110 from 9 to 19 consecutive pyrimidines increased the overall efficiency of splicing in vivo dramatically, but was less effective than the strong BP in overriding the restriction on splicing imposed by high growth temperatures. Finally, decreasing gradually the overall size of the intron unexpectedly reduced splicing efficiency at growth temperatures permissive for splicing, suggesting that non-conserved sequences within the intron of MuSVts110 participate in splicing regulation as well. Taken together, these results suggest a mechanism of control in which MuSVts110 splicing is modulated by the entire intron, but principally by suboptimal signals at the splice acceptor site. Furthermore, this retroviral system provides a powerful genetic method for selection and analysis of mutations that affect splicing. ^

Regulation of pyridoxal $5\prime$-phosphate biosynthesis in {\it Escherichia coli\/} K-12

Vitamin B$\sb6$ (or pyridoxal 5$\sp\prime$-phosphate, PLP) is an essential, ubiquitous coenzyme that affects many aspects of amino acid and cellular metabolism in all organisms. The goal of this thesis is to examine the regulation of PLP biosynthesis in Escherichia coli K-12. First, PdxH oxidase is a PLP biosynthetic enzyme, which uses molecular oxygen as an electron acceptor under aerobic assay conditions. To test if facultative anaerobic E. coli uses another enzyme to replace the function of PdxH oxidase anaerobically, suppressors of a pdxH null mutant were isolated anaerobically after 2-aminopurine or spontaneous mutagenesis. Only one specific bypass mutation in another PLP biosynthetic gene pdxJ was found, suggesting that PdxH oxidase is able to function anaerobically and PdxT utilizes D-1-deoxyxyulose as a substrate. Second, regulation of the serC (pdxF)-aroA operon, which is involved the biosynthesis of L-serine, PLP and aromatic compounds was examined. A serC (pdxF) single gene transcript and a serC (pdXf)-aroA cotranscript initiated at P$\sb{serC\ (pdxF)}$ upstream of serC (pdxF) were detected. The expression of the operon is activated by leucine responsive regulatory protein (LRP) and repressed by cAMP receptor protein-cAMP complex (CRP$\cdot$cAMP) at the transcriptional level. LRP activates the operon by directly binding to the upstream consensus box. Binding of CRP$\cdot$cAMP to the upstream CRP box diminishes the activation effect of LRP. However, deletion of the CRP box did not affect the repression of CRP$\cdot$cAMP, suggesting that CRP$\cdot$cAMP may repress the operon indirectly by stimulating the activity or level of an unidentified repressor. The overall effect of this regulation is to maximize the expression of the operon when the cells are growing in minimal-glucose medium. In addition, the binding and the transcription of P$\sb{serC\ (pdxF)}$ by RNA polymerase require a supercoiled circular DNA, indicating that DNA supercoiling affects the transcription of the operon. Third, regulation of another PLP biosynthetic gene gapB was also examined. gapB is activated by CRP$\cdot$cAMP and repressed by catabolic repressor activator protein (CRA). However, the activation of CRP$\cdot$cAMP is epistatic to the repression of CRA. Due to the CRA repression, gapB was expressed at a low level in all the media tested, suggesting that it may be the rate-limiting step of PLP biosynthesis. In summary, unlike genes in many biosynthetic pathways, PLP biosynthetic genes are regulated by global regulators that are important for carbon and amino acid metabolism, instead of the end product(s) of the pathway. ^

Integrin signaling in the regulation of lymphocyte morphology

In normal lymphocytes an “inside-out” signal up-regulating integrin adhesion is followed by a ligand mediated “outside-in” signal for cell spreading. Although PKC mediates both events, distinct roles were found for different PLCs. The inhibition of phosphatidylinositol specific PLC decreased both cell adhesion and spreading on fibronectin in T cell receptor/CD28 activated peripheral blood T cells. However, inhibition of phosphatidylcholine specific PLC only blocked cell spreading and did not affect adhesion, indicating that “inside-out” signaling for the integrin α4β1 proceeds through phosphatidylinositol specific PLC and PKC, while the “outside-in” signal utilizes phosphatidylcholine specific PLC and PKC. Furthermore, β1 integrin chain mediated morphological changes in the T lymphocytic cell line HPB-ALL directly paralleled PKA activation, treatment of these cells with an inhibitory anti-β1 antibody blocked PKA activation and cell spreading, and this inhibition could be overcome by activating adenylate cyclase. Furthermore, inhibition of PKA was found to decrease the overall strength of cell adhesion or cellular avidity without affecting individual receptor affinity for soluble ligand. ^ When HPB-ALL cells interact with immobilized FN, two separate morphological phenotypes can be induced. Some cells flattened their cell body into a triangular shape and begin to migrate, while others extended a pseudopod from their stationary cell body. This second morphology recapitulates the shape changes observed during transendothelial migration. During these morphological changes, α4β1 integrins are internalized into endocytic vesicles that ultimately accumulate at the juncture between the cell body and an extending pseudopod. From this juncture, they are rapidly transported down the length of the pseudopod to its most distal end. ^ In addition to an accumulation of integrin containing vesicles, the pseudopod base was found to have increased amounts of the small GTPase RhoA and active PKA. The inhibition of PKA or RhoA resulted in lymphocytes with similar aberrant stellate morphologies. Furthermore, inhibition of PKA blocked the α4β1 mediated phosphorylation of RhoA. The co-localization of active PKA, RhoA and integrin containing endocytic vesicles indicates that integrin triggering can cause the rapid redistribution and activation of key signaling intermediates and raises the possibility that regulation of lymphocyte morphology by PKA and RhoA is through adhesion receptor recycling. ^

Characterization of cytochrome P450 4F subfamily: Response in traumatic brain injury and gene regulation

CYP4F enzymes metabolize endogenous molecules including arachidonic acid, leukotrienes and prostaglandins. The involvement of these eisosanoids in inflammation has led to the hypothesis that CYP4Fs may modulate inflammatory conditions after traumatic brain injury (TBI). In rat, TBI elicited changes in mRNA expression of CYP4Fs as a function of time in the cerebrum region. These changes in CYP4F mRNA levels inversely correlated with the cerebral leukotriene B4 (LTB4) level following injury at the same time points. TBI also resulted in changes in CYP4F protein expression and localization around the injury site, where CYP4F1 and CYP4F6 immunoreactivity increased in surrounding astrocytes and CYP4F4 immunoreactivity shifted from endothelia of cerebral vessels to astrocytes. The study with rat primary astrocytes indicated that pro-inflammatory cytokines TNFα and IL-1β could affect the transcription of CYP4Fs to a certain degree, whereas the changing pattern in the primary astrocytes appeared to be different from that in the in vivo TBI model.^ In addition, the regulation of CYP4F genes has been an unsolved issue although factors including cytokines and fatty acids appear to affect CYP4Fs expression in multiple models. In this project, HaCaT cells were used as an in vitro cellular model to define signaling pathways involved in the regulation of human CYP4F genes. Retinoic acids inhibited CYP4F11 expression, whereas cytokines TNFα and IL-1β induced transcription of CYP4F11 in HaCaT cells. The induction of CYP4F11 by both cytokines could be blocked by a JNK specific inhibitor, indicating the involvement of the JNK pathway in the up-regulation of CYP4F11. Retinoic acids are known to function in gene regulation through nuclear receptors RARs and RXRs. The RXR agonist LG268 greatly induced transcription of CYP4F11, whereas RAR agonist TTNPB obviously inhibited CYP4F11 transcription, indicating that the down-regulation of CYP4F11 by retinoic acid was mediated by RARs, and that inhibition of CYP4F11 by retinoic acid may also be related to the competition for RXR receptors. Thus, the CYP4F11 gene is regulated by signaling pathways including the RXR pathway and the JNK pathway. In contrast, the regulation mechanism of other CYP4Fs by retinoic acids appears to be different from that of CYP4F11.^

Mouse retinal development: Role of cell adhesion and mechanism of gene regulation

Cell differentiation and pattern formation are fundamental processes in animal development that are under intense investigation. The mouse retina is a good model to study these processes because it has seven distinct cell types, and three well-laminated nuclear layers that form during embryonic and postnatal life. β-catenin functions as both the nuclear effector for the canonical Wnt pathway and a cell adhesion molecule, and is required for the development of various organs. To study the function of β-catenin in retinal development, I used a Cre-loxP system to conditionally ablate β-catenin in the developing retina. Deletion of β-catenin led to disrupted laminar structure but did not affect the differentiation of any of the seven cell types. Eliminating β-catenin did not reduce progenitor cell proliferation, although enhanced apoptosis was observed. Further analysis showed that disruption of cell adhesion was the major cause of the observed patterning defects. Overexpression of β-catenin during retinal development also disrupted the normal retinal lamination and caused a transdifferentiation of neurons into pigmented cells. The results indicate that β-catenin functions as a cell adhesion molecule but not as a Wnt pathway component during retinal neurogenesis, and is essential for lamination but not cell differentiation. The results further imply that retinal lamination and cell differentiation are genetically separable processes. ^ Sonic hedgehog (shh) is expressed in retinal ganglion cells under the control of transcription factor Pou4f2 during retinal development. Previous studies identified a phylogenetically conserved region in the first intron of shh containing a Pou4f2 binding site. Transgenic reporter mice in which reporter gene expression was driven by this region showed that this element can direct gene expression specifically in the retina, but expression was not limited to the ganglion cells. From these data I hypothesized that this element is required for shh expression in the retina but is not sufficient for specific ganglion cell expression. To further test this hypothesis, I created a conditional allele by flanking this region with two loxP sites. Lines carrying this allele will be crossed with retinal-specific Cre lines to remove this element in the retina. My hypothesis predicts that alteration in shh expression and subsequent retinal defects will occur in the retinas of these mice. ^

Policy analysis of Texas Senate Bill 532 and the regulation of nurse practitioners in the convenience care model

Background. Retail clinics, also called convenience care clinics, have become a rapidly growing trend since their initial development in 2000. These clinics are coupled within a larger retail operation and are generally located in "big-box" discount stores such as Wal-mart or Target, grocery stores such as Publix or H-E-B, or in retail pharmacies such as CVS or Walgreen's (Deloitte Center for Health Solutions, 2008). Care is typically provided by nurse practitioners. Research indicates that this new health care delivery system reduces cost, raises quality, and provides a means of access to the uninsured population (e.g., Deloitte Center for Health Solutions, 2008; Convenient Care Association, 2008a, 2008b, 2008c; Hansen-Turton, Miller, Nash, Ryan, Counts, 2007; Salinsky, 2009; Scott, 2006; Ahmed & Fincham, 2010). Some healthcare analysts even suggest that retail clinics offer a feasible solution to the shortage of primary care physicians facing the nation (AHRQ Health Care Innovations Exchange, 2010). ^ The development and performance of retail clinics is heavily dependent upon individual state policies regulating NPs. Texas currently has one of the most highly regulated practice environments for NPs (Stout & Elton, 2007; Hammonds, 2008). In September 2009, Texas passed Senate Bill 532 addressing the scope of practice of nurse practitioners in the convenience care model. In comparison to other states, this law still heavily regulates nurse practitioners. However, little research has been conducted to evaluate the impact of state laws regulating nurse practitioners on the development and performance of retail clinics. ^ Objectives. (1). To describe the potential impact that SB 532 has on retail clinic performance. (2). To discuss the effectiveness, efficiency, and equity of the convenience care model. (3). To describe possible alternatives to Texas' nurse practitioner scope of practice guidelines as delineated in Texas Senate Bill 532. (4). To describe the type of nurse practitioner state regulation (i.e. independent, light, moderate, or heavy) that best promotes the convenience care model. ^ Methods. State regulations governing nurse practitioners can be characterized as independent, light, moderate, and heavy. Four state NP regulatory types and retail clinic performance were compared and contrasted to that of Texas regulations using Dunn and Aday's theoretical models for conducting policy analysis and evaluating healthcare systems. Criteria for measurement included effectiveness, efficiency, and equity. Comparison states were Arizona (Independent), Minnesota (Light), Massachusetts (Moderate), and Florida (Heavy). ^ Results. A comparative states analysis of Texas SB 532 and alternative NP scope of practice guidelines among the four states: Arizona, Florida, Massachusetts, and Minnesota, indicated that SB 532 has minimal potential to affect the shortage of primary care providers in the state. Although SB 532 may increase the number of NPs a physician may supervise, NPs are still heavily restricted in their scope of practice and limited in their ability to act as primary care providers. Arizona's example of independent NP practice provided the best alternative to affect the shortage of PCPs in Texas as evidenced by a lower uninsured rate and less ED visits per 1,000 population. A survey of comparison states suggests that retail clinics thrive in states that more heavily restrict NP scope of practice as opposed to those that are more permissive, with the exception of Arizona. An analysis of effectiveness, efficiency, and equity of the convenience care model indicates that retail clinics perform well in the areas of effectiveness and efficiency; but, fall short in the area of equity. ^ Conclusion. Texas Senate 532 represents an incremental step towards addressing the problem of a shortage of PCPs in the state. A comparative policy analysis of the other four states with varying degrees of NP scope of practice indicate that a more aggressive policy allowing for independent NP practice will be needed to achieve positive changes in health outcomes. Retail clinics pose a temporary solution to the shortage of PCPs and will need to expand their locations to poorer regions and incorporate some chronic care to obtain measurable health outcomes. ^

Regulation of myogenesis by growth signals -growth factors, oncogenes and protein kinases

Establishment of a myogenic phenotype involves antagonism between cell proliferation and differentiation. The recent identification of the MyoD family of muscle-specific transcription factors provides opportunities to dissect at the molecular level the mechanisms through which defined cell type-specific transcription factors respond to environmental cues and regulate differentiation programs. This project is aimed at elucidation of the molecular mechanism whereby growth factors repress myogenesis. Initial studies demonstrated that nuclear oncogenes such as c-fos, junB and c-jun are immediate early genes that respond to serum and TGF-$\beta$. Using the muscle creatine kinase (MCK) enhancer linked to the reporter gene CAT as a marker for differentiation, we showed that transcriptional function of myogenin can be disrupted in the presence of c-Fos, JunB and cjun. In contrast, JunD, which shares DNA-binding specificity with JunB and c-Jun but is expressed constitutively in muscle cells, failed to show the inhibition. The repression by Fos and Jun is targeted at KE-2 motif, the same sequence that mediates myogenin-dependent activation and muscle-specific transactivation. Deletion analysis indicated that the transactivation domain of c-Jun at the N-terminus is responsible for the repression. Considering that myogenin is a phosphoprotein and cAMP and TPA are able to regulate myogenesis, we examined whether constitutively active protein kinase C (PKC) and protein kinase A (PKA) could substitute for exogenous growth factors and prevent transcription activation by myogenin. Indeed, the basic region of myogenin is phosphorylated by PKC at a threonine that is conserved in all members of the MyoD family. Phosphorylation at this site attenuates DNA binding activity of myogenin. Protein kinase A can also phosphorylate myogenin in a region adjacent to the DNA binding domain. However, phosphorylation at this site is insufficient to abrogate myogenin's DNA binding capacity, suggesting that PKA and PKC may affect myogenin transcriptional activity through different mechanisms. These findings provide insight into the mechanisms through which growth factor signals negatively regulate the muscle differentiation program and contribute to an understanding of signal transducing pathways between the cell membrane and nucleus. ^