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PubMed2001

How to build a pathway program from scratch.

Starting a pathway program from the ground up requires specific building blocks and certain personality traits: persistence, flexibility, and the ability to work with others. Carol Freeborn, RN, started a pathway program in the early 1990s at Mercy Hospital in Toledo, OH. She shares her experience and advice for others who are starting pathway programs in their own facilities.

Case Management↗
PubMed2005

Inhibiting the expression of CD28 costimulatory molecule on human lymphocytes by special siRNA.

BACKGROUND: The B7/CD28 pathway provides critical costimulatory signals for complete T cell activation, and members of this pathway have served as useful targets for immunotherapeutic strategies. In this study, we investigated the RNA interference (RNAi) effect induced by small interfering RNA (siRNA) targeting CD28 mRNA on human lymphocytes and its specificity. METHODS: According to CD28 gene sequence, we designed and synthysized three different siRNAs (siRNA-1, siRNA-2, siRNA-3) containing 21 bases using Silencertrade mark siRNA construction kit. These siRNAs were transfected into freshly isolated human lymphocytes with Lipofectamine 2000 reagent. At 24-hour, 48-hour and 72-hour post transfection, these cells were collected and analyzed. The changes of surface expression of CD28 gene were detected by flow cytometry, and the changes of CD28 mRNA levels were determined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). The cell viability of transfected lymphocytes was determined by methyl thiazolyl tetrazolium (MTT) assay and trypan blue dye exclusion assay. RESULTS: Three siRNAs (siRNA-1, siRNA-2, siRNA-3) specifically targeting CD28 mRNA were successfully designed and constructed. Flow cytometry analysis showed that a decrease in CD28 expression was detectable at 24-hour post transfection. Different siRNA showed different inhibition effects on CD28 expression. At 48-hour post transfection, the degrees of reduction with siRNA-1, siRNA-2 and siRNA-3 were 22.10% +/- 1.63%, 73.50% +/- 1.02% and 42.90% +/- 0.89% respectively compared with the control (P < 0.001). Neither of the groups transfected only with siRNA or lipo showed marked reduction in CD28 expression (3.15% +/- 0.75% and 4.55% +/- 0.80%) (P > 0.05). Moreover, lymphocytes treated with siRNA-co showed no marked reduction in CD28 expression (5.07% +/- 0.96%) (P > 0.05). The results of semi-quantitative RT-PCR assay indicated CD28 mRNA level was inhibited after transfection of specific siRNAs. At least 4-fold of reduction in siRNA-2 group occurred at 48-hour post transfection compared with the control (P < 0.001). MTT assay and trypan blue dye exclusion assay demonstrated that the viable cell rations of transfected lymphocytes were significantly reduced in siRNA-1, siRNA-2 and siRNA-3 groups at 48-hour post transfection (P < 0.01). The control groups showed no marked reduction in cell viability (P > 0.05). CONCLUSIONS: Three different siRNAs were synthesized and transfected into lymphocytes. They could reduce the expression of CD28 and the CD28 mRNA level. siRNA-2 was the most efficient. The cell viability reduced correspondingly. Therefore, the silencing effect on CD28 mRNA induced by siRNA may contribute to costimulatory blockade. This result show that siRNA may be useful for further study on graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (allo-BMT).

Adolescent↗
PubMed2005

Inhibition of either phosphatidylinositol 3-kinase/Akt or the mitogen/extracellular-regulated kinase, MEK/ERK, signaling pathways suppress growth of breast cancer cell lines, but MEK/ERK signaling is critical for cell survival.

The phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen/extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathways are important integrators of growth and survival signals originating from extracellular stimuli. We assessed the importance of these signaling pathways in the growth and survival of 8 breast cell lines (MCF10A, an immortalized line; and 7 cancer cell lines). The cell lines expressed variable levels of both phosphorylated ERK and phosphorylated Akt, but these were unchanged by incubation in serum-free medium. Despite continued activity of these pathways, the cells arrested growth in the absence of serum demonstrating that additional pathways are required for growth. Incubation with the PI3K inhibitor LY294002 suppressed growth of all cell lines, but most remained viable for at least 7-14 days. This long-term survival may be attributable to recovery of phospho-Akt by 24-48 h despite the continued presence of active LY294002, suggesting that alternate pathways may be activating Akt. In contrast, incubation with the MEK inhibitor U0126 not only arrested growth, but also killed all the cell lines within 2-4 days in the absence of serum; the presence of serum only slighted extended viability, except in MCF10A and MDA-MB-468 cells, in which serum provided significantly greater protection. It is likely that these signaling pathways control the level of pro-and anti-apoptotic proteins, yet assessment of Bcl-2 and Bcl-X showed dramatic reduction in level only when large numbers of cells were dead suggesting this may be a consequence rather than cause of death. Overall, the results demonstrate that the MEK/ERK pathway represents the more critical pathway for cell survival of these breast cancer cell lines, and suggest this pathways represents the better target for cancer therapy.

Blotting, Western↗
PubMed1987

Effect of 6-hydroxydopamine-induced lesions to ascending and descending noradrenergic pathways on morphine analgesia.

A systematic study of the role of descending, ascending and both aspects of noradrenergic pathways in the analgesic action of morphine was undertaken. The neurotoxin 6-hydroxydopamine (6-OHDA) was microinjected into the medullary A1 region, the dorsal bundle (DB), locus coeruleus (LC) or the cerebral ventricles (i.c.v.) to deplete noradrenaline (NA) in these pathways. The analgesic effect of systemically administered morphine 7-15 mg/kg was generally tested 7-12 days postlesion, and at the end of the experiment, brain and spinal cord regions were extracted and NA and dopamine (DA) measured by HPLC to verify the placement of lesions. Medullary A1 lesions profoundly depleted spinal cord NA with only a modest effect on mesencephalic levels. Such lesions inhibited the effect of morphine in a pressure test, but not in thermal tests (tail flick and hot plate) for nociception. DB lesions reduced NA in the cortex, hippocampus, hypothalamus and midbrain, but not in the spinal cord, and potentiated morphine analgesia in thermal tests for nociception. DA levels in the striatum were normal in this group. In another group where different stereotaxic coordinates were used, the pattern of NA depletion was similar, but DA levels in the striatum were reduced. In this group, potentiation of analgesia was no longer observed. LC lesions depleted NA throughout the neuraxis and potentiated morphine analgesia in both pressure and thermal tests for nociception. I.c.v. 6-OHDA depleted NA to a comparable degree to LC lesions, but striatal DA levels also were reduced and potentiation of morphine analgesia was not observed. These results indicate that central NA pathways are critical to the expression of morphine analgesia. The effect of depletion of NA in both ascending and descending aspects is the same as depletion in ascending pathways only, suggesting that this pathway is an important mediator of morphine analgesia. Simultaneous depletion of DA in the striatum can reverse the potentiating action of NA depletion indicating a critical role for DA with respect to NA pathways and mechanisms of analgesia.

Analgesia↗
PubMed1999

Influence of critical storage temperatures on degradative pathways of pigments in green beans (Phaseolus vulgaris cvs. Perona and boby).

In this work a study of critical storage temperatures on pigment degradation of green beans (Phaseolus vulgaris, cvs. Perona and Boby) was conducted. In this way, green beans kept better quality at 4 degrees C than either 8 or 12 degrees C, maintaining a bright green color and good texture. Nevertheless, temperatures of 4 degrees C induced chilling injury (CI) after eight days of storage, which became evident when the pods were transferred to 20 degrees C. Cold storage temperatures, 12, 8, and 4 degrees C, produced different changes on the green beans chlorophyll profile. Green beans of both cultivars, Perona and Boby, stored at 4 and 12 degrees C showed a continuous degradation of chlorophyll pigments during storage, while samples stored at 8 degrees C showed an increase of chlorophyll content at the first 15 days. Carotenoid pigments also suffered different changes during cold storage. Perona was the green beans cultivar which maintained the higher level of lutein, mainly when samples were stored at the most suitable temperature (8 degrees C).

Fabaceae↗
PubMed2002

Critical role of Rho-kinase pathway for cardiac performance and remodeling in failing rat hearts.

OBJECTIVES: Rho and Rho-kinase play a critical role in the regulation of cellular functions such as proliferation and migration. To elucidate the molecular mechanisms that regulate cardiac function and cardiovascular remodeling, we determined whether the signaling pathway through Rho is involved in Dahl salt-sensitive hypertensive rats with congestive heart failure (CHF) using a specific Rho-kinase inhibitor, Y-27632. METHODS: Y-27632 was administered from the left ventricular hypertrophy stage (11 weeks) to the CHF stage (18 weeks) for 7 weeks. The left ventricular end-systolic pressure-volume relationship (contractility: E(es)) was evaluated using a conductance catheter. RESULTS: Downregulated E(es) in the CHF stage was significantly ameliorated by Y-27632 treatment. Increased RhoA protein, Rho-kinase gene expression and myosin light chain phosphorylations in CHF rats were suppressed by Y-27632. Upregulated proto-oncogene c-fos gene expression in CHF rats was decreased by inhibiting Rho-kinase. In contrast, Y-27632 showed no effect on upregulated extracellular signal-regulated kinases (ERK) and p70S6 kinase phosphorylations, which were reported to be involved in protein synthesis. In the CHF stage, Y-27632 effectively inhibited vascular lesion formation such as medial thickness and perivascular fibrosis. CONCLUSIONS: These results suggest that differential activation of the Rho-Rho-kinase and the ERK-p70S6 kinase pathways may play a critical role in CHF, and the Rho-Rho-kinase pathway is involved in the pathogenesis of cardiac dysfunction and cardiovascular remodeling. Thus, inhibition of the Rho-kinase pathway may be at least a potential therapeutic strategy for CHF.

Amides↗
PubMed2002

A critical residue in the folding pathway of an integral membrane protein.

Although a number of common diseases are a direct consequence of membrane protein misfolding, studies of membrane protein folding and misfolding lag well behind those of soluble proteins. Here it is shown that an interfacial residue, Tyr16, of the integral membrane protein diacylglycerol kinase (DAGK) plays a critical role in the folding pathway of this protein. Properly folded Y16C exhibits kinetic parameters and stability similar to wild-type DAGK. However, when unfolded and then allowed to spontaneously fold in the presence of model membranes, Y16C exhibits dramatically lower rates and efficiencies of functional assembly compared to the wild-type protein. The Y16C mutant represents a class of mutations which may be commonly found in disease-related membrane proteins.

Amino Acid Sequence↗
PubMed2001

The casein kinase I family in Wnt signaling.

The canonical Wnt-signaling pathway is critical for many aspects of development, and mutations in components of the Wnt pathway are carcinogenic. Recently, sufficiency tests identified casein kinase Iepsilon (CKIepsilon) as a positive component of the canonical Wnt/beta-catenin pathway, and necessity tests showed that CKIepsilon is required in vertebrates to transduce Wnt signals. In addition to CKIepsilon, the CKI family includes several other isoforms (alpha, beta, gamma, and delta) and their role in Wnt sufficiency tests had not yet been clarified. However, in Caenorhabditis elegans studies, loss-of-function of a CKI isoform most similar to alpha produced the mom phenotype, indicative of loss-of-Wnt signaling. In this report, we examine the ability of the various CKI isoforms to activate Wnt signaling and find that all the wild-type CKI isoforms do so. Dishevelled (Dsh), another positive component of the Wnt pathway, becomes phosphorylated in response to Wnt signals. All the CKI isoforms, with the exception of gamma, increase the phosphorylation of Dsh in vivo. In addition, CKI directly phosphorylates Dsh in vitro. Finally, we find that CKI is required in vivo for the Wnt-dependent phosphorylation of Dsh. These studies advance our understanding of the mechanism of Wnt action and suggest that more than one CKI isoform is capable of transducing Wnt signals in vivo.

Adaptor Proteins, Signal Transducing↗