Reproducibility of Serum Potassium Values in Serum From Blood Samples Stored for Increasing Times Prior to Centrifugation and Analysis.

GOALS: The goal of this work was to determine if immediate versus postponed centrifugation of samples affects the levels of serum potassium. METHODS: Twenty participants donated normal venous blood that was collected in four serum separator tubes per donor, each of which was analyzed at 0, 1, 2, or 4 hr on the Siemens Advia 1800 autoanalyzer. RESULTS: Coefficients of variation (CVs) for potassium levels ranged from 0% to 7.6% with a mean of 3 ± 2%. ANOVA testing of the means for all 20 samples showed a P-value of 0.72 (>0.05) indicating that there was no statistically significant difference between the means of the samples at the four time points. Sixteen samples were found to have CVs that were ≤5%. Two samples showed increases of potassium from the reference range to levels higher than the upper reference limit, one of which had a 4-hr value that was within the reference or normal range (3.5-5 mEq/l). Overall, most samples were found to have reproducible levels of serum potassium. CONCLUSIONS: Serum potassium levels from stored whole blood collected in serum separator tubes are, for the most part, stable at room temperature for at least 4 hr prior to analysis. However, some samples can exhibit significant fluctuations of values.

Poptosis or Peptide-Induced Transmembrane Pore Formation: A Novel Way to Kill Cancer Cells without Affecting Normal Cells.

Recent advances in cancer treatment like personalized chemotherapy and immunotherapy are aimed at tumors that meet certain specifications. In this review, we describe a new approach to general cancer treatment, termed peptide-induced poptosis, in which specific peptides, e.g., PNC-27 and its shorter analogue, PNC-28, that contain the segment of the p53 transactivating 12-26 domain that bind to HDM-2 in its 1-109 domain, bind to HDM-2 in the membranes of cancer cells, resulting in transmembrane pore formation and the rapid extrusion of cancer cell contents, i.e., tumor cell necrosis. These peptides cause tumor cell necrosis of a wide variety of solid tissue and hematopoietic tumors but have no effect on the viability and growth of normal cells since they express at most low levels of membrane-bound HDM-2. They have been found to successfully treat a highly metastatic pancreatic tumor as well as stem-cell-enriched human acute myelogenous leukemias in nude mice, with no evidence of off-target effects. These peptides also are cytotoxic to chemotherapy-resistant cancers and to primary tumors. We performed high-resolution scanning immuno-electron microscopy and visualized the pores in cancer cells induced by PNC-27. This peptide forms 1:1 complexes with HDM-2 in a temperature-independent step, followed by dimerization of these complexes to form transmembrane channels in a highly temperature-dependent step parallel to the mode of action of other membranolytic but less specific agents like streptolysin. These peptides therefore may be effective as general anti-cancer agents.

Anti-Cancer Peptide PNC-27 Kills Cancer Cells by Unique Interactions with Plasma Membrane-Bound hdm-2 and with Mitochondrial Membranes Causing Mitochondrial Disruption.

OBJECTIVE: We have previously shown that the anti-cancer peptide PNC-27 kills cancer cells by co-localizing with membrane-expressed HDM-2, resulting in transmembrane pore formation causing extrusion of intracellular contents. We have also observed cancer cell mitochondrial disruption in PNC-27-treated cancer cells. Our objectives are to determine: 1. if PNC-27 binds to the p53 binding site of HDM-2 (residues 1-109) in the cancer cell membrane and 2. if this peptide causes selective disruption of cancer cell mitochondria. METHODS: For aim 1, we incubated MIA-PaCa-2 human pancreatic carcinoma cells with PNC-27 in the presence of a monoclonal antibody against the amino terminal p53 binding site of HDM-2 to determine if it, but not negative control immune serum, blocks PNC-27-induced tumor cell necrosis. For the second aim, we incubated these cells with PNC-27 in the presence of two specific dyes that highlight normal organelle function: mitotracker for mitochondria and lysotracker for lysosomes. We also performed immuno-electron microscopy (IEM) with gold-labeled anti-PNC-27 antibody on the mitochondria of these cells treated with PNC-27. RESULTS: Monoclonal antibody to the p53 binding site of HDM-2 blocks PNC-27-induced cancer cell necrosis, whereas negative control immune serum does not. The mitochondria of PNC-27-treated cancer cells fail to retain mitotracker dye while their lysosomes retain lysotracker dye. IEM of the mitochondria cancer cells reveals gold particles present on the mitochondrial membranes. CONCLUSIONS: PNC-27 binds to the p53 binding site of HDM-2 (residues 1-109) inducing transmembrane pore formation and cancer cell necrosis. Furthermore, this peptide enters cancer cells and binds to the membranes of mitochondria, resulting in their disruption.

Stability of BUN and creatinine determinations on the Siemens Advia 1800 analyzer.

BACKGROUND: Serum creatinine values of patients tend to change as a result of the use of different blanks used for creatinine determinations on the Advia 1650. After upgrading the analyzer to the Advia 1800, creatinine values tended to be more reproducible. As part of a quality assurance investigation to test the reproducibilities of creatinine values, we determined serial creatinine values in the sera of 13 patients whose initial values were either in the reference range or elevated (range 0.58-7.8 mg/dl). These values were determined concurrently with serum blood urea nitrogen (BUN) determinations (range 6.0-84.4 mg/dl) as these two analytes are used together in evaluation of renal function. METHODS: We determined BUN and creatinine values, using the glutamate dehydrogenase lined enzyme assay system and the Jaffe method, respectively. RESULTS: We find that all values for creatinine on samples stored at 4 °C were reproducible as were the corresponding BUN values, which is revealed by low values for the coefficients of variation (CVs), that is, mean CV of 4.55% for creatinine and 2.52% for BUN. One sample with relatively high CV (10.6%) for creatinine was found to have an initial value of 1.1 mg/dl, in the reference range; but, on repeat determinations, the obtained levels were as high as 1.5 mg/dl, above the reference range. BUN values for this sample remained in the reference range, suggesting that no renal disease was present. CONCLUSION: We conclude that creatinine and BUN determinations are stable, but occasional spurious creatinine values can occur on the Advia 1800 analyzer.

Anticancer peptide PNC-27 adopts an HDM-2-binding conformation and kills cancer cells by binding to HDM-2 in their membranes.

The anticancer peptide PNC-27, which contains an HDM-2-binding domain corresponding to residues 12-26 of p53 and a transmembrane-penetrating domain, has been found to kill cancer cells (but not normal cells) by inducing membranolysis. We find that our previously determined 3D structure of the p53 residues of PNC-27 is directly superimposable on the structure for the same residues bound to HDM-2, suggesting that the peptide may target HDM-2 in the membranes of cancer cells. We now find significant levels of HDM-2 in the membranes of a variety of cancer cells but not in the membranes of several untransformed cell lines. In colocalization experiments, we find that PNC-27 binds to cell membrane-bound HDM-2. We further transfected a plasmid expressing full-length HDM-2 with a membrane-localization signal into untransformed MCF-10-2A cells not susceptible to PNC-27 and found that these cells expressing full-length HDM-2 on their cell surface became susceptible to PNC-27. We conclude that PNC-27 targets HDM-2 in the membranes of cancer cells, allowing it to induce membranolysis of these cells selectively.

Peptides That Block RAS-p21 Protein-Induced Cell Transformation.

This is a review of approaches to the design of peptides and small molecules that selectively block the oncogenic RAS-p21 protein in ras-induced cancers. Single amino acid substitutions in this protein, at critical positions such as at Gly 12 and Gln 61, cause the protein to become oncogenic. These mutant proteins cause over 90 percent of pancreatic cancers, 40-50 percent of colon cancers and about one third of non-small cell cancers of the lung (NSCCL). RAS-p21 is a G-protein that becomes activated when it exchanges GDP for GTP. Several promising approaches have been developed that target mutant (oncogenic) RAS-p21 proteins in these different cancers. These approaches comprise: molecular simulations of mutant and wild-type proteins to identify effector domains, for which peptides can be made that selectively inhibit the oncogenic protein that include PNC-1 (ras residues 115-126), PNC-2 (ras residues 96-110) and PNC7 (ras residues 35-47); the use of contiguous RAS-p21 peptide sequences that can block ras signaling; cyclic peptides from large peptide libraries and small molecule libraries that can be identified in high throughput assays that can selectively stabilize inactive forms of RAS-p21; informatic approaches to discover peptides and small molecules that dock to specific domains of RAS-p21 that can block mitogenic signal transduction by oncogenic RAS-p21; and the use of cell-penetrating peptides (CPPs) that are attached to the variable domains of the anti-RAS-p21 inactivating monoclonal antibody, Y13 259, that selectively enters oncogenic RAS-p21-containing cancer cells, causing these cells to undergo apoptosis. Several new anti-oncogenic RAS-p21 agents, i.e., Amgen's AMG510 and Mirati Therapeutics' MRTX849, polycyclic aromatic compounds, have recently been FDA-approved and are already being used clinically to treat RAS-p21-induced NSCCL and colorectal carcinomas. These new drugs target the inactive form of RAS-p21 bound to GDP with G12C substitution at the critical Gly 12 residue by binding to a groove bordered by specific domains in this mutant protein into which these compounds insert, resulting in the stabilization of the inactive GDP-bound form of RAS-p21. Other peptides and small molecules have been discovered that block the G12D-RAS-p21 oncogenic protein. These agents can treat specific mutant protein-induced cancers and are excellent examples of personalized medicine. However, many oncogenic RAS-p21-induced tumors are caused by other mutations at positions 12, 13 and 61, requiring other, more general anti-oncogenic agents that are being provided using alternate methods.

Ketone Bodies Induce Unique Inhibition of Tumor Cell Proliferation and Enhance the Efficacy of Anti-Cancer Agents.

The ketone bodies, sodium and lithium salts of acetoacetate (AcAc) and sodium 3-hydroxybutyrate (3-HB; commonly called beta-hydroxybutyrate) have been found to inhibit the proliferation of cancer cells. Previous studies have suggested that lithium itself may be an inhibiting agent but may be additive or synergistic with the effect of AcAc. We previously found that sodium acetoacetate (NaAcAc) inhibits the growth of human colon cancer cell line SW480. We report here similar results for several other cancer cell lines including ovarian, cervical and breast cancers. We found that NaAcAc does not kill cancer cells but rather blocks their proliferation. Similar inhibition of growth was seen in the effect of lithium ion alone (as LiCl). The effect of LiAcAc appears to be due to the combined effects of acetoacetate and the lithium ion. The ketone bodies, when given together with chemotherapeutic agents, rapamycin, methotrexate and the new peptide anti-cancer agent, PNC-27, substantially lowers their IC50 values for cancer cell, killing suggesting that ketone bodies and ketogenic diets may be powerful adjunct agents in treating human cancers.

Stability of electrolyte determinations on the Siemens Advia 1800 analyzer.

BACKGROUND: It is sometimes necessary for the laboratory to re-test samples for critical serum electrolyte levels. It is important to assure reproducibility of results when testing is performed on stored, refrigerated samples. We have tested the reproducibility of results for the critical electrolytes, Na, K, Cl and Ca, from ten randomly selected patients'sera over our maximum storage period of nine (9) days on the Siemens Advia 1800 analyzer. The ranges for each electrolyte were 131-150 meq/L (Na), 3.4-5.2 meq/L (K), 101-123 meq/L (Cl) and 7.3-9.9 mg/dL (Ca). METHODS: We used ion-selective electrodes for Na, K and Cl and the ortho-cresolphthalein dye method for Ca. RESULTS: We find that the reproducibility of determinations for all of these electrolytes was excellent, i.e. the coefficients of variation for each electrolyte determination for each patient were low. CONCLUSION: The methods of measurement for these electrolytes on the Advia 1800 are reliable and reproducible.

PNC-27, a Chimeric p53-Penetratin Peptide Binds to HDM-2 in a p53 Peptide-like Structure, Induces Selective Membrane-Pore Formation and Leads to Cancer Cell Lysis.

PNC-27, a 32-residue peptide that contains an HDM-2 binding domain and a cell-penetrating peptide (CPP) leader sequence kills cancer, but not normal, cells by binding to HDM-2 associated with the plasma membrane and induces the formation of pores causing tumor cell lysis and necrosis. Conformational energy calculations on the structure of PNC-27 bound to HDM-2 suggest that 1:1 complexes form between PNC-27 and HDM-2 with the leader sequence pointing away from the complex. Immuno-scanning electron microscopy was carried out with cancer cells treated with PNC-27 and decorated with an anti-PNC-27 antibody coupled to 6 nm gold particles and an anti-HDM-2 antibody linked to 15 nm gold particles. We found multiple 6 nm- and 15 nm-labeled gold particles in approximately 1:1 ratios in layered ring-shaped structures in the pores near the cell surface suggesting that these complexes are important to the pore structure. No pores formed in the control, PNC-27-treated untransformed fibroblasts. Based on the theoretical and immuno-EM studies, we propose that the pores are lined by PNC-27 bound to HDM-2 at the membrane surface with the PNC-27 leader sequence lining the pores or by PNC-27 bound to HDM-2.

Molecular Targeting of H/MDM-2 Oncoprotein in Human Colon Cancer Cells and Stem-like Colonic Epithelial-derived Progenitor Cells.

BACKGROUND/AIM: We have tested whether the anticancer peptide, PNC-27, that kills cancer cells but not normal cells by binding to cancer cell membrane HDM-2 forming pores, kills CD44+ colon cancer stem cells. MATERIALS AND METHODS: Flow cytometry determined the CD44 and HDM-2 expression on six-colon cancer cell lines and one normal cell line (CCD-18Co). MTT, LDH release, annexin V binding and caspase 3 assays were used to assess PNC-27-induced cell death. Bioluminescence imaging measured PNC-27 effects on in vivo tumor growth. RESULTS: High percentages of cells in all six tumor lines expressed CD44. PNC-27 co-localized with membrane HDM-2 only in the cancer cells and caused total cell death (tumor cell necrosis, high LDH release, negative annexin V and caspase 3). In vivo, PNC-27 caused necrosis of tumor nodules but not of normal tissue. CONCLUSION: PNC-27 selectively kills colon cancer stem cells by binding of this peptide to membrane H/MDM-2.

Unique Features of Prostate Cancer in African American and West Indian Patients Including Diagnosis of High Grade Cancers Using Only Elevated Serum Levels of Prostate Specific Antigen.

OBJECTIVE: We have studied the occurrence and nature of prostate cancer in 330 African American patients with respect to its frequency of occurrence, the prevalence of high grade cancers (Gleason score ≥7), distribution of prostate specific antigen (PSA) levels, whether serum PSA levels correlate with Gleason scores, and whether tumor grade correlates with tumor extension and/or metastasis. METHODS: We reviewed the medical charts of patients at the University Hospital of SUNY Downstate Medical Center for whom prostate biopsies or excisions were performed (2015-2019). We then computed the prevalence of prostate cancer and high grade tumors. To determine if there was a quantitative relationship between PSA and Gleason score, we used linear regression analysis. We further used the Fisher exact test to determine if there exists a serum PSA level beyond which the diagnosis of high-grade prostate cancer is definitive. RESULTS: The prevalence of prostate cancer was high at 75.8%; of these cancers, 70% were found to be high grade. Ninety two percent of PSA values were ≥ 4.0ng/mL; the sensitivity was 94%; the positive predictive value was 80%. There was a poor correlation between PSA and Gleason score (R2=0.1), but almost 30 percent of PSA values were >20 ng/mL, and almost all of these corresponded to high grade tumors (Fisher exact test, p<0.00001, α=0.05). Fifteen cancers extended beyond the capsule or metastasized; all were high grade tumors. CONCLUSIONS: These patients presented with a high frequency of high-grade prostate cancer and elevated PSA values, such that PSA> 20ng/mL are virtually diagnostic of high-grade prostate cancer. Since the average age, 65, of screening for biopsy in this population is the same as for other demographic groups, screening should be performed at younger ages in this population.

Low Energy Conformations for Endogenous Mu-Receptor-Specific Peptides.

We have computed the low energy minima for the two endomorphin peptides, N-acetyl-Tyr-Pro-Trp-Phe-NHCH3 (endomorphin 1) and Tyr-Pro-Phe-Phe-NHCH3 (endomorphin 2) in aqueous solution. These peptides block pain without inducing the harmful side effects of the opiates that bind to the same mu opiate receptor but have short half lives. From over 1000 starting conformations for each peptide, we find less than 200 low energy structures whose conformational energies were ≤ 5 kcal/mole of the energy of the global minimum. The most probable conformations calculated using the Boltzmann distribution for both peptides were similar to one another. Using the letter representation for backbone conformational states, these most probable structures were D A E E for endomorphin 1 and E A E E for endomorphin 2. Both of these structures form reverse turns at Pro 2-Trp (Phe) 3 resulting in the juxtaposition of the aromatic rings of Tyr 1 and Phe 4. The Trp residue of endomorphin 1 points to the back of the reverse turn. These features may be useful in the design of non-peptide analogues that will have longer half-lives than the peptides.

Targeting Membrane HDM-2 by PNC-27 Induces Necrosis in Leukemia Cells But Not in Normal Hematopoietic Cells.

BACKGROUND/AIM: Anticancer peptide PNC-27 binds to HDM-2 protein on cancer cell membranes inducing the formation of cytotoxic transmembrane pores. Herein, we investigated HDM-2 membrane expression and the effect of PNC-27 treatment on human non-stem cell acute myelogenous leukemia cell lines: U937, acute monocytic leukemia; OCI-AML3, acute myelomonocytic leukemia and HL60, acute promyelocytic leukemia. MATERIALS AND METHODS: We measured cell surface membrane expression of HDM-2 using flow cytometry. Cell viability was assessed using MTT assay while direct cytotoxicity was measured by lactate dehydrogenase (LDH) release and induction of apoptotic markers annexin V and caspase-3. RESULTS: HDM-2 is expressed at high levels in membranes of U937, OCI-AML3 and HL-60 cells. PNC-27 can bind to membrane HDM-2 to induce cell necrosis and LDH release within 4 h. CONCLUSION: Targeting membrane HDM-2 can be a potential strategy to treat leukemia. PNC-27 targeting membrane HDM-2 demonstrated significant anti-leukemia activity in a variety of leukemic cell lines.

Anti-Cancer Tumor Cell Necrosis of Epithelial Ovarian Cancer Cell Lines Depends on High Expression of HDM-2 Protein in Their Membranes.

OBJECTIVE: Patients with epithelial ovarian cancers experience the highest fatality rates among all gynecological malignancies which require development of novel treatment strategies. Tumor cell necrosis was previously reported in a number of cancer cell lines following treatment with a p53-derived anti-cancer peptide called PNC-27. This peptide induces necrosis by transmembrane pore formation with HDM-2 protein that is expressed in the cancer cell membrane. We aimed to extend these studies further by investigating expression of membrane HDM-2 protein in ovarian cancer as it relates to susceptibility to PNC-27. PROCEDURES: Herein, we measured HDM-2 membrane expression in two ovarian cancer cell lines (SKOV-3 and OVCAR-3) and a non-transformed control cell line (HUVEC) by flow cytometric and western blot analysis. Immunofluorescence was used to visualize colocalization of PNC-27 with membrane HDM-2. Treatment effects with PNC-27 and control peptide were assessed using a MTT cell proliferation assay while direct cytotoxicity was measured by lactate dehydrogenase (LDH) release and induction of apoptotic markers; annexin V and caspase-3. RESULTS: HDM-2 protein was highly expressed and frequently detected in the membranes of SKOV-3 and OVCAR-3 cells; a prominent 47.6 kDa HDM-2 plasma membrane isoform was present in both cell lines whereas 25, 29, and 30 kDa isoforms were preferentially expressed in OVCAR-3. Notably, PNC-27 colocalized with HDM-2 in the membranes of both cancer cell lines that resulted in rapid cellular necrosis. In contrast, no PNC-27 colocalization and cytotoxicity was observed with non-transformed HUVEC demonstrating minimal expression of membrane HDM-2. CONCLUSIONS: Our results suggest that HDM-2 is highly expressed in the membranes of these ovarian cancer cell lines and colocalizes with PNC-27. We therefore conclude that the association of PNC-27 with preferentially expressed membrane HDM-2 isoforms results in the proposed model for the formation of transmembrane pores and epithelial ovarian cancer tumor cell necrosis, as previously described in a number of solid tissue and hematologic malignancies.

Conformational effects of a common codon 751 polymorphism on the C-terminal domain of the xeroderma pigmentosum D protein.

AIM: The xeroderma pigmentosum D (XPD) protein is a DNA helicase involved in the repair of DNA damage, including nucleotide excision repair (NER) and transcription-coupled repair (TCR). The C-terminal domain of XPD has been implicated in interactions with other components of the TFIIH complex, and it is also the site of a common genetic polymorphism in XPD at amino acid residue 751 (Lys->Gln). Some evidence suggests that this polymorphism may alter DNA repair capacity and increase cancer risk. The aim of this study was to investigate whether these effects could be attributable to conformational changes in XPD induced by the polymorphism. MATERIALS AND METHODS: Molecular dynamics techniques were used to predict the structure of the wild-type and polymorphic forms of the C-terminal domain of XPD and differences in structure produced by the polymorphic substitution were determined. RESULTS: The results indicate that, although the general configuration of both proteins is similar, the substitution produces a significant conformational change immediately N-terminal to the site of the polymorphism. CONCLUSION: These results provide support for the hypothesis that this polymorphism in XPD could affect DNA repair capability, and hence cancer risk, by altering the structure of the C-terminal domain.

The penetratin sequence in the anticancer PNC-28 peptide causes tumor cell necrosis rather than apoptosis of human pancreatic cancer cells.

BACKGROUND: PNC-27 and PNC-28 are p53-derived peptides from the human double minute (hdm-2) binding domain attached to penetratin. These peptides induce tumor cell necrosis of cancer cells, but not normal cells. The anticancer activity and mechanism of PNC-28 (p53 aa17-26-penetratin) was specifically studied against human pancreatic cancer. METHODS: MiaPaCa-2 cells were treated with PNC-28. Necrosis was determined by measuring lactate dehydrogenase (LDH) and apoptosis as assayed for measuring elevation of proapoptotic proteins. PNC-29, an unrelated peptide, and hdm-2-binding domain p53 aa12-26 without penetratin (PNC-26) were used as controls. Since there is evidence that penetratin is required for tumor cell necrosis, we tested "naked" p53 peptide without penetratin by transfecting a plasmid that encodes p53 aa17-26 segment of PNC-28 into MiaPaCa-2 and an untransformed rat pancreatic acinar cell line, BMRPA1. Time-lapse electron microscopy was employed to further elucidate anticancer mechanism. RESULTS: Treatment with PNC-28 does not result in the elevation of proapoptotic proteins found in p53-induced apoptosis, but elicits rapid release of LDH, indicative of tumor cell necrosis. Accordingly, we observed membrane pore formation and dose-dependent killing. In direct contrast, transfected MiaPaCa-2 cells underwent apoptosis, and not necrosis, as evidenced by expression of high levels of caspases-3 and 7 and annexin V with background levels of LDH. CONCLUSION: These results suggest that PNC-28 may be effective in treating human pancreatic cancer. The penetratin sequence appears to be responsible for the fundamental change in the mechanism of action, inducing rapid necrosis initiated by membrane pore formation. Cancer cell death by apoptosis was observed in the absence of penetratin.

Two peptides derived from ras-p21 induce either phenotypic reversion or tumor cell necrosis of ras-transformed human cancer cells.

PURPOSE: We investigated the effects of two peptides from the ras-p21 protein, corresponding to residues 35-47 (PNC-7) and 96-110 (PNC-2), on two ras-transformed human cancer cell lines, HT1080 fibrosarcoma and MIAPaCa-2 pancreatic cancer cell lines. In prior studies, we found that both peptides block oncogenic, but not insulin-activated wild-type, ras-p21-induced oocyte maturation. When linked to a transporter penetratin peptide, these peptides induce reversion of ras-transformed rat pancreatic cancer cells (TUC-3) to the untransformed phenotype. METHODS: These peptides and a control peptide, linked to a penetratin peptide, were incubated with each cell lines. Cell counts were obtained over several weeks. The cause of cell death was determined by measuring caspase as an indicator of apoptosis and lactate dehydrogenase (LDH) as marker of necrosis. Since both peptides block the phosphorylation of jun-N-terminal kinase (JNK) in oocytes, we blotted cell lysates of the two cancer cell lines for the levels of phosphorylated JNK to determine if the peptides reduced these levels. RESULTS: We find that both peptides, but not control peptides linked to the penetratin sequence, induce phenotypic reversion of the HT-1080 cell line but cause tumor cell necrosis of the MIA-PaCa-2 cell line. On the other hand, neither peptide has any effect on the viability of an untransformed pancreatic acinar cell line, BMRPA1. We find that, while total JNK levels remain constant during peptide treatment, phosphorylated JNK levels decrease dramatically, consistent with the mechanisms of action of these peptides. CONCLUSION: We conclude that these peptides block tumor but not normal cell growth likely by blocking oncogenic ras-p21-induced phosphorylation of JNK, an essential step on the oncogenic ras-p21-protein pathway. These peptides are therefore promising as possible anti-tumor agents.

IgG anti-cardiomyocyte antibodies in giant cell myocarditis.

Giant cell myocarditis, a rare, fatal, and poorly understood cause of myocarditis, requires pathological examination for diagnosis. It is considered to be an autoimmune disease and is frequently associated with other conditions, in particular thymoma and myasthenia gravis. The typical patient with giant cell myocarditis is young and has severe, progressive congestive cardiac failure that is unresponsive to standard medical therapy and ultimately requires cardiac transplantation. Hence giant cell myocarditis is the most dangerous form of myocarditis. Here we report an unusual presentation of giant cell myocarditis, which mimicked acute myocardial infarction in an elderly woman with myasthenia gravis and a previous diagnosis of thymoma. This patient had evidence of anti-myocyte antibodies, consistent with an autoimmune mechanism.

Site-specific phosphorylation of raf in cells containing oncogenic ras-p21 is likely mediated by jun-N-terminal kinase.

In a study of interactions between the raf-MEK-MAPK (ERK) and JNK-jun pathways, we found previously that JNK can induce phosphorylation of raf but not vice versa. In this study, we investigate the nature of the JNK-induced phosphorylation of raf. In in vitro experiments in which immunobead-bound raf is phosphorylated by activated JNK, we find strong phosphorylation signals at raf-Ser259 and Ser338. The Ser259 phosphorylation is surprising since it is associated with inhibition of migration of raf to the cell membrane where it can interact with ras-p21. We also find that in oocytes induced to mature with oncogenic ras-p21, which induces high levels of phosphorylated JNK and MAPK, the same pattern of phosphorylation of raf occurs. In contrast, in oocytes induced to mature with insulin, which requires activation of wild-type ras-p21, phosphorylation of raf-Ser338 but not raf-Ser259 occurs. In oncogenic ras-transformed human pancreatic cancer MIA-PaCa-2 cells, phosphorylation of both raf serines occurs. Treatment of these cells with the ras peptide, PNC-2 attached to a penetrating sequence that blocks JNK and MAPK phosphorylation and induces tumor cell necrosis, results in a marked decrease in phosphorylation of raf-Ser259, but not that of raf-Ser338. These results suggest that oncogenic ras-p21 induces phosphorylation of both raf-Ser259 and Ser338 and that raf-Ser 259 phosphorylation may be effected by activated JNK.

Low Energy Conformations for S100 Binding Peptide from the Negative Regulatory Domain of p53.

We have computed the low energy conformations of the negative regulatory domain of p53, residues 374-388 using Empirical Conformational Energies of Peptides Program including solvation and computed the statistical weights of distinct conformational states. We find that there are two high probability conformations, one an α-helix from Lys 374-Lys 381, followed by another helical structure involving Lys 382-Glu 388 (statistical weight of 0.48) and an all-α-helix for the entire sequence (statistical weight of 0.23). Both structure are superimposable on the NMR structure of this sequence bound to the S100 protein. The global minimum structure (statistical weight of 0.014) is a beta structure from Gly 374-Arg 379 followed by an α-helix from His 380-Glu 388. Based on these results, we propose a possible strategy for enhancement of p53 anti-tumor activity in cancer cells. Since the structure of this sequence bound to the sirtuin protein, Sir2, is a ß-sheet, we further propose that the global minimum may be an intermediate on the α-ß structure transition.