Structure-based design of 3-carboxy-substituted 1,2,3,4-tetrahydroquinolines as inhibitors of myeloid cell leukemia-1 (Mcl-1).

Mcl-1 has recently emerged as an attractive target to expand the armamentarium in the war on cancer. Using structure-based design, 3-carboxy-substituted 1,2,3,4-tetrahydroquinolines were developed as a new chemotype to inhibit the Mcl-1 oncoprotein. The most potent compound inhibited Mcl-1 with a Ki of 120 nM, as determined by a fluorescence polarization competition assay. Direct binding was confirmed by 2D (1)H-(15)N HSQC NMR spectroscopy with (15)N-Mcl-1, which indicated that interactions with R263 and T266, and occupation of the p2 pocket are likely responsible for the potent binding affinity. The short and facile synthetic chemistry to access target molecules is expected to mediate lead optimization.

Hair analysis versus conventional methods of drug testing in substance abusers seeking organ transplantation.

As substance abusers need to demonstrate abstinence prior to transplant, valid/reliable drug tests are needed. Patients may deny use, fearing surgery will be delayed. Breath, blood and urine tests have brief detection windows that allow patients to evade detection. Routine laboratory tests do not include all substances of abuse. Hair analysis overcomes these barriers, increasing the likelihood that active users will be identified. This study compared results for alcohol, opioids and cocaine based on 445 self-report, breath, urine and hair samples from 42 patients who had been denied a transplant due to recent substance abuse. Compared to hair toxicology, sensitivity for conventional drug tests was moderate for cocaine and opioids, but poor for alcohol. Of positive hair tests, only half were corroborated through other tests. In contrast, specificity was high across tests and substances, with positive findings from conventional tests confirmed through hair toxicology. Based on a 90-day detection window for hair analysis, two negative tests suggest 6 months of continuous abstinence. Hair testing should be considered as an alternative approach for monitoring substance use in the transplant population, either as a routine procedure or when the veracity of findings from conventional tests is in doubt.

Tyrosine-phosphorylated extracellular signal--regulated kinase associates with the Golgi complex during G2/M phase of the cell cycle: evidence for regulation of Golgi structure.

Phosphorylation of the extracellular signal-regulated kinases (ERKs) on tyrosine and threonine residues within the TEY tripeptide motif induces ERK activation and targeting of substrates. Although it is recognized that phosphorylation of both residues is required for ERK activation, it is not known if a single phosphorylation of either residue regulates physiological functions. In light of recent evidence indicating that ERK proteins regulate substrate function in the absence of ERK enzymatic activity, we have begun to examine functional roles for partially phosphorylated forms of ERK. Using phosphorylation site--specific ERK antibodies and immunofluorescence, we demonstrate that ERK phosphorylated on the tyrosine residue (pY ERK) within the TEY activation sequence is found constitutively in the nucleus, and localizes to the Golgi complex of cells that are in late G2 or early mitosis of the cell cycle. As cells progress through metaphase and anaphase, pY ERK localization to Golgi vesicles is most evident around the mitotic spindle poles. During telophase, pY ERK associates with newly formed Golgi vesicles but is not found on there after cytokinesis and entry into G1. Increased ERK phosphorylation causes punctate distribution of several Golgi proteins, indicating disruption of the Golgi structure. This observation is reversible by overexpression of a tyrosine phosphorylation--defective ERK mutant, but not by a kinase-inactive ERK2 mutant that is tyrosine phosphorylated. These data provide the first evidence that pY ERK and not ERK kinase activity regulates Golgi structure and may be involved in mitotic Golgi fragmentation and reformation.

Activation of the MKK/ERK pathway during somatic cell mitosis: direct interactions of active ERK with kinetochores and regulation of the mitotic 3F3/2 phosphoantigen.

The mitogen-activated protein (MAP) kinase pathway, which includes extracellular signal-regulated protein kinases 1 and 2 (ERK1, ERK2) and MAP kinase kinases 1 and 2 (MKK1, MKK2), is well-known to be required for cell cycle progression from G1 to S phase, but its role in somatic cell mitosis has not been clearly established. We have examined the regulation of ERK and MKK in mammalian cells during mitosis using antibodies selective for active phosphorylated forms of these enzymes. In NIH 3T3 cells, both ERK and MKK are activated within the nucleus during early prophase; they localize to spindle poles between prophase and anaphase, and to the midbody during cytokinesis. During metaphase, active ERK is localized in the chromosome periphery, in contrast to active MKK, which shows clear chromosome exclusion. Prophase activation and spindle pole localization of active ERK and MKK are also observed in PtK1 cells. Discrete localization of active ERK at kinetochores is apparent by early prophase and during prometaphase with decreased staining on chromosomes aligned at the metaphase plate. The kinetochores of chromosomes displaced from the metaphase plate, or in microtubule-disrupted cells, still react strongly with the active ERK antibody. This pattern resembles that reported for the 3F3/2 monoclonal antibody, which recognizes a phosphoepitope that disappears with kinetochore attachment to the spindles, and has been implicated in the mitotic checkpoint for anaphase onset (Gorbsky and Ricketts, 1993. J. Cell Biol. 122:1311-1321). The 3F3/2 reactivity of kinetochores on isolated chromosomes decreases after dephosphorylation with protein phosphatase, and then increases after subsequent phosphorylation by purified active ERK or active MKK. These results suggest that the MAP kinase pathway has multiple functions during mitosis, helping to promote mitotic entry as well as targeting proteins that mediate mitotic progression in response to kinetochore attachment.

Megakaryocytic differentiation induced by constitutive activation of mitogen-activated protein kinase kinase.

The K562 erythroleukemia cell line was used to study the molecular mechanisms regulating lineage commitment of hematopoietic stem cells. Phorbol esters, which initiate megakaryocyte differentiation in this cell line, caused a rapid increase in extracellular-signal-regulated kinase (ERK), which remained elevated for 2 h and returned to near-basal levels by 24 h. In the absence of extracellular stimuli, ERK could be activated by expression of constitutively active mutants of mitogen-activated protein (MAP) kinase kinase (MKK), resulting in cell adhesion and spreading, increased cell size, inhibition of cell growth, and induction of the platelet-specific integrin alphaIIb beta3, all hallmarks of megakaryocytic differentiation. In contrast, expression of wild-type MKK had little effect. In addition, constitutively active MKK suppressed the expression of an erythroid marker, alpha-globin, indicating the ability to suppress cellular responses necessary for alternative cell lineages. The MKK inhibitor PD98059 blocked MKK/ERK activation and cellular responses to phorbol ester, demonstrating that activation of MKK is necessary and sufficient to induce a differentiation program along the megakaryocyte lineage. Thus, the MAP kinase cascade, which promotes cell growth and proliferation in many cell types, instead inhibits cell proliferation and initiates lineage-specific differentiation in K562 cells, establishing a model system to investigate the mechanisms by which this signal transduction pathway specifies cell fate and developmental processes.

Extracellular signal-regulated kinase activates topoisomerase IIalpha through a mechanism independent of phosphorylation.

The mitogen-activated protein (MAP) kinases, extracellular signal-related kinase 1 (ERK1) and ERK2, regulate cellular responses by mediating extracellular growth signals toward cytoplasmic and nuclear targets. A potential target for ERK is topoisomerase IIalpha, which becomes highly phosphorylated during mitosis and is required for several aspects of nucleic acid metabolism, including chromosome condensation and daughter chromosome separation. In this study, we demonstrated interactions between ERK2 and topoisomerase IIalpha proteins by coimmunoprecipitation from mixtures of purified enzymes and from nuclear extracts. In vitro, diphosphorylated active ERK2 phosphorylated topoisomerase IIalpha and enhanced its specific activity by sevenfold, as measured by DNA relaxation assays, whereas unphosphorylated ERK2 had no effect. However, activation of topoisomerase II was also observed with diphosphorylated inactive mutant ERK2, suggesting a mechanism of activation that depends on the phosphorylation state of ERK2 but not on its kinase activity. Nevertheless, activation of ERK by transient transfection of constitutively active mutant MAP kinase kinase 1 (MKK1) enhanced endogenous topoisomerase II activity by fourfold. Our findings indicate that ERK regulates topoisomerase IIalpha in vitro and in vivo, suggesting a potential target for the MKK/ERK pathway in the modulation of chromatin reorganization events during mitosis and in other phases of the cell cycle.

Activation of extracellular signal-regulated kinase (ERK) in G2 phase delays mitotic entry through p21CIP1.

Extracellular signal-regulated kinase activity is essential for mediating cell cycle progression from G(1) phase to S phase (DNA synthesis). In contrast, the role of extracellular signal-regulated kinase during G(2) phase and mitosis (M phase) is largely undefined. Previous studies have suggested that inhibition of basal extracellular signal-regulated kinase activity delays G(2)- and M-phase progression. In the current investigation, we have examined the consequence of activating the extracellular signal-regulated kinase pathway during G(2) phase on subsequent progression through mitosis. Using synchronized HeLa cells, we show that activation of the extracellular signal-regulated kinase pathway with phorbol 12-myristate 13-acetate or epidermal growth factor during G(2) phase causes a rapid cell cycle arrest in G(2) as measured by flow cytometry, mitotic indices and cyclin B1 expression. This G(2)-phase arrest was reversed by pre-treatment with bisindolylmaleimide or U0126, which are selective inhibitors of protein kinase C proteins or the extracellular signal-regulated kinase activators, MEK1/2, respectively. The extracellular signal-regulated kinase-mediated delay in M-phase entry appeared to involve de novo synthesis of the cyclin-dependent kinase inhibitor, p21(CIP1), during G(2) through a p53-independent mechanism. To establish a function for the increased expression of p21(CIP1) and delayed cell cycle progression, we show that extracellular signal-regulated kinase activation in G(2)-phase cells results in an increased number of cells containing chromosome aberrations characteristic of genomic instability. The presence of chromosome aberrations following extracellular signal-regulated kinase activation during G(2)-phase was further augmented in cells lacking p21(CIP1). These findings suggest that p21(CIP1) mediated inhibition of cell cycle progression during G(2)/M phase protects against inappropriate activation of signalling pathways, which may cause excessive chromosome damage and be detrimental to cell survival.

Lithium and inositol: effects on brain water homeostasis in the rat.

RATIONALE: Since its earliest use in psychiatry, lithium has been known to alter body water homeostasis. Although lithium is also known to decrease the concentration of inositol, an important brain osmolyte, little is known of the effects of lithium on brain water homeostasis. OBJECTIVE: To determine whether lithium alters brain water homeostasis, and, if so, whether the mechanism involves changes in inositol concentration. MATERIALS AND METHODS: Rats were fed regular food or regular food plus lithium chloride for either 11 days or 5 weeks. Brains were dissected and assayed for tissue water by the wet-dry method and for inositol by gas chromatography-mass spectrometry. RESULTS: We found a statistically significant (p=0.05, corrected) 3.1% mean elevation in frontal cortex tissue water in 5-week lithium-fed rats (86.7+/-3.9%), compared to control rats (83.6+/-2.6%). Inositol concentration correlated inversely with percent tissue water (r=-0.50, p=0.003, corrected) in pooled samples of 5-week lithium-fed rats, and was significantly lower in frontal cortex and hippocampus of 5-week lithium-fed rats, compared to controls. Rats fed lithium for 11 days did not differ significantly from controls on either variable. CONCLUSIONS: This is the first report of a lithium-induced increase in brain tissue water. Although the mechanism is unclear, it does not appear to result from changes in brain inositol concentration or blood sodium concentration. This finding may have implications for the therapeutic or toxic effects of lithium on brain, because increased tissue water can augment cell excitability.

Effect of innervation on heart rate response to mental stress.

Heart transplant recipients provide a useful model for study of the autonomic control of the cardiovascular response to mental stress. Utilizing the innervated native atrial tissue of heart transplant recipients as an internal control exposed to the same circulatory milieu as the denervated graft heart was exposed to, the effect of innervation on the heart rate response to a mentally stressful arithmetic task was examined in eight subjects. Compared with the graft, the innervated atrial tissue manifested a larger heart rate increase during the task, larger heart rate decrease after the task, and more rapid rate of change in heart rate during the task and recovery periods. Thus, cardiac denervation results in a chronotropic response to mental arithmetic-induced stress that is blunted and more gradual than that of the innervated heart but not completely eliminated. The cardiac chronotropic response to mental arithmetic stress is dependent on both humoral factors and, predominantly, its direct autonomic innervation.

Nortriptyline treatment of depressed cardiac transplant recipients.

The safety of tricyclic antidepressants in cardiac transplant recipients has not been established. The author used nortriptyline to treat major depressive episodes in eight cardiac transplant recipients. Nortriptyline therapy was associated with increased QRS interval and heart rate but did not significantly affect other hemodynamic or ECG variables or cyclosporine dose requirements. It appears that nortriptyline may be used safely in depressed cardiac transplant patients.

Depression and the course of coronary artery disease.

Literature and folk wisdom have long linked depression and death; however, only recently have scientific studies examined the relation between them. Beginning in the 1970s, investigators compared mortality among patients treated for major depression and the general population. Nine of ten studies found an increased mortality from cardiovascular disease among depressed patients. However, such studies confound the relation between depression and its treatment. Community surveys circumvent this difficulty, but as these studies began to appear, other investigations revealed the strong association between depression and cigarette smoking, which made obvious a need to control for smoking. The first study to do this appeared in 1993, and not only did a relation between depression and mortality persist, but a relation between depression and the development of ischemic disease was revealed. In the past 2 years, six more community surveys have followed populations initially free of disease, and five have observed an increased risk of ischemic heart disease among depressed persons. Another research strategy is to start with subjects who have preexisting cardiovascular disease. Here, too, depression has consistently been associated with a worse outcome. In one well-designed study, patients with depression in the period immediately after a myocardial infarction were 3.5 times more likely to die than nondepressed patients. The basis of this association remains speculative. However, it is likely that the changes in the autonomic nervous system and platelets that are seen in depression account for a substantial portion of the association.

Cardiac denervation and cardiovascular reactivity to psychological stress.

OBJECTIVE: This study investigated the mechanisms responsible for increases in heart rate and blood pressure during psychological stress, which are incompletely understood. Since cardiac transplant patients have denervated hearts, they provide a unique model for isolating central versus peripheral influences on the cardiovascular response to stress. METHODS: The authors compared the responses to two laboratory stressors of 20 ambulatory heart transplant recipients and two groups of normal subjects, one whose ages were matched to the ages of the transplant patients (mean = 46 years) and one whose ages were matched to the ages of the heart donors (mean = 27 years). The three groups of subjects performed mental arithmetic and reaction time tasks. RESULTS: Heart rate increase during the mental arithmetic task was significantly attenuated in the transplant recipients. During stress, stroke volume increased in the transplant recipients but decreased in both groups of comparison subjects. The difference in age between the heart recipients and donors did not account for the difference in reactivity between the heart transplant patients and the normal subjects. CONCLUSIONS: Direct neural stimulation of the heart is more important than peripherally circulating factors in producing tachycardia during psychological stress. Cardiac but not vascular responses to psychological stress are altered by cardiac denervation.

The histologic spectrum of mycosis fungoides/Sézary syndrome (cutaneous T-cell lymphoma). A review of 222 biopsies, including newly described patterns and the earliest pathologic changes.

We studied 222 skin biopsies of mycosis fungoides and Sézary syndrome (cutaneous T-cell lymphoma [CTCL]) to document the huge histologic spectrum and to evaluate the earliest histologic changes. Our results indicate that CTCL produces practically all of the patterns used for diagnosing inflammatory skin disease: superficial or superficial and deep perivascular without epidermal changes; spongiotic; psoriasiform, with or without a lichenoid infiltrate; interface, including lichenoid without vacuolar alteration, lichenoid with vacuolar alteration, and vacuolar alteration without a lichenoid infiltrate; follicular, with or without mucin; nodular and diffuse; vasculitis; vesicular; and panniculitis. Unusual examples resembling granuloma annulare, gyrate erythema, lichen planus, and pityriasis lichenoides were seen. To further document the spectrum within each pattern, we analyzed many variables, such as lymphocytic atypia, epidermotropism, epidermal contour, and composition of the dermal infiltrate. Common clues to the diagnosis of CTCL include epitheliotropism with little spongiosis; lymphocytes lined up along the basal layer; hyperconvoluted lymphocytes; and broad areas of slight hyperorthokeratosis that is compact or laminated, with subtle interspersed parakeratosis. Less common clues include Pautrier's microabscesses; granulomatous foci; coexistence of plasma cells and eosinophils; and rounded, hyperplastic rete ridges adjacent to flattened rete. The earliest changes of CTCL appear to be a sparse, superficial perivascular infiltrate with slight or no epidermal hyperplasia and with rare lymphocytes in the lower epidermis, especially the basal layer, often with hyperconvoluted nuclei. Our findings support the hypothesis that CTCL develops sui generis, rather than from another chronic dermatosis.

Pagetoid intraepidermal spread in Merkel cell (primary neuroendocrine) carcinoma of the skin.

Pagetoid intraepidermal spread of neoplastic cells was noted in six cases of Merkel (primary neuroendocrine) cell carcinoma of the skin. In two cases, the volume of the intraepidermal portion of the neoplasm was either equal to or more extensive than the dermal component. The intraepidermal component in all six cases was remarkable because of the following findings: the presence of cells with scant cytoplasm arranged both individually and as nests, sometimes along the dermoepidermal junction; splaying of the apical portions of basal keratinocytes by solitary neoplastic cells; incomplete rims of compressed basal keratinocytes at the peripheries of some junctional nests; and occasional contiguity of neuroendocrine carcinoma cells with those of Bowen's disease or solar keratosis. These features can be used to distinguish these Merkel cell carcinomas from other lesions that have a pagetoid pattern, even in superficial biopsies, and immunohistochemistry can confirm the diagnosis or resolve problematic cases. The occurrence of cutaneous neuroendocrine carcinoma situated largely in the epidermis raises the possibility that some of these tumors may arise from intraepidermal Merkel cells.

Psychosocial evaluation and prediction of compliance problems and morbidity after heart transplantation.

We examined prospectively determined psychosocial evaluation data in 125 consecutive adult patients undergoing heart transplantation from January 1992 to April 1994 to determine their associations with morbidity, mortality, and compliance. Prospective ratings included age, sex, weight, education, social support, living arrangements, motivation, knowledge and expectations about transplantation, intercurrent social stressors, substance abuse, personality disorder, cognitive impairment, other psychiatric disorders, and the evaluating psychiatrist's global assessment of psychosocial risk. Additional variables evaluated were support group attendance and waiting list time. We examined outcomes including patient survival, compliance, episodes of rejection and infection, development of transplant coronary artery disease, number of missed appointments, and maintenance of ideal body weight. The posttransplant follow-up period was 13.8 +/- 9.9 months (mean +/- SD). In univariate analyses, compliance problems were associated with substance abuse history (P = .0007), personality disorder (P = .007), living arrangements (P = .02), and global psychosocial risk (P = .001). The number of rejection episodes was associated with global psychosocial risk (P = .029), and transplant coronary artery disease was inversely associated with education (P = .01). Survival was not associated with any of the predictor variables. In stepwise multivariate analyses, the significant predictors of compliance were substance abuse (odds ratio 3.69, confidence limits 1.07-12.71) and global psychosocial risk (odds ratio 3.76, confidence intervals 1.18-11.97). These findings suggest that pretransplant evaluation of psychosocial risk factors can identify patients with increased risk of postoperative noncompliance and morbidity.

Facial characteristics of children who breathe through the mouth.

There are many claims that abnormal breathing patterns alter facial growth; however, there are limited controlled data to confirm these claims. Thirty children with allergy, aged 6 to 12 years, who had moderate-to-severe nasal mucosal edema on physical examination and who appeared to breathe predominantly through the mouth and 15 children without allergy who had normal findings from nasal examination and who appeared to breathe predominantly through the nose were evaluated. All subjects received an intraoral clinical examination and cephalometric radiograph analysis. In comparison with children who breathed through the nose, children who breathed through the mouth had longer faces with narrower maxillae and retruded jaws. This supports the hypothesis that children with nasal obstruction and who appear to breathe through the mouth have distinctive facial characteristics.

The use of antidepressant drugs in patients with heart disease.

Both depression and cardiovascular disease are common as people age and are, therefore, likely to coexist. It has become evident recently that the rate of this comorbidity exceeds substantially what is expected by chance. A major problem arises in that there is increasing evidence that the tricyclic antidepressants (TCAs) carry more risk than originally thought in patients with ischemic heart disease. This risk increases the importance of understanding both the safety and efficacy of the serotonin selective reuptake inhibitors (SSRIs) in this population. Three recent studies on safety data in patients with overt heart disease are now available: although the total of 94 patients limits the ability to make generalizations, the data that are available give little evidence of harm and even suggest that SSRIs may have beneficial effects in ischemic heart disease.

Automated quantitation of hemoglobin-based blood substitutes in whole blood samples.

It is necessary to develop methods for accurate monitoring of cell-free hemoglobin in circulation. Routine monitoring of circulating cell-free hemoglobin will be useful for evaluating the efficacy of blood substitute administration andfor determining the clearance rates of the blood substitute from circulation. In addition, discriminating between cell-free hemoglobin and cell-associated hemoglobin will enable accurate determination of RBC indices, mean cell hemoglobin and mean corpuscular hemoglobin concentration, in individuals receiving hemoglobin-based blood substitutes. As colorimetric methods used by hematology analyzers to quantitate the hemoglobin value of a blood sample cannot distinguish between cell-associated and cell-free hemoglobin, it is currently not feasible to quantitate the levels of hemoglobin substitutes in circulation. The advent of a technology that measures volume and hemoglobin concentration of individual RBCs provides an alternative strategy for quantitating the cell-associated hemoglobin in a blood sample. We document that the combined use of cell-based and colorimetric hemoglobin measurements provides accurate discrimination between cell-associated and cell-free hemoglobin over a wide range of hemoglobin levels. This strategy should enable rapid and accurate monitoring of the levels of cell-free hemoglobin substitutes in the circulation of recipients of these blood substitutes.

Energy deposition and ionization fluctuations induced by ions in small sites--an analytical approach.

A concise, analytical approach is developed for calculating energy deposition and ionization fluctuations in volumes within ion-irradiated media which have dimensions as small as 1 nm. The method accounts for both direct ion interactions with the site and interactions of secondary electrons which are produced by ions in the surrounding medium. Particular attention is given to the way the contributions of the two types of events are combined. Since energy deposition fluctuations are simply related to the fundamental quantities ZD and yD employed in microdosimetry theory, this new approach provides a convenient means to obtain these parameters. Results obtained with the analytical method show good agreement with Monte Carlo charged-particle track-structure calculations of yD for 0.5 to 20 MeV protons incident on spherical sites of water vapor with diameters ranging from 1 nm to 10 microns. In contrast to Monte Carlo techniques, the analytical method does not depend on knowing the intricacies of single ion and electron interactions with the target and can therefore be adapted to calculations with heavier incident ions and different target materials, including those of the condensed state.

Endoscopic removal of submucosal colonic lipomas.

Although lipomas of the colon are second only to adenomatous polyps in frequency of benign lesions of the large bowel, they occur relatively rarely. These lesions are reported to be symptomatic in 15% to 91% of cases. Endoscopically, the lipoma is soft and pliable and appears globular and covered by pale "normal" tunica mucosa. The treatment of choice is colonoscopy and snare excision because of the potential hazards of bleeding and bowel obstruction and because the diagnosis can be confirmed only by histologic examination.