Activation of hepatic guanylate cyclase by nitrosyl-heme complexes: Comparison of unpurified and partially purified enzyme

The objective of this study was to evaluate the effects of several agents on activation of both unpurified and partially purified hepatic soluble guanylate cyclase by performed NO (nitric oxide or nitrosyl)-heme complexes. Guanylate cyclase was activated by NO complexes of the heme compounds, hematin, hemoglobin, myoglobin, catalase and cytochrome c, and also by the reaction product of NO and ferredoxin, a non-heme, iron sulfur electron transfer protein. NO-lipoxygenase, which contains non-heme iron, did not activate guanylate cyclase. NO-heme complexes activated unpurified enzyme almost equally well in the presence of either Mg²⁺ or Mn²⁺. However, activation of purified (350- to 750-fold) guanylate cyclase was markedly greater with Mg²⁺ than with Mn²⁺. At concentrations that did not alter basal enzymatic activity, Ca²⁺ markedly inhibited guanylate cyclase activation in the presence of Mg²⁺ but not of Mn²⁺. Hemoproteins inhibited activation of unpurified and purified enzyme by NO-heme complexes, and increasing the concentrations of the latter overcame the inhibition. Gel filtration studies indicated that uncomplexed and NO-complexed hematin bind to common or adjacent sites on guanylate cyclase. Whereas dl-dithiothreitol enhanced activation, ferricyanide, cystine, o-iodosobenzoic acid and ethacrynic acid inhibited activation of guanylate cyclase by NO-heme complexes. The data indicate that the effects of these diverse agents on guanylate cyclase activation by preformed NO-heme are similar to their effects on enzyme activation by NO and nitroso compounds, both of which readily form NO-heme complexes. Therefore, the effects of these diverse agents may be on guanylate cyclase rather than on NO-heme formation.     

Depressive symptomatology, exposure to violence, and the role of social capital among African American adolescents

Focusing on the role of capital as both personal and social resources for adolescents, the authors examined depressive symptomatology among a sample of 10- to 18-year-old African American youths (N=1,538). In addition to gender and age differences, adolescents exposed to threatening environments (school, neighborhood, home) reported more depressive symptoms. Social capital had a significant inverse relationship with adolescent depression; self-esteem and a social capital index were negatively related to depressive symptomatology. Furthermore, the interaction effects of gender with social capital, age with self-esteem, and age with grades were significant, indicating the presence of a buffering effect. These findings suggest the importance of interrelationships among violence exposure, capital, and well-being for adolescents.     

Neutrophil-specific chemokines are produced by astrocytic cells but not by neuronal cells

BACKGROUND: Neutrophils have a central role in the inflammatory conditions of the central nervous system (CNS). ELR chemokines direct neutrophil migration, but the source of chemokines in the CNS is unclear. We quantified chemokine production using cell-line models of astrocytic and neuronal cells, specifically NT2.N cells, a human line with characteristics of immature neurons, and NT2.A cells, a line with characteristics of astrocytes. OBJECTIVE: In NT2.N and NT2.A cells, and their parent cell line NT2, we sought to: (1) quantify ELR chemokines, (2) determine receptor (CXCR-1 and CXCR-2) expression, and (3) measure the function of the chemokines generated from these cells. DESIGN/METHODS: NT2 cells were differentiated into NT2.N cells and NT2.A cells with all trans retinoic acid and mitosis inhibitors. Chemokine concentrations in culture supernatants were determined by ELISA. Immunofluorescence was used to detect CXCR-1 and CXCR-2. RT-PCR was used to determine chemokine and chemokine receptor mRNA. Chemotaxis assays were used to assess function. RESULTS: ELR chemokines were not detected in supernatants of NT2 or NT2.N cells, although mRNA for GRO-gamma/CXCL3 was found in both. In contrast, in NT2.A cells, mRNA and protein were present for GCP-2/CXCL6, GRO-alpha/CXCL1, GRO-gamma/CXCL3, and IL-8/CXCL8. CXCR-1 and CXCR-2 were expressed on NT2, NT2.N, and NT2.A cells detected by immunofluorescent staining and RT-PCR. Supernatants of NT2.A cells resulted in neutrophil chemotactic function of 30.5 +/- 3.9%, greater than NT2 cells (12.3 +/- 1.6%, mean +/- SEM, P < 0.01). CONCLUSIONS: We speculate that astrocytes are a source of ELR chemokines in the human CNS and that neurons and astrocytes can respond to those chemokines.     

Blood treatment influences the yield of apoptotic lymphocytes after maximal exercise

PURPOSE: No systematic investigation has been reported assessing the effect of cell isolation processes on postexercise apoptosis. Therefore, the effect of cell isolation procedures on apoptosis was evaluated in this study. METHODS: Untrained healthy individuals participated (N=13). Blood samples obtained at rest and immediately after an incremental exercise test to exhaustion were partitioned into three treatments: 1) whole blood smears made immediately after the sample was obtained (WB), 2) cells subjected to density-gradient isolation before smears were made (ISO), and 3) samples allowed to sit at room temperature (i.e., time-treated) before centrifugation and smearing (TT). Blood smears were stained using the May-Grünwald Giemsa procedure and lymphocytes were evaluated under a light microscope for characteristic features of apoptosis. Data were analyzed using a 2x3 ANOVA. RESULTS: A significant interaction effect existed (P<0.0001) such that at rest, no difference was detected in the amount of lymphocyte apoptosis among WB, ISO, or TT samples. However, after exhaustive exercise, the amount of apoptotic lymphocytes was significantly greater in WB compared with ISO and TT samples (P<0.0001). CONCLUSION: Lymphocyte isolation results in a significant decrease in the percent of apoptotic lymphocytes after exhaustive exercise. This reduction is likely due to the time needed to isolate cells, rather than the isolation process itself. Because apoptosis is a time-sensitive process that occurs within minutes rather than hours, the length of time from initial sampling to the preparation of cells for assessment of apoptosis is critical and should be considered in future exercise studies.     

Effects of enterally administering granulocyte colony-stimulating factor to suckling mice

Gastrointestinal (GI) tract development is influenced by multiple growth factors, some of which are delivered directly to the GI lumen, as they are swallowed constituents of amniotic fluid, colostrum, and milk. Granulocyte colony-stimulating factor (G-CSF), traditionally known as a granulocytopoietic growth factor, is an example of one such factor. However, it is not clear whether the large amounts of G-CSF that are normally swallowed by the fetus and neonate have systemic effects on circulating neutrophils or local effects in the developing intestine. To assess this, we administered either active or heat-denatured (control) recombinant human G-CSF to 5- to 7-d-old C57BL/6 x 129SvJ mice. Pups received either a low dose (3 ng) that was calculated to approximate the amount of G-CSF swallowed in utero from amniotic fluid or an isovolemic high dose 100 times larger (300 ng). Oral dosing was performed daily for either 3 or 7 d, after which pups were killed and measurements were made on the blood and the GI tract. Absolute blood neutrophil counts and immature to total neutrophil ratios did not differ from controls in any of the test groups. However, intestinal villus area, perimeter, length, crypt depth, and proliferating cell nuclear antigen index increased significantly among those that were treated with active G-CSF. Thus, in suckling mice, enterally administered G-CSF had no effect on the concentration of circulating neutrophils but had trophic effects on the intestine. We speculate that the G-CSF present in amniotic fluid, colostrum, and milk acts as a topical intestinal growth factor and has little or no granulocytopoietic action.     

Imaging of bioluminescent LNCaP-luc-M6 tumors: a new animal model for the study of metastatic human prostate cancer

BACKGROUND: Animal experiments examining hormone-sensitive metastatic prostate cancer using the human LNCaP cell line have been limited to endpoint analyses. To permit longitudinal studies, we generated a luciferase-expressing cell line and used bioluminescent imaging (BLI) to non-invasively monitor the in vivo growth of primary LNCaP tumors and metastasis. METHODS: LNCaP.FGC cells were transfected to constitutively express firefly luciferase. LNCaP-luc-M6 cells were tested for bioluminescent signal intensity and hormone responsiveness in vitro. The cells were implanted in subcutaneous and orthotopic sites in SCID-bg mice and imaged over time. RESULTS: The LNCaP-luc-M6 cells formed subcutaneous and orthotopic tumors in SCID-bg mice, and nearly all tumor-bearing animals developed pulmonary metastases. Early detection and temporal growth of primary tumors and metastatic lesions was successfully monitored by BLI. CONCLUSIONS: The LNCaP-luc-M6 cell line is a bioluminescent, hormone-sensitive prostate cancer cell line applicable for BLI studies to non-invasively monitor subcutaneous and orthotopic prostate tumor growth and metastasis in vivo.     

Pontine hypoplasia in Carey-Fineman-Ziter (CFZ) syndrome

We describe an infant with multiple congenital anomalies including cleft palate and micrognathia, M?bius sequence, developmental delay, myopathy, hydronephrosis, and bilateral clubfeet. These features are consistent with Carey-Fineman-Ziter (CFZ) syndrome (MIM 254940), which has been previously reported in six children (including two sibling pairs). Cranial magnetic resonance imaging (MRI) revealed an unusually small pons, a finding not previously described in CFZ syndrome.     

Benign B-cell precursors (hematogones) are the predominant lymphoid population in the bone marrow of preterm infants

Bone marrow (BM) findings in 3rd-trimester stillborns and full-term living neonates have been previously described. However, there is no information regarding BM composition in living preterm infants. Specifically, it is unknown whether the BM lymphocytosis seen in full-term infants at 1-4 weeks of age also occurs in preterm infants. Furthermore, the lineage of these cells has never been investigated. We used a panel of immunohistochemical stains to characterize the BM composition in 11 neonates (8 living and 3 deceased). Unlike in the other age groups, immature B cells (hematogones) were the most common lymphoid population, accounting for 10-60% (mean 34%) of all cells. In two additional cases (both living patients), flow cytometry revealed a level of 3.8% of immature B cells in a <1-week-old neonate and 25.7% in a 19-week-old infant. Immature B cells were not identified in 6 peripheral blood samples from preterm neonates. These findings are pertinent for the interpretation of BM and peripheral blood samples in this age group as survival improves and diagnostic samples become more common.