Behavioral and neurochemical changes in the dopaminergic system after repeated cocaine administration

In order to determine whether repeated cocaine administration produced persistent changes in dopamine (DA) receptor binding and release consistent with behavioral sensitization, rats were treated with either cocaine (25 mg/kg ip) or saline twice daily for 14 consecutive days followed by a 3-d withdrawal period. The DA transporter site was assayed using [³H]GBR 12935, whereas D₁ and D₂ sites were assayed using [³H]SCH 23390 and [³H]spiperone, respectively. The density (B _max) of the DA transporter binding sites in the ST of the cocaine-treated group increased significantly (p<0.05) over controls 3 d after the last injection, whereas the density of striatal D₁ and D₂ binding sites remained unchanged. The DA transporter in the nucleus accumbens (NA) was also studied with [³H]GBR 12935 and was unchanged following drug treatment. D₁ and D₂ binding parameters for the NA were not determined in this study. Furthermore, cocaine administration did not affect the affinities (K _d ) of the radioligands used to label the transporter, D₁, or D₂ sites in any of the studies performed. In addition, striatal DA release was measured using in vivo microdialysis in anesthetized rats. Linear regression analysis on maximal decreases in DA release after apomorphine (0.02, 0.2, and 2.0 mg/kg sc) injection showed no difference in the functional capacity of the ST to modulate DA transmission between control and treated groups. Moreover, animals pretreated with cocaine showed a significant (p<0.01) decrease in locomotor activity (LA) after a presynaptic, autoregulating dose of apomorphine (0.03 mg/kg sc) was given. These results suggest that the effects seen after repeated exposure to cocaine may be regulated, in part, by changes in striatal DA transporter binding site densities and not necessarily by DA-releasing mechanisms or D₁ and D₂ receptor modification.     

Protein kinase C (PKC)-induced phosphorylation of ROMK1 is essential for the surface expression of ROMK1 channels

We carried out in vitro phosphorylation assays to determine whether ROMK1 is a substrate of protein kinase C (PKC) and used the two-electrode voltage clamp method to investigate the role of serine residues 4, 183, and 201, the three putative PKC phosphorylation sites, in the regulation of ROMK1 channel activity. Incubation of the purified His-tagged ROMK1 protein with PKC and radiolabeled ATP resulted in (32)P incorporation into ROMK1 detected by autoradiography. Moreover, the in vitro phosphorylation study of three synthesized peptides corresponding to amino acids 1-16, 174-189, and 196-211 of ROMK1 revealed that serine residues 4 and 201 of ROMK1 were the two main PKC phosphorylation sites. In contrast, (32)P incorporation of peptide 174-189 was absent. In vitro phosphorylation studies with ROMK1 mutants, R1S4/201A, R1S4/183A, and R1S183/201A, demonstrated that the phosphorylation levels of R1S4/201A were significantly lower than those of the other two mutants. Also, the Ba(2+)-sensitive K(+) current in oocytes injected with green fluorescent protein (GFP)-R1S4/201A was only 5% of that in oocytes injected with wild type GFP-ROMK1. In contrast, the K(+) current in oocytes injected with GFP-ROMK1 mutants containing either serine residue 4 or 201 was similar to those injected with wild type ROMK1. Confocal microscope imaging shows that the surface expression of the K(+) channels was significantly diminished in oocytes injected with R1S4/201A and completely absent in oocytes injected with R1S4/183/201A. Furthermore, the biotin labeling technique confirmed that the membrane fraction of ROMK channels was almost absent in HEK293 cells transfected with either R1S4/201A or R1S4/183/201A. However, when serine residues 4 and 201 were mutated to aspartate, the K(+) currents and the surface expression were completely restored. Finally, addition of calphostin C in the incubation medium significantly decreased the K(+) current in comparison with that under control conditions. Biotin labeling technique further indicated that inhibition of PKC decreases the surface ROMK1 expression in human embryonic kidney (HEK) cells transfected with ROMK1. We conclude that ROMK1 is a substrate of PKC and that serine residues 4 and 201 are the two main PKC phosphorylation sites that are essential for the expression of ROMK1 in the cell surface.     

MFR, a Putative Receptor Mediating the Fusion of Macrophages

We had previously identified a macrophage surface protein whose expression is highly induced, transient, and specific, as it is restricted to actively fusing macrophages in vitro and in vivo. This protein is recognized by monoclonal antibodies that block macrophage fusion. We have now purified this protein and cloned its corresponding cDNA. This protein belongs to the superfamily of immunoglobulins and is similar to immune antigen receptors such as the T-cell receptor, B-cell receptor, and viral receptors such as CD4. We have therefore named this protein macrophage fusion receptor (MFR). We show that the extracellular domain of MFR prevents fusion of macrophages in vitro and therefore propose that MFR belongs to the fusion machinery of macrophages. MFR is identical to SHPS-1 and BIT and is a homologue of P84, SIRPα, and MyD-1, all of which have been recently cloned and implicated in cell signaling and cell-cell interaction events.     

Molecular markers in patients with chronic wounds to guide surgical debridement

Chronic wounds, such as venous ulcers, are characterized by physiological impairments manifested by delays in healing, resulting in severe morbidity. Surgical debridement is routinely performed on chronic wounds because it stimulates healing. However, procedures are repeated many times on the same patient because, in contrast to tumor excision, there are no objective biological/molecular markers to guide the extent of debridement. To develop bioassays that can potentially guide surgical debridement, we assessed the pathogenesis of the patients' wound tissue before and after wound debridement. We obtained biopsies from three patients at two locations, the nonhealing edge (prior to debridement) and the adjacent, nonulcerated skin of the venous ulcers (post debridement), and evaluated their histology, biological response to wounding (migration) and gene expression profile. We found that biopsies from the nonhealing edges exhibit distinct pathogenic morphology (hyperproliferative/hyperkeratotic epidermis; dermal fibrosis; increased procollagen synthesis). Fibroblasts deriving from this location exhibit impaired migration in comparison to the cells from adjacent nonulcerated biopsies, which exhibit normalization of morphology and normal migration capacity. The nonhealing edges have a specific, identifiable, and reproducible gene expression profile. The adjacent nonulcerated biopsies have their own distinctive reproducible gene expression profile, signifying that particular wound areas can be identified by gene expression profiling. We conclude that chronic ulcers contain distinct subpopulations of cells with different capacity to heal and that gene expression profiling can be utilized to identify them. In the future, molecular markers will be developed to identify the nonimpaired tissue, thereby making surgical debridement more accurate and more efficacious.     

Plasma BDNF and PDGF-AA levels are associated with high TCD velocity and stroke in children with sickle cell anemia

Sickle cell anemia (SCA) associated cerebrovascular disease includes vascular remodeling, abnormal cerebral blood flow (CBF) and infarction. We studied the relationships between plasma brain derived neurotropic factor (BDNF), platelet derived growth factors (PDGF-AA and -AB/BB) and high trans-cranial Doppler (TCD) velocity, an indication of CBF velocity. Baseline plasma samples from 39 children (19 SCA with abnormal/high TCD [SATCD], 13 SCA with normal TCD [SNTCD] and 7 healthy non-SCA), were assayed for BDNF, PDGF-AA and -AB/BB plus 11 other cytokines. The sensitivity, specificity and usefulness of these biomarkers for stroke prediction was investigated. All subject groups were of similar age and gender distribution. Mean BDNF was significantly higher among SATCD than SNTCD (p=0.004) as was mean PDGF-AA (p=0.001). Similarly, mean PDGF-AA was higher among SCA subjects who developed stroke than those who did not (p=0.012). Elevated BDNF and PDGF-AA were good predictors of the presence of abnormally high CBF velocity and were both associated with severity of anemia. Elevated PDGF-AA predicted risk for stroke development. Stroke incidence and high TCD velocity were associated with elevated BDNF and PDGF-AA. These findings suggest a role for BDNF and PDGF-AA in the patho-physiological mechanism of cerebrovascular disease in SCA.     

Cervical Cancer and Pap Smear Awareness and Utilization of Pap Smear Test among Federal Civil Servants in North Central Nigeria

Cervical cancer is a leading cause of cancer death in women in developing countries. A key factor linked to the relatively high levels of cervical cancer in these populations is the lack of awareness and access to preventive methods. This study aimed to determine the level of awareness of cervical cancer and Papanicolaou test (Pap smear test) and factors associated with the utilization of Pap test among female civil servants in Jos. Data was obtained from female workers (n = 388) aged 18–65 years in a Nigerian Federal establishment. Participants were randomly approached and instructed to complete validated questionnaires. Data was analyzed using Chi-square, t-tests and logistic regression analysis to determine if there was an association between variables and identify any predictors of awareness and utilization of the Pap test. Cervical cancer and Pap smear test awareness was 50.9% and 38.6% respectively, with the media as the major source of information. Pap smear test utilization rate was 10.2%, with routine antenatal care (ANC) as the major reason for getting screened. Personal barriers to screening include the lack of awareness, and belief that cervical cancer is not preventable. Opportunistic screening, mass media campaigns and ANC education were suggested as ways of improving awareness and utilization of cervical cancer screening services.     

Inhibition of protein-tyrosine phosphatase stimulates the dynamin-dependent endocytosis of ROMK1.

We have previously shown that inhibiting protein-tyrosine kinase increased whereas inhibiting protein-tyrosine phosphatase (PTP) decreased renal outer medullary potassium channel 1 (ROMK1) channel activity (1). We have now used confocal microscopy, the patch clamp technique, and biotin labeling to further examine the role of tyrosine phosphorylation in regulating ROMK1 trafficking. Human embryonic kidney 293 cells were cotransfected with c-Src and green fluorescent protein-ROMK1, which has the same biophysical properties as those of ROMK1. Patch clamp studies have shown that phenylarsine oxide (PAO), an inhibitor of PTP, decreased the activity of ROMK1. Moreover, addition of PAO reduced the cell surface localization of green fluorescent protein-ROMK1 detected by confocal microscopy and diminished the surface ROMK1 density by 65% measured by biotin labeling. Also, PAO treatment significantly increased the phosphorylation of ROMK1. The notion that the effect of PAO is mediated by stimulating tyrosine phosphorylation-induced endocytosis of ROMK1 has also been supported by findings that mutating the tyrosine residue 337 of ROMK1 to alanine abolished the effect of PAO. Finally, the inhibitory effect of PAO on ROMK1 was completely blocked in the cells co-transfected with dominant negative dynamin (dynaminK44A). This indicates that the tyrosine phosphorylation-induced endocytosis of ROMK1 is dynamin-dependent. We conclude that inhibiting PTP increases ROMK1 phosphorylation and results in a dynamin-dependent internalization of the channel.     

An electrophilic affinity ligand based on (+)-MK801 distinguishes PCP site 1 from PCP site 2

The electrophilic affinity ligand, (+)-3-isothiocyanato-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine hydrochloride {(+)-MK801-NCS} was characterized for its ability to acylate phencyclidine (PCP) and sigma binding sites in vivo. Initial studies, conducted with mouse brain membranes, characterized the binding sites labeled by [³H]1-[1-(2-thienyl)cyclohexyl]piperidine ([³H]TCP). The Kd values of [³H]TCP for PCP site 1 (MK801-sensitive) and PCP site 2 (MK801-insensitive) were 12 nM and 68 nM, with Bmax values of 1442 and 734 fmol/mg protein, respectively. Mice were sacrificed 18–24 hours following intracerebroventricular administration of the acylator. The administration of (+)-MK801-NCS increased [³H]TCP binding to site 2, but not to site. 1. Although (+)-MK801-NCS decreased [³H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d; cbcyclohepten-5,10-imine maleate ([³H](+)-MK801) binding to site 1, it had no effect on [³H]TCP binding to site 1. Viewed collectively with other published data, these data support the hypothesis that PCP sites 1 and 2 are distinct binding sites, and that [³H]TCP and [³H](+)-MK801 label different domains of the PCP binding site associated with the NMDA receptor.Abbreviations ((+)-MK801) (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine - ((+)-MK801-NCS) (+)-3-isothiocyanato-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine hydrochloride - (PCP) 1-(1-phencyclohexyl)piperidine - (TCP) 1-{1-(2-thienyl)cyclohexyl}piperidine - (DTG) (2-(tllyl)guanidine - (metaphit) (1-(1-(3-isothiocyanatophenyl)-cyclohexyl)piperidine) - (NMDA) N-methyl-D-aspartate - (HEPPSO) (N-[2-hydroxyethyl]piperazine-N-[2-hydroxypropanesulfoni c acid] - ((+)-MK801-NCS) (+)-5-methyl(3-isothiocyanatophenyl)-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine - (NMDA) N-methyl-D-aspartate Address reprint requests to Dr. Rothman, Phone (410)550-1487.FAX 410-550-2997     

MPTP lesions of the nigrostriatal dopaminergic projection decrease [3H]1-[1-(2-thienyl)cyclohexyl]-piperidine binding to PCP site 2: Further evidence that PCP site 2 is associated with the biogenic amine reuptake complex

Our previous studies have demonstrated that, using membranes of guinea pig brain, [³H]1-[1-(2-thienyl)cyclohexyl]piperidine ([³H]TCP) labels not only the phencyclidine binding site associated with the NMDA receptor (PCP site 1), but also a second high affinity binding site which is associated with the biogenic amine reuptake carrier (termed PCP site 2). To test this hypothesis, the binding of [³H]GBR12935 to the dopamine transporter, and [³H]TCP binding to PCP sites 1 and 2 were measured in caudates harvested from control MPTP-treated and reserpine-treated dogs. MPTP treatment decreased dopamine levels by over 99%, decreased [³H]GBR12935 binding by over 90%, decreased [³H]TCP binding to PCP site 2 by about 50%, and had no significant effect on [³H]TCP binding to PCP site 1. These data are consistent with hypothesis that a portion of PCP site 2 is associated with dopaminergic nerve, terminals in dog caudate.     

Effect of Chronic Blood Transfusion on Biomarkers of Coagulation Activation and Thrombin Generation in Sickle Cell Patients at Risk for Stroke

Hypercoagulability in sickle cell disease (SCD) is associated with multiple SCD phenotypes, association with stroke risk has not been well described. We hypothesized that serum levels of biomarkers of coagulation activation correlate with high transcranial Doppler ultrasound velocity and decreases with blood transfusion therapy in SCD patients. Stored serum samples from subjects in the Stroke Prevention in Sickle Cell Anemia (STOP) trial were analyzed using ELISA and protein multiplexing techniques. 40 subjects from each treatment arm (Standard Care [SC] and Transfusion [Tx]) at three time points—baseline, study exit and one year post-trial and 10 each of age matched children with SCD but normal TCD (SNTCD) and with normal hemoglobin (HbAA) were analyzed. At baseline, median vWF, TAT and D-dimer levels were significantly higher among STOP subjects than either HbAA or SNTCD. At study exit, median hemoglobin level was significantly higher while median TCD velocity was significantly lower in Tx compared to SC subjects. Median vWF (409.6 vs. 542.9 μg/ml), TAT (24.8 vs. 40.0 ng/ml) and D-dimer (9.2 vs. 19.1 μg/ml) levels were also significantly lower in the Tx compared to the SC group at study exit. Blood levels of biomarkers coagulation activation/thrombin generation correlated positively with TCD velocity and negatively with number of blood transfusions. Biomarkers of coagulation activation/thrombin generation were significantly elevated in children with SCD, at high risk for stroke. Reduction in levels of these biomarkers correlated with reduction in stroke risk (lower TCD velocity), indicating a possible role for hypercoagulation in SCD associated stroke.