A C-terminal fragment of Agouti-related protein increases feeding and antagonizes the effect of alpha-melanocyte stimulating hormone in vivo

Agouti-related protein (Agrp) is present in rat and human hypothalamus and is structurally related to agouti protein. Overexpression of either of these proteins results in obesity. However the effect of exogenous Agrp and its in vivo interaction with alpha-melanocyte stimulating hormone (alphaMSH), the likely endogenous melanocortin 3 and 4 receptor (MC3-R and MC4-R) agonist, have not been demonstrated. We report that 1 nmol of Agrp(83-132), a C-terminal fragment of Agrp, when administered intracerebroventricularly (ICV) into rats, increased food intake over a 24-h period (23.0+/-1.4 g saline vs 32.9+/-2.3 g Agrp, p<0.05). The hyperphagia was similar to that seen when 1 nmol of the synthetic MC3-R and MC4-R antagonist SHU9119 was given i.c.v. (19.6+/-1.8 g saline vs 32.5+/-1.7 g SHU9119, p<0.001). Both Agrp(83-132) and SHU9119 blocked the reduction in 1-h food intake of i.c.v. alphaMSH at the beginning of the dark phase. This effect occurred independently of whether the antagonists were administered simultaneously, or nine hours prior, to the alphaMSH. We have also shown Agrp(83-132) is an antagonist at the MC3-R and MC4-R, with similar inhibition of cAMP activation to that previously reported for the full length peptide. In conclusion, Agrp(83-132) administered i.c.v. increases feeding with long lasting effects and is able to inhibit the action of alphaMSH. This interaction may be mediated by the MC3-R and/or MC4-R.     

Neurofilament phosphorylation is increased in ventral horn neurons of neonatal rat spinal cord exposed to cerebrospinal fluid from patients with amyotrophic lateral sclerosis

Aberrant neurofilament (NF) phosphorylation in the soma of the ventral horn neurons of neonatal rat spinal cord is observed following exposure to cerebrospinal fluid (CSF) of patients suffering from Amyotrophic Lateral Sclerosis (ALS). CSF samples from ALS and non-ALS neurological patients were injected into the spinal subarachnoid space of 3 day old rat pups. After 48 h, sections of spinal cords were stained for the presence of phosphorylated NF epitopes with SMI-31 antibody. The number of neuronal soma staining with this antibody in the ventral and dorsal horns sides of the spinal cord was counted. There was a significant 3-fold increase in the number of soma stained with SMI-31 antibody in the ventral horns of rat spinal cords exposed to CSF of patients with ALS compared to cords from rats exposed to CSF of non-ALS patients and those which were not exposed to any CSF samples. Such an increase in staining of neuronal soma was not observed in the dorsal horns. Hyperphosphorylation of neuronal soma suggests an initial stage of degenerative changes occurring in the motor (ventral horn) neurons following exposure to circulating factor(s) in the CSF of patients with ALS.     

Activation and adherence of lyophilized human platelets on canine vessel strips in the Baumgartner perfusion chamber

The Baumgartner perfusion technique was used to test the ability of rehydrated lyophilized human platelets to adhere to the thrombogenic surface of a denuded arterial vessel segment and to undergo platelet activation under conditions of high shear. Twenty preparations of washed platelets were stabilized by 1-hour or 2-hour exposure to paraformaldehyde before freeze-drying in a Virtis 600 lyophilizer. The response of these fixed-dried preparations was compared with that of non-fixed platelets in fresh citrated whole blood. The outcome of each perfusion experiment was quantified in photomicrographs by morphometric estimation of the percent area of the vessel segment covered by adherent platelets after immunofluorescent staining with monoclonal antibodies to glycoprotein Ib (CD42) or glycoprotein IIbIIIa (CD41a). Evidence of activation in nonadherent platelets was examined by flow cytometry for CD62 and GP53 expression. In addition, thromboxane B2 was measured by radioimmunoassay as an index of platelet responsiveness to activation conditions during perfusion. The percent vessel coverage observed with lyophilized platelets in recombined whole blood was somewhat less than that of platelets in fresh whole blood (39% vs 73%, respectively). In other perfusion experiments performed with non-denuded vessel segments, the percent coverage was reduced by half or more for both types of platelet preparation. Scanning electron microscopy confirmed that the lyophilized platelets did not adhere to areas of intact endothelium. Furthermore, the lyophilized platelets showed a small-but-significant rise in CD62 or CD63 activation antigen expression and generated thromboxane B2 at about one third the rate of fresh platelets in these perfusion experiments. The thromboxane generation during perfusion was inhibited in fresh or lyophilized platelet preparations by pretreatment with indomethacin or PGE-1. We interpret these data as evidence of the ability of our lyophiilized platelet preparations to respond at least partially to physiologic stimuli and to adhere to appropriate thrombogenic sites to support hemostasis.     

The Challenges of Ensuring Participant Consent in Internet-based Sex Studies: A Case Study of the Men's INTernet Sex (MINTS-I and II) Studies

This study documents our experience in designing, testing, and refining human subjects' consent protocol in 3 of the first NIH-funded online studies of HIV/STI sexual risk behavior in the USA. We considered 4 challenges primary: a) designing recruitment and enrollment procedures to ensure adequate attention to subject considerations; b) obtaining and documenting subjects' consent; c) establishing investigator credibility through investigator-participant interactions; d) enhancing confidentiality during all aspects of the study. Human consent in online studies appears more relative, continuous, inherent, tenuous, and diverse than in offline studies. Reasons for declining consent appear related to pragmatic concerns not human subjects' risks. Reordering the consent process, and short, chunked, stepwise, tailored consent procedures may enhance communicating information and documenting consent.     

EFFECT OF HIGH-INTENSITY PULSED ELECTRIC FIELDS ON SURVIVAL OF ESCHERICHIA COLI K-12 SUSPENDED IN MEAT INJECTION SOLUTIONS

Survival of Escherichia coli K12 suspended in solutions used for enhancing meat products after high‐intensity pulsed electric field (PEF) treatments was examined. Solutions were prepared to provide 0.3% salt, 0.3% phosphate and/or 0, 1, 2 or 3% sodium lactate in a finished product enhanced to 110% of initial weight. Therefore, enhancement solutions contained water, 3% NaCl, 3% phosphate and/or 0, 10, 20 or 30% sodium lactate. Samples containing 0.1% NaCl were run as controls. Single PEF strengths used for PEF treatments were 12.5, 7.0, 6.0, 5.0 and 3.5 kV/cm. For 12.5, 7.0 and 6.0 kV/cm, two levels of pulse controller resistance were used, ∝ and 200 ohm; for 5 and 3.5 kV/cm, only 200 ohm was tested. Above 7 kV/cm, arcing occurred which limited the application of this technology in these solutions. Electrical field strength at 7 kV/cm with a pulse controller resistance of 200 ohm resulted in a reduction of about 2 log cfu/mL when cells were suspended in the enhancement solution containing no lactate. Lower electrical field strengths (≥6 kV/cm) were generally ineffective.     

Treatment of refractory and relapsed acute myelogenous leukemia with combination chemotherapy plus the multidrug resistance modulator PSC 833 (Valspodar)

A potential mechanism of chemotherapy resistance in acute myeloid leukemia (AML) is the multidrug resistance (MDR-1) gene product P-glycoprotein (P-gp), which is often overexpressed in myeloblasts from refractory or relapsed AML. In a multicenter phase II clinical trial, 37 patients with these poor risk forms of AML were treated with PSC 833 (Valspodar; Novartis Pharmaceutical Corporation, East Hanover, NJ), a potent inhibitor of the MDR-1 efflux pump, plus mitoxantrone, etoposide, and cytarabine (PSC-MEC). Pharmacokinetic (PK) interactions of etoposide and mitoxantrone with PSC were anticipated, measured in comparison with historical controls without PSC, and showed a 57% decrease in etoposide clearance (P =.001) and a 1.8-fold longer beta half-life for mitoxantrone in plasma (P <.05). The doses of mitoxantrone and etoposide were substantially reduced to compensate for these interactions and clinical toxicity and in Cohort II were well tolerated at dose levels of 4 mg/m2 mitoxantrone, 40 mg/m2 etoposide, and 1 g/m2 C daily for 5 days. Overall, postchemotherapy marrow hypoplasia was achieved in 33 patients. Twelve patients (32%) achieved complete remission, four achieved partial remission, and 21 failed therapy. The PK observations correlated with enhanced toxicity. The probability of an infectious early death was 36% (4 of 11) in patients with high PK parameters for either drug versus 5% (1 of 20) in those with lower PK parameters (P =.04). P-gp function was assessed in 19 patients using rhodamine-123 efflux and its inhibition by PSC. The median percentage of blasts expressing P-gp was increased (49%) for leukemic cells with PSC-inhibitable rhodamine efflux compared with 17% in cases lacking PSC-inhibitable efflux (P =.004). PSC-MEC was relatively well tolerated in these patients with poor-risk AML, and had encouraging antileukemic effects. The Eastern Cooperative Oncology Group is currently testing this regimen versus standard MEC chemotherapy in a phase III trial, E2995, in a similar patient population.     

Uterine rhabdomyosarcoma metastatic to mediastinal lymph nodes: diagnosis by transbronchial needle aspiration

An inherited tendency to hypercoagulability has been suggested as a cause of vascular thrombosis resulting in Legg-Calvé-Perthes disease (LCPD). Here we carried out an investigation of the most common inherited risk factors for hypercoagulability including the mutation in the factor V gene (factor V Leiden), the transition 20.210G-->A in the prothrombin gene, and also the homozygosity for the 677C-->T transition in the methylenetetrahydrofolate reductase gene (MTHFR). The investigation was carried out among 61 Brazilian children with LCPD, who were compared with 296 individuals from the general population. The prevalence of the factor V Leiden mutation was higher in LCPD patients than in the controls (4.9 vs. 0.7%; p = 0.03). However, no patient had the prothrombin gene variant, and no difference was found between patients and controls when homozygosity for MTHFR-T (3.2 vs. 2.6%: p = 0.64) was determined. These data suggest that in our population, the heterozygosity for factor V Leiden was the only inherited risk factor associated with the development of LCPD.     

Variation of P-QRS relation during atrioventricular node reentry tachycardia

OBJECTIVES: The main objective of this study was to characterize the phenomenon of variation in the P-QRS relation during atrioventricular node reentry tachycardia. BACKGROUND: Variation of P-QRS relation during tachycardia has been observed occasionally in atrioventricular node reentry tachycardia. However, the incidence, the characteristics and the mechanisms of this phenomenon have not been investigated previously. METHODS: Retrospective analysis was performed in 311 consecutive patients with slow-fast form and 108 patients with atypical or multiple form of atrioventricular node reentry tachycardia to examine whether variation of P-QRS relation with changes in AH, HA and AH/HA (A = atria; H = His bundle) ratio occurred during tachycardia. RESULTS: A total of 28 patients, 8 with slow-fast and 20 with atypical or multiple tachycardias, were found to manifest this phenomenon. There were 6 males and 22 females, with an average age of 38+/-16 years. In 10 patients, this phenomenon occurred transiently following electrical induction of the tachycardia. In 15 patients, changes in AH, HA and AH/HA ratio were associated with the occurrence of Wenckebach or 2:1 block proximal to the His bundle (H) recording site without interruption of the tachycardia. In nine patients, three with nonsustained tachycardia and six after administration of adenosine triphosphate, this phenomenon was observed at the termination of the tachycardia. This phenomenon was usually accompanied by a mild lengthening of the tachycardia cycle length. CONCLUSIONS: Variation of P-QRS relation with or without block may occur during atrioventricular node reentry tachycardia, especially in atypical or multiple-form tachycardias. It was postulated that decremental conduction in the distal common pathway, which exists between the distal link of the reentry circuit and the H, is primarily responsible for this phenomenon.