Excess of hMLH1 germline mutations in Swiss families with hereditary non-polyposis colorectal cancer

This article describes a high-performance liquid chromatographic (HPLC) method for the measurement of azithromycin (AZI) and two of its metabolites, 9a-N-desmethylazithromycin (ADES) and N-desmethylazithromycin (NDES), in human tears and plasma. The drug, metabolites, and internal standard (n-propylazithromycin [IS]) were detected electrochemically after injection of the extracted sample into the HPLC system. The peak height ratio (AZI, ADES, or NDES to IS) varied linearly, with concentrations in the ranges of 0.1 mg/L to 2.0 mg/L (tears) and 0.01 mg/L to 2.0 mg/L (plasma) of AZI, ADES, and NDES; the correlation coefficient (r) was more than 0.994 mg/L for all of the compounds (n=6). The analysis of tear samples collected at different intervals within 12 hours to 144 hours after a dose of 20 mg/kg of AZI from a trachoma patient yielded concentrations ranging from 1.52 mg/L to 0.34 mg/L for AZI, 0.79 mg/L to 0.27 mg/L for ADES, and 1.99 mg/L to less than 0.20 mg/L for NDES. The concentration of AZI in plasma ranged from 0.15 mg/L to 0.01 mg/L, whereas ADES and NDES were undetectable.     

Naltrexone, naltrindole, and CTOP block cocaine-induced sensitization to seizures and death

I.c.v. injection for 9 days of either naltexone (NTX) (5, 10, 20, 40 micrograms/rat) or a selective mu peptide (CTOP) (0.125, 0.25, 0.5, 1, 3, 6 micrograms/rat) or delta (naltrindole) (NLT) (5, 10, 20 micrograms/rat) subtype opioid receptor antagonist affected sensitization to cocaine (COC) (50 mg/kg, i.p.) administered 10 min after. NTX (5 and 40 micrograms/rat), NLT (10 and 20 micrograms/rat), and the peptide CTOP (0.25-0.5 microgram/rat) attenuated seizure parameters (percent of animals showing seizures, mean score and latency) in a day-related manner. The DD50 (days to reach 50% of death) value for COC was 2.69, whereas it was 9.67 and 7.27 for NTX 5 and 40 micrograms/rat, 8.59 for NLT (10 micrograms/rat), and 6.11, 5.95, and 4.30 for CTOP (0.25, 0.5, and 1 microgram/rat respectively). These findings suggest a concurrent involvement of mu- and delta-opioid receptor subtype in COC-induced sensitization to toxic effects.     

Chemical and swelling evaluations of amino group crosslinking in gelatin and modified gelatin matrices

PURPOSE: To determine the extent of amino group crosslinking in gelatin matrices by chemical assay, and to compare these results to crosslinking evaluations from swelling measurements. METHODS: Matrices crosslinked with a water soluble carbodiimide (EDC/G), glutaraldehyde (GTA/G), as well as a GTA crosslinked matrix prepared from gelatin modified to contain 230% greater crosslinking sites (GTA/Mod) were evaluated. Crosslinking extent, Xc, was determined by a UV assay of uncrosslinked amino groups before and after crosslinking, and was used to obtain crosslinking densities. Equilibrium swelling ratios, Qm, at 37 degrees C in isotonic pH 7.4 were used to calculate crosslinking degree from the Flory equation for swelling of ionic polymers for comparison to the chemically determined crosslinking densities. RESULTS: Of the original 33 x 10(-5) moles epsilon-amino groups/g gelatin, 91 to 95% were crosslinked in EDC/G and GTA/G. GTA/Mod lost 95% of the original 108 x 10(-5) moles amino groups/g gelatin. Crosslinking densities were 4.1 x 10(-4) and 4.2 x 10(-4) moles/mL for EDC/G and GTA/G, respectively. The value for GTA/Mod increased to 14.2 x 10(-4) moles/mL. Values of Qm followed the same trend. The Flory crosslinking degrees for both gelatin matrices were 12 x 10(-4) and 13 x 10(-4) moles/mL, respectively. The value for the more extensively crosslinked GTA/Mod was 280 x 10(-4) moles/mL. CONCLUSIONS: The swelling and chemical evaluations of crosslinking are in general agreement for matrices with the lower of two crosslinking levels. The chemical determination appears suitable for evaluating amino group crosslinking in gelatin and it may be suitable for other proteinaceous materials.     

Cyclophosphamide and 4-Hydroxycyclophosphamide/aldophosphamide kinetics in patients receiving high-dose cyclophosphamide chemotherapy

Using a recently developed gas chromatography and mass spectrometry method to determine whole-blood cyclophosphamide (CP) and 4-hydroxycyclophosphamide/aldophosphamide (4-HO-CP/AP) concentrations, we investigated their pharmacokinetics in women receiving CP therapy. Patients (n = 18) received one or two courses of CP: (a) a 90-min i.v. infusion (4 g/m2) followed by a 96-h i.v. infusion (6 g/m2) in combination with high-dose thiotepa; or (b) a 96-h i.v. infusion (6 g/m2) in combination with high-dose thiotepa. Whole-blood exposures to CP [area under the whole blood concentration versus time curve (AUCCP)] and 4-HO-CP/AP (AUC4HOCP) between courses 1 and 2 were compared after normalization to dose (g/m2). A nonproportional increase was observed for the AUCCP between the first course [1112 micrometer. h/g/m2 +/- 14% coefficient of variation (CV)] and the second course (1579 micrometer . h/g/m2 +/- 28% CV) (P < 0.001). In contrast, the AUC4HOCP (27 micrometer . h/g/m2 +/- 25% CV) determined for the first course was 29% higher than the AUC4HOCP (21 micrometer . h/g/m2 +/- 26% CV) for the second course (P < 0.01). The interpatient whole-blood exposures to both CP and 4-HO-CP/AP were remarkably consistent in this patient population with percent CVs ranging from 14 to 28%. Because thiotepa (800 mg/m2) was administered simultaneously with CP during the second course of treatment, possible inhibition of CP metabolism by thiotepa was investigated using human liver microsomes in vitro. IC50 values determined for inhibition of CP metabolism in three individual liver donors ranged from 1.0 to 40 micrometer. However, the clinical relevance of this observation has not been established.     

Effect of prednisone on response to influenza virus vaccine in asthmatic children

OBJECTIVE: To evaluate the immunogenicity of the influenza virus vaccine in children receiving short-course (a burst) prednisone therapy for acute asthmatic exacerbations. DESIGN: Prospective cohort study. SETTING: Outpatient pediatric clinic of a military medical center. PATIENTS: Children aged 6 months to 18 years requiring the 1996 influenza virus vaccine were eligible for the study. A total of 58 children were enrolled initially. The control group included 37 asthmatic children requiring less than 900 microg/d of inhaled prednisone and their siblings. The prednisone group included 21 children vaccinated at the beginning of a course of prednisone prescribed to treat an asthma exacerbation. Thirty-one control subjects (84%) and 19 patients in the prednisone group (90%) completed the study. Dropout was due to failure to come in for the postvaccination serum sampling. INTERVENTIONS: All study patients underwent immunization with the 1996-1997 trivalent subvirion influenza virus vaccine (FluShield; Wyeth Laboratories Inc, Marietta, Pa) containing 15-microg hemagglutinin antigens each of A/Texas/36/91 (H1N1) (A/H1), A/Wuhan/359/95 (H3N2)(A/H3), and B/Beijing/184/93 (B). The prednisone cohort received a burst of oral prednisone therapy (2 mg/kg per day for 5 days). MAIN OUTCOME MEASURES: To assess the immunogenicity of the vaccine between both groups, at least a 4-fold rise in titer and end titers of at least 1:40 to each of the 3 antigens were compared. Mean changes in geometric titers to the 3 antigens were also compared. RESULTS: Proportion of patients in each group with at least a 4-fold rise in titer to each of the influenza antigens was as follows: for A/H3N3 antigen, 15 patients (79%) in the prednisone group vs 22 controls (71%) (P = .74); for A/ H1N1 antigen, 16 patients in the prednisone group (84%) vs 20 controls (64%) (P = .20); and for B antigen, 7 patients in the prednisone group (37%) vs 8 controls (26%) (P = .53). Proportion of patients in each group with an end titer of at least 1:40 to each of the antigens was as follows: for A/ H3N2 antigen, 18 patients in the prednisone group (95%) vs 28 controls (90%) (P = .69); for A/H1N1 antigen, 17 patients in the prednisone group (89%) vs 26 controls (84%) (P = .99); and for B antigen, 7 patients in the prednisone group (37%) vs 13 controls (42%) (P = .99). There were also no significant differences between groups in the mean changes in geometric titers to any of the 3 antigens. CONCLUSIONS: Prednisone bursts did not diminish the response of asthmatic children to the 1996 influenza virus vaccine, compared with controls. Children can be effectively vaccinated against influenza virus while they are receiving prednisone therapy bursts for asthmatic exacerbations.     

Maximum rate of change in oesophageal pressure assessed from unoccluded breaths: an option where mouth occlusion pressure is impractical

A nested polymerase chain reaction (PCR) assay was used to determine the levels of cytomegalovirus (CMV) genomes in cells of CSF from 19 patients with AIDS and 12 human immunodeficiency virus type I (HIV-1) seronegative individuals with various neurologic disorders. Five AIDS patients had autopsy-proven CMV encephalitis (CMVE) and 14 patients had no evidence of CMV-related CNS manifestations. CSF cells from AIDS patients with confirmed CMVE harbored viral genomes at a median value of 3,333/10(5) cells (range, 1,667 to 5,333/10(5) cells; mean, 3,558/10(5) cells) compared with a median value of 125/10(5) cells (range, 9 to 1,000/10(5) cells; mean, 281/10(5) cells) for AIDS patients with CMV-unrelated symptoms and a median value of 1.9/10(5) cells (range, 0 to 562/10(5) cells; mean, 52/10(5) cells) for HIV-1 seronegative control subjects. A subset of CSF samples was assessed using a modified single round amplification PCR with a detection limit of 500 viral copies. CMV DNA was detected in all four specimens from AIDS patients with proven CMVE, in two of five AIDS patients without CMVE, and in none of five seronegative control subjects. Quantitation of CMV genomes in CSF cells is indicative of latent or productive CMV infection and is a reliable means for diagnosis of CMVE in patients with AIDS. Detection of a cutoff value of cellular CMV genomes by means of nonquantitative PCR may identify patients at risk for CMV infection of the CNS.     

Chimeric purine transporters of Aspergillus nidulans define a domain critical for function and specificity conserved in bacterial, plant and metazoan homologues

In Aspergillus nidulans, purine uptake is mediated by three transporter proteins: UapA, UapC and AzgA. UapA and UapC have partially overlapping functions, are 62% identical and have nearly identical predicted topologies. Their structural similarity is associated with overlapping substrate specificities; UapA is a high-affinity, high-capacity specific xanthine/uric acid transporter. UapC is a low/moderate-capacity general purine transporter. We constructed and characterized UapA/UapC, UapC/UapA and UapA/UapC/UapA chimeric proteins and UapA point mutations. The region including residues 378-446 in UapA (336-404 in UapC) has been shown to be critical for purine recognition and transport. Within this region, we identified: (i) one amino acid residue (A404) important for transporter function but probably not for specificity and two residues (E412 and R414) important for UapA function and specificity; and (ii) a sequence, (F/Y/S)X(Q/E/P) NXGXXXXT(K/R/G), which is highly conserved in all homologues of nucleobase transporters from bacteria to man. The UapC/UapA series of chimeras behaves in a linear pattern and leads to an univocal assignment of functional domains while the analysis of the reciprocal and 'sandwich' chimeras revealed unexpected inter-domain interactions. cDNAs coding for transporters including the specificity region defined by these studies have been identified for the first time in the human and Caenorhabditis elegans databases.     

Preclinical and phase I clinical studies with the nonclassical antifolate thymidylate synthase inhibitor nolatrexed dihydrochloride given by prolonged administration in patients with solid tumors

PURPOSE: A phase I, multicenter trial of the thymidylate synthase (TS) inhibitor THYMITAQ (nolatrexed dihydrochloride; Agouron Pharmaceuticals, Inc, San Diego, CA) given by 5-day continuous infusion was performed to establish the maximum-tolerated dose (MTD) and to investigate pharmacokinetics, pharmacodynamics, and antitumor effects. METHODS: In vitro and in vivo preclinical studies demonstrated increased activity with prolonged nolatrexed exposure. In 32 patients, nolatrexed was given as a 5-day infusion at 96 to 1,040 mg/m2/d for 5 days. Pharmacokinetics were determined from high-performance liquid chromatography (HPLC) analyses of plasma and urine. In addition to studying toxicity, plasma deoxyuridine (UdR) elevations were measured as a marker of TS inhibition. RESULTS: The MTD was 904 mg/m2/d for 5 days and the recommended phase II dose is 800 mg/m2/d for 5 days. The dose-limiting toxicity was neutropenia with clinically significant thrombocytopenia and mucositis. These antiproliferative toxicities of nolatrexed were predictable and reversible. A partial response that lasted 3 months occurred in a patient with metastatic colorectal cancer. Pharmacokinetics were nonlinear, with the median plasma clearance (CI) decreasing from 151 mL/min/m2 (range, 124 to 211) at 96 mg/m2/d for 5 days to 49 mL/min/m2 (range, 30 to 84) at 768 mg/ m2/d for 5 days. The half-life (t1/2) was 173 minutes (range, 43 to 784) and 18% (range, 9% to 35%) of the dose was excreted unchanged in the urine. Plasma UdR increased, but returned to pretreatment levels after the end of infusion. Hematologic toxicity was significantly related to nolatrexed plasma concentrations and dose. CONCLUSION: Nolatrexed can be safely administered to patients at a dose of 800 mg/m2/d over 5 days by continuous intravenous infusion and this schedule is associated with antitumor effects. The phase II evaluation of nolatrexed is ongoing.     

Clinical evaluation of a reagent for detection of DNA of Mycobacterium tuberculosis complex using the ligase chain reaction (LCR) method

We evaluated the clinical efficacy of LCR MTB, a reagent developed by Abbott in the USA, in the full automatic ligase chain reaction (LCR) for detection of DNA of M. tuberculosis complex using a thermostable ligase. Using 458 samples isolated from patients with tuberculosis, LCR was compared with a smear method and with a culture method, and was also compared with two other methods of gene amplification, MTD and Amplicor, using 340 and 200 of the 458 samples, respectively. The LCR method detected M. tuberculosis in 49.8% (228/458) of the samples, and was superior to the smear method (31.9%, 146/458) and the culture method (39.1%, 179/458) in sensitivity. The LCR method was also superior to the MTD and Amplicor methods; sensitivity were 37.9% (129/340) for MTD vs. 47.6% (162/340) for LCR, and 56.5% (113/200) for Amplicor vs. 59.5% (119/200) for LCR. These favorable results and the convenience of the LCR method, which enables rapid detection of target genes with a high degree of sensitivity, strongly suggest that LCR MTB is useful as a reagent for detection of M. tuberculosis using nucleic acid amplification.     

Pharmacokinetic analysis of gentamicin thrice and single daily dosage in pediatric cancer patients

Fifty-two children suffering from different types of malignancies were included and evaluated for the pharmacokinetics of gentamicin thrice or single daily dosage protocols. All the study population received a total dose of 5 mg/kg daily. Thirty children received gentamicin thrice daily, and 22 were treated using the single daily protocol; all had fever and neutropenia when included. The individual pharmacokinetic parameters were calculated using a one-compartment model for two blood gentamicin samples. The mean (SD) t 1/2 (h), clearance (mL/min/BSA), Vss (L/kg), Cmax (micrograms/mL), and Cmin (micrograms/mL) were 3.05 (1.0) and 3.9 (0.6) h, 136 (61.3) and 99.9 (65.3) mL/min/BSA, 0.4035 (0.167) and 0.457 (0.17) L/kg, 5.2 (2.0) and 11.5 (4.2) micrograms/mL, 0.8 (0.6) and 0.18 (0.1) microgram/mL for thrice and single daily dosage schedules, respectively. The single gentamicin daily dose protocol had a significantly longer t 1/2, shorter clearance normalized to BSA, higher Cmax, and lower Cmin in comparison with the thrice daily schedule. We recommend the use of gentamicin at 5 mg/kg daily delivered as a single daily dose for pediatric cancer patients with fever and neutropenia, in spite of the measured pharmacokinetic differences, which in our opinion have no clinical significance.     

Nicotine as a conditioned stimulus: Impact of attention-deficit/hyperactivity disorder medications.

People diagnosed with attention-deficit/hyperactivity disorder (ADHD) are at an increased risk to start smoking and have greater difficulty quitting. Nicotine, one of the principal addictive components of tobacco smoke, functioned as a conditioned stimulus (CS) for intermittent sucrose delivery in a Pavlovian drug discrimination task with rats. This study compared the ability of commonly prescribed ADHD medications (i.e., methylphenidate, atomoxetine, and bupropion) and additional dopamine reuptake inhibitors (i.e., cocaine and GBR 12909) to substitute for the CS effects of nicotine. Atomoxetine was also used to antagonize these CS effects. Rats acquired the discrimination as evidenced by increased dipper entries in nicotine (0.2 mg base/kg) sessions as compared with saline sessions. Nicotine generalization was dose dependent. Bupropion (10 and 20 mg/kg), methylphenidate (10 mg/kg), and cocaine (5 and 10 mg/kg) partially substituted for the 0.2 mg/kg nicotine CS. Atomoxetine did not substitute for the nicotine CS; however, atomoxetine (1 to 10 mg/kg) partially blocked nicotine's CS effects. These results suggest that atomoxetine, bupropion, and/or methylphenidate may be effective treatments for people diagnosed with ADHD and addicted to nicotine. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Genetic and electrophysiological studies of Drosophila syntaxin-1A demonstrate its role in nonneuronal secretion and neurotransmission

Development of a new method for the determination of Cr(III) and Cr(VI) is described. Anion-exchange high-performance liquid chromatography (HPLC) was used to separate Cr(III) and Cr(VI) with on-line detection by inductively coupled plasma atomic emission spectroscopy (ICP-AES) at 2766 A in preliminary studies, and inductively coupled plasma mass spectrometry (ICP-MS) with single-ion monitoring at m/z 52 and m/z 53 for final work. A mobile phase consisting of ammonium sulfate and ammonium hydroxide was used, and a simple chelation procedure with EDTA was followed to stabilize the Cr(III) species in standard solutions. ICP-MS results indicated the feasibility of using chromium isotope m/z 53 instead of the more abundant m/z 52 isotope due to a high mobile-phase background most significantly from the SO+ polyatomic interference. The absolute detection limits based on peak-height calculations were 40 pg for Cr(III) and 100 pg for Cr(VI) in aqueous media by HPLC-ICP-MS. The linear dynamic range extended from 5 ppb (ng/ml) to 1 ppm (micrograms/ml) for both species. By HPLC-ICP-AES, detection limits were 100 ng for Cr(III) and 200 ng for Cr(VI). Cr(III) was detected in NIST-SRM 1643c (National Institute of Standards and Technology-Standard Reference Material, Trace Elements in Water) by HPLC-ICP-MS at the 20 ppb level.     

Pharmacokinetics of proguanil in malaria patients treated with proguanil plus atovaquone

Clinical studies have shown atovaquone (ATQ), a new blood schizontocidal drug, in combination with proguanil (PROG) to be very effective in the treatment of acute multidrug-resistant falciparum malaria. The multiple dose pharmacokinetics of PROG were determined in Thai patients with acute falciparum malaria given PROG alone (200 mg PROG twice a day for 3 days, n = 4) and concurrently PROG and ATQ (200 mg PROG and 500 mg ATQ twice a day for 3 days, n = 12). There were no statistical differences (p > 0.05) in the area under the plasma drug concentration-time curve (AUC), apparent oral clearance (CL/F) and elimination half-life (t1/2) of PROG between patients given PROG alone and PROG/ ATQ. The median (range) kinetic values of PROG in patients given PROG alone and PROG/ATQ were respectively: CL/F = 1.25 l/h/kg (0.99-1.45) and 0.95 (0.73-1.32) l/h/kg, and t1/2 = 14.2 hours (9.3-16.8) and 13.6 hours (9.1-17.6). The CL/F and t1/2 of PROG in the Thai patients treated with the 2 treatment regimens were also comparable to values reported in healthy Thai volunteers given a standard prophylactic dose (200 mg PROG). The results of this preliminary study suggest that ATQ is unlikely to affect the pharmacokinetics of PROG to a clinically important extent at an ATQ dosage of 500 mg twice a day for 3 days in malaria infected patients.     

A method for a selective local application of radioactive precursors of nucleic acids to the cow ovary

OBJECTIVE: To determine whether genetic polymorphism of cytochrome P450 (CYP) 2C9 affects the in vivo metabolism of warfarin enantiomers. METHODS: Eighty-six Japanese patients heart disease who were given warfarin participated in the study. Plasma unbound concentrations of warfarin enantiomers and urinary (S)-7-hydroxywarfarin concentrations were measured by means of a chiral HPLC and ultrafiltration technique to calculate the unbound oral clearance (CLpo,u) for the enantiomers and the formation clearance (CLm) for (S)-warfarin 7-hydroxylation. Genotyping for CYP2C9 (the wild type [wt], Arg144/Cys, and I1e359/Leu) and for CYP2C19 (wt, ml, and m2) was performed with a polymerase chain reaction method. RESULTS: Three patients were heterozygous for the CYP2C9 Leu359 mutation but none were homozygous for the mutation (the allele frequency of 0.017). None had a CYP2C9 Cys144 allele. The medians for (S)-warfarin CLpo,u and its 7-hydroxylation CLm obtained from heterozygotes of CYP2C9 Leu359 were significantly less than those obtained from homozygotes of the wt allele, as follows: 234 ml/min (range, 156 to 269 ml/min) versus 632 ml/min (range, 180 to 2070 ml/min) (p < 0.001) and 0.20 ml/min (range, 0.05 to 0.77 ml/min) versus 0.80 ml/min (range, 0.05 to 14.9 ml/min) (p < 0.05), respectively. In contrast, no difference was observed in (R)-warfarin CLpo,u between the groups. The allele frequencies for CYP2C19 m1 and CYP2C19 m2 were 0.26 and 0.14, respectively, indicating 15% of patients were genotypically poor metabolizers of CYP2C19. No difference in CLpo,u for warfarin enantiomers was observed between the assumed CYP2C19 phenotypes. CONCLUSION: Heterozygotes for CYP2C9 I1e359/Leu allele have reduced in vivo metabolism of (S)-warfarin but not (R)-warfarin. Because (S)-warfarin has a greater anticoagulant potency than its (R)-congener, the genetic polymorphism of CYP2C9 may partly account for the large interpatient variability in therapeutic dosages of warfarin.     

Developmental exposure to Aroclor 1254 produces low-frequency alterations in adult rat brainstem auditory evoked responses

Developmental exposure of Long-Evans rats to 0, 1, 4, or 8 mg/kg/day Aroclor 1254 (A1254) from Gestational Day 6 through Postnatal Day 21 produces an elevated behavioral threshold for a 1-kHz tone. Brainstem auditory evoked responses (BAERs) were assessed in a subset of these animals (about 1 year old) using filtered clicks at 1 (65 and 80 dB SPL), 4 (60 and 80 dB SPL), 16 (40 and 80 dB SPL), and 32 (40 and 80 dB SPL) kHz. Aroclor 1254 decreased BAER amplitudes at 1 and 4 kHz, but not at 16 or 32 kHz. A dose-related decrease in the baseline-to-peak P1A amplitude was observed for the 1-kHz (80-dB) stimulus. Doses of 1, 4, or 8 mg/kg/day A1254 decreased the peak-to-peak amplitude of both P1AN1 and P1BN1 for a 1-kHz (80-dB) stimulus. Doses of 4 and 8 mg/kg/day A1254 decreased the peak-to-peak amplitude of N1P2 and P2N2 for a 4-kHz (60-dB) or 1-kHz (80-dB) stimulus. At 8 mg/kg/day, A1254 also increased the latency of peak P4 at 1 kHz (65 dB). The decreases in peak P1A amplitudes are consistent with a dysfunction of the cochlea and/or auditory nerve. Together, the data confirm that developmental exposure of rats to A1254 produces a permanent low- to mid-frequency auditory dysfunction and suggest a cochlear and/or auditory nerve site of action.     

Assessment of basal sound identification skills and communication abilities in profoundly deaf children fitted with hearing aids or a cochlear implant

Recent in vitro findings suggest that bisphosphonates, potent inhibitors of osteoclastic bone resorption, may also have a direct action on osteoblasts. The purpose of this study was to search for potential effects of etidronate and alendronate on the formation of early and late osteoblastic cell precursors by measuring the number of colony-forming units for fibroblasts (CFU-F) and colony-forming units for osteoblasts (CFU-OB) in murine and human bone marrow cultures. In murine marrow cultures, etidronate (10(-5) to 10(-9) mol/L) significantly stimulated the formation of CFU-F with a maximal effect at 10(-5) mol/L (mean increase over control values+/-SD: 106+/-17%;p < 0.001), whereas alendronate had a biphasic effect, being stimulatory at concentrations below 10(-7) mol/L (78+/-5%; p < 0.001), and inhibitory at higher doses. The formation of CFU-OB was also inhibited by both bisphosphonates at the highest concentrations (10(-5) mol/L and 10(-6) mol/L), but it was significantly stimulated at lower concentrations (from 10(-7) to 10(-9) mol/L for etidronate and 10(-7) to 10(-10) mol/I, for alendronate; p < 0.001). In human bone marrow cultures, alendronate (10(-8) to 10-(12) mol/L) increased CFU-F formation with a maximal effect at 10(-10) mol/L (161+/-12 %; p < 0.01). CFU-OB formation, observed only in the presence of dexamethasone (10(-8) mol/L), was markedly stimulated by alendronate at the above concentrations with a maximal increase at 10(-10) mol/L (133+/-34%; p < 0.001). The in vivo short-term effects of bisphosphonates on the formation of early osteoblast precursors were also studied in bone marrow cultures from young female mice treated with weekly subcutaneous injections of etidronate (0.3, 3, and 30 mg/kg) or alendronate (0.3, 3, and 30 microg/kg) and from aging female mice treated with the two lowest doses of both drugs. After 1 month of treatment, etidronate (0.3 and 3 mg/kg) and alendronate (0.3 and 3 microg/kg) significantly increased the number of CFU-F colonies in the bone marrow from young and old animals, whereas the highest dose of both drugs had no effect in young mice. Our results, together with previously reported observations of bone-forming effects in osteoporosis, suggest that bisphosphonates may have, in vivo, a potentially relevant influence on cells of the osteoblastic lineage, distinct from their inhibitory action on osteoclasts.     

Effect of continuous rotational therapy on intracranial pressure in the severely brain-injured patient

Deoxycytidine kinase activity (dCk) was monitored in cell lines from a rat acute myeloid leukemia model of acquired resistance to cytosine arabinoside (AraC) and decitabine (DAC). In both AraC-resistant cell lines (RCL/A and its subclone RA/7), as well as in a DAC-resistant cell line (RCL/D) which we generated from the drug-sensitive RCL/0 cell line, a total deficiency of dCk activity and a cross-resistance for AraC and DAC was demonstrated. Furthermore, the metabolization of deoxycytidine (dC) was severely impaired in all these cell lines. Km values for dC (9.4 microM in RCL/0 cells) had increased 70- to 100-fold in RCL/D (Km = 673.2 microM), in RCL/A (Km = 947.2 microM) and in RA/7 (Km = 817.5 microM). Vmax values were unaltered in RCL/D and RA/7, and twofold increased in RCL/A. Addition of hydroxyurea (HU) to cell cultures stimulated dCk salvage pathway activity in RCL/0 cells for dC, AraC, and DAC by increasing Vmax values approximately 160% leaving Km constants unchanged. In all resistant cell lines, HU pre-incubation did not influence the level of dCk activity, leaving Km and Vmax values unaltered. These data indicate that deficiency of dCk activity is crucial in the mechanism of drug resistance in this model.     

Recombinant fusion proteins for the industrial production of disulfide bridge containing peptides: purification, oxidation without concatamer formation, and selective cleavage

The relative contributions of protein tyrosine kinases (PTKs) and protein kinase C isoenzymes (PKCs), a family of serine/threonine kinases, in integrin alpha(IIb)beta3 (glycoprotein IIb/IIIa) exposure are the subject of much controversy. In the present study we measured the effect of the PTK inhibitor herbimycin A and the PKC inhibitor bisindolylmaleimide I on 125I-fibrinogen binding to alpha(IIb)beta3 and on aggregation/secretion induced by different agonists. Dose-response studies showed complete inhibition of alpha(IIb)beta3 exposure by 30 micromol/L (ADP stimulation) and 35 to 40 micromol/L (alpha-thrombin stimulation) herbimycin A. In contrast, inhibition of exposure by bisindolylmaleimide I varied from none (for ADP and epinephrine), to 30% (for platelet-activating factor), and to approximately 80% (for alpha-thrombin). Studies with a submaximal dose of herbimycin A (approximately 50% inhibition of the ADP-response) and a maximal dose of bisindolylmaleimide I showed that optical aggregation had a similar sensitivity to the inhibitors as alpha(IIb)beta3 exposure with minimal interference by secreted ADP. Thus, the relative contributions of tyrosine and serine/threonine kinases in alpha(IIb)beta3 exposure and aggregation differ among the different agonists, with an exclusive role for PTKs in ADP- and epinephrine-induced responses and a role for both PTKs and PKCs in responses induced by platelet-activating factor and alpha-thrombin.     

Predatory behavior in laboratory mice: Strain and sex comparisons.

Measured latency to attack live crickets in 26 male and 26 female naive, non-food-deprived laboratory mice from a genetically heterogenous stock (HS/Ibg) and in 7 inbred strains (10 male and 10 female Ss for each strain). The HS/Ibg, Is/Crgl, C57BL/Crgl, and C3H/Crgl Ss had the shortest latencies to attack crickets. The RIII/Crgl and BALB/cCrgl Ss were intermediate, while DBA/2Crgl and A/Crgl were slowest. Among the inbred strains sex differences were nonexistent or inconsistent from strain to strain. However, HS/Ibg males were more likely to attack than were females. Enough females attack for cricket killing to be a useful form of attack behavior for genetic experiments. In nearly all Ss latency decreased from the 1st to the 2nd trial, indicating that cricket killing is rapidly learned in adult mice of a wide variety of genotypes. (16 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

HBED: the continuing development of a potential alternative to deferoxamine for iron-chelating therapy

To further examine the potential clinical usefulness of the hexadentate phenolic aminocarboxylate iron chelator N, N'-bis(2-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid (HBED) for the chronic treatment of transfusional iron overload, we performed a subchronic toxicity study of the HBED monosodium salt in rodents and have evaluated the iron excretion in primates induced by HBED. The HBED-induced iron excretion was determined for the monohydrochloride dihydrate that was first dissolved in a 0.1-mmol/L sodium phosphate buffer at pH 7.6 and administered to the primates either orally (PO) at a dose of 324 micromol/kg (149.3 mg/kg, n = 5), subcutaneously (sc) at a dose of 81 micromol/kg (37.3 mg/kg, n = 5), sc at 324 micromol/kg (n = 5), and sc at 162 micromol/kg (74.7 mg/kg) for 2 consecutive days for a total dose of 324 micromol/kg (n = 3). In addition, the monosodium salt of HBED in saline was administered to the monkeys sc at a single dose of 150 micromol/kg (64.9 mg/kg, n = 5) or at a dose of 75 micromol/kg every other day for three doses, for a total dose of 225 micromol/kg (n = 4). For comparative purposes, we have also administered deferoxamine (DFO) PO and sc in aqueous solution at a dose of 300 micromol/kg (200 mg/kg). In the iron-loaded Cebus apella monkey, whereas the PO administration of DFO or HBED even at a dose of 300 to 324 micromol/kg was ineffective, the sc injection of HBED in buffer or its monosodium salt, 75 to 324 micromol/kg, produced a net iron excretion that was nearly three times that observed after similar doses of sc DFO. In patients with transfusional iron overload, sc injections of HBED may provide a much needed alternative to the use of prolonged parenteral infusions of DFO. Note: After the publication of our previous paper (Blood, 91:1446, 1998) and the completion of the studies described here, it was discovered that the HBED obtained from Strem Chemical Co (Newburyport, MA) that was labeled and sold as a dihydrochloride dihydrate was in fact the monohydrochloride dihydrate. Therefore, the actual administered doses were 81, 162, or 324 micromol/kg; not 75, 150, or 300 micromol/kg as was previously reported. The new data have been recalculated accordingly, and the data from our earlier study, corrected where applicable, are shown in parentheses.