Serial cytological assay of micronucleus induction: a new tool to predict human cancer radiosensitivity

The role of the propeptide sequence and a disulfide bridge between sites 1 and 122 in chymotrypsin has been examined by comparing enzyme activities of wild-type and mutant enzymes. The kinetic constants of mutants devoid of the Cys1-Cys122 disulfide-linked propeptide show that this linkage is not important either for activity or substrate specificity. However this linkage appears to be the major factor in keeping the zymogen stable against non-specific activation. A comparison of zymogen stabilities showed that the trypsinogen propeptide is ten times more effective than the chymotrypsinogen propeptide in preventing non-specific zymogen activation during heterologous expression and secretion from yeast. This feature can also be transferred in trans to chymotrypsinogen; i.e. the chymotrypsin trypsin propeptide chimera forms a stable zymogen.     

Experimental fetal tracheal ligation reverses the structural and physiological effects of pulmonary hypoplasia in congenital diaphragmatic hernia

Infants with congenital diaphragmatic hernia (DH) and profound pulmonary hypoplasia are currently unsalvageable. The authors previously demonstrated that tracheal ligation (TL) accelerates fetal lung growth and reverses the pulmonary hypoplasia of fetal nephrectomy. The purpose of this study was to determine if the pulmonary hypoplasia of experimental DH could be similarly reversed and, if so, whether the resulting lungs would show better function than those of their DH counterparts. Eighteen fetal lambs were divided into three experimental groups of six animals each. In group 1, DH was created at 90 days' gestation. In group 2, DH was created at 90 days' gestation and TL performed during the same operation. Group 3 consisted of sham-operated controls. These animals were delivered near full-term, and their lungs analyzed by standard morphometric techniques. Ten additional fetal lambs were divided into two experimental groups of five animals each. In group 4, DH was created at 90 days' gestation. In group 5, DH was created at 90 days' gestation and TL performed 20 days later, at 110 days' gestation. These animals were pressure-ventilated via tracheostomy over a 2-hour period in which PaO2, PaCO2, and compliance were measured. Intratracheal pressure (ITP) was measured at the time of delivery in all groups. Upon retrieval, DH animals had abdominal viscera in the chest and small lungs; in contrast, DH/TL animals had the herniated viscera reduced from the chest by enlarged lungs. DH/TL lungs showed markedly increased growth, with significant increases in lung volume:body weight ratio (LV:BW; P = .0001), alveolar surface area (ALV.SA; P = .0001), and alveolar number (ALV#) (P = .0001) when compared with those of the DH or control group. This growth was associated with a normal maturation pattern based on histological appearance, normal airspace fraction, and normal alveolar numerical density. ITP in the DH/TL group was increased when compared with that of DH and control animals (P = .0001). Total lung DNA and protein were both elevated in the DH/TL animals (P = .0001). However, the DNA:protein ratio remained normal, suggesting lung growth had occurred through cell proliferation, not by hypertrophy. When ventilated over a range of settings, DH/TL lungs were more compliant (P = .0001) and achieved higher PaO2s (P < .003) and lower PaCO2s (P = .0001) than their DH counterparts. From these data, the authors conclude: (1) Experimental fetal DH produces hypoplastic lungs that are not capable of adequate gas exchange with conventional ventilation.(ABSTRACT TRUNCATED AT 400 WORDS)     

An in vitro model for toxin-mediated vascular leak syndrome: ricin toxin A chain increases the permeability of human endothelial cell monolayers

Vascular leak syndrome (VLS) is the dose-limiting toxicity observed in clinical trials of immunotoxins containing ricin toxin A chain (RTA). RTA itself is thought to cause VLS by damaging vascular endothelial cells, but the exact mechanism remains unclear. This is partially due to the paucity of appropriate models. To study VLS, we developed an in vitro model in which human umbilical vein-derived endothelial cells were first grown to confluence on microporous supports and then cultured under low pressure in the presence or absence of RTA. Endothelial cell barrier function was assessed by measuring the volume of fluid that passed through each monolayer per unit time. We found that RTA significantly increased monolayer permeability at times and concentrations consistent with the onset of VLS in patients treated with RTA-based immunotoxins. Scanning electron microscopy showed that intercellular gaps formed in endothelial monolayers exposed to RTA. Intercellular gap formation followed endothelial cell death caused by the enzymatic activity of RTA. We conclude that RTA is directly toxic to endothelial cells in vitro and speculate that this contributes to VLS in vivo.     

Identification and activity of cytosol creatine phosphokinase enzymes in normal and diseased skin

Phosphocreatine molecules (PCR) in skin regenerate adenosine triphosphate and help cutaneous tissue survive ischemia associated with skin flaps, grafts, and hair transplantation procedures. In addition, PCR concentration in psoriasis is elevated many times above normal, indicating either overproduction of PCR by mitochondrial creatine phosphokinase (CPK) enzymes or a defect in cytosol CPK enzymatic activity. Skin CPK isoenzymes, before this study, have not been identified. Herein, for the first time, cytosol CPK enzymatic activity was measured in normal and psoriatic, involved and uninvolved skin, skin tumors, and mouse skin and keratinocyte cell cultures. Creatine phosphokinase MM is the major isoenzyme in normal, uninvolved psoriatic and mouse skin. Total CPK enzymatic activity was increased in psoriasis and skin tumors. These data clearly indicate that increased PCR concentration in a psoriatic skin is not a result of decreased cytosol CPK enzymatic activity.     

Determinants of genital human papillomavirus infection among cytologically normal women attending the University of New Mexico student health center

OBJECTIVES: To confirm the risk factors for genital human papillomavirus (HPV) infection. GOAL OF THIS STUDY: To investigate risk factors for HPV detection apart from the correlated risk factors for cervical neoplasia. STUDY DESIGN: Cervical human papillomavirus (HPV) DNA was assessed in 357 cytologically normal women attending the University of New Mexico student health center. Cervical swab samples were obtained for HPV DNA detection and typing using a PCR-based DNA amplification system. Possible determinants of cervical HPV were examined including age, ethnicity, history of sexually transmitted disease, oral contraceptive use, smoking, age at first intercourse, lifetime number of sex partners, marital status, and history of pregnancy. RESULTS: A 44.3% overall prevalence of cervical HPV was observed. On univariate analysis, factors associated with increasing HPV prevalence included higher lifetime number of sex partners and single marital status. After adjustment for potential confounding variables, we found that HPV prevalence increased with higher lifetime number of sexual partners. CONCLUSION: These findings, along with those from the companion reports in this issue of the journal, support the sexual route of transmission of the virus.     

High-performance liquid chromatographic separation of carminic acid, alpha- and beta-bixin, and alpha- and beta-norbixin, and the determination of carminic acid in foods

During a study of natural food colours, a simple and reliable high-performance liquid chromatography system was developed for use with cochineal and annato. An isocratic mobile phase, consisting of methanol and 6% aqueous acetic acid, resolved bixin and norbixin, while a gradient system was used to separate carminic acid and the annato compounds. The carminic acid contents of cochineal extract, carmine and carmine hydrosoluble were determined using an isocratic mobile phase (40:60, v/v). The detection limit for carminic acid in the various products was approximately 100 ng/g. Carminic acid was determined quantitatively in fruit beverages, yogurt and candies. It was demonstrated that, because of decomposition, carminic acid was not suitable for use in candies when manufacturing temperatures above 100 degrees C were required. Most membrane filters are not suitable for use with cochineal solutions, but a cellulose membrane filter did not adsorb carminic acid and was used successfully to remove impurities from water-based cochineal products and food extracts containing carminic acid.     

Dexamethasone inhibition of interferon-alpha 2-induced stimulation of cortisol and growth hormone secretion in chronic myeloproliferative syndrome

This study investigated the acute effects of interferon-alpha 2 (IFN-alpha 2) on hormonal secretion in adult patients affected by a chronic myeloproliferative syndrome and tried to shed some light on the mechanism by which IFN-alpha 2 stimulates cortisol and GH secretion in humans. We compared the pattern of IFN-alpha 2-induced cortisol and GH release with that elicited after the same challenge given subsequent to pretreatment with dexamethasone (Dex). We studied eight patients affected by a chronic myeloproliferative syndrome (thrombocythemia) who had been selected for treatment with IFN-alpha 2. Four sets of experiments were performed: 1) 2 mL iv saline was given at 0800 h in eight cases; 2) 3 x 10(6) IU iv IFN-alpha 2 was given at 0800 h in eight cases; 3) 3 x 10(6) IU iv IFN-alpha 2 was given at 0800 h after pretreatment with 1.5 mg Dex (1 mg at midnight the previous night and 0.5 mg at 0700 h on the day of the test) in six cases; and 4) 2 mL iv saline was given at 0800 h after the same Dex pretreatment in four cases. Cortisol and GH were measured in plasma samples drawn at 30-min intervals between 0800 and 1300 h. Acute iv administration of IFN-alpha 2 stimulated the release of both cortisol and GH in each patient with a significant increment vs. control values, as assessed by areas under the curve. The administration of Dex significantly decreased basal plasma cortisol secretion and abolished cortisol response to IFN-alpha 2 administration. These data suggest that the stimulatory action of IFN-alpha 2 on cortisol release is mediated via a modulation of the activity of the hypothalamic-pituitary axis rather than through a direct effect at the level of the adrenal cortex. After Dex plus saline administration, no significant effect was observed on plasma GH levels, which remained low. Dex administration significantly decreased GH response to IFN-alpha 2. These data suggest that a hypothalamic or pituitary stimulation (or both) is involved in the mechanism of IFN-alpha 2-induced GH secretion. It remains to be established whether IFN-alpha 2 directly stimulates pituitary somatotropic cells or whether the cytokine exerts a stimulatory action on GH secretion by indirectly modulating the hypothalamic or pituitary activity. In conclusion, acute iv administration of IFN-alpha 2 represents a potent stimulus for cortisol and GH secretion in adult human subjects.(ABSTRACT TRUNCATED AT 400 WORDS)     

Optimisation of antibiotic therapy in cystic fibrosis patients. Pharmacokinetic considerations

Antibiotic therapy plays a central role in the medical management of patients with cystic fibrosis. While totally convincing efficacy data are lacking, antibiotics probably have a pronounced beneficial effect on both morbidity and mortality. Much has been learned in the past 20 years about antibiotic use in this population. At the same time, new antimicrobial agents with the potential to treat this condition have become available for use. The pharmacokinetics of a number of antibiotic classes, including beta-lactams, aminoglycosides and quinolones, are altered in this patient population. Increased total body clearance is a common occurrence but is not always changed enough to warrant altered dosages. Nonetheless, in light of altered pharmacokinetics in the cystic fibrosis population, appropriate dosage and monitoring parameters for a number of antibiotics have been determined.     

Common region of ALL-1 gene disrupted in epipodophyllotoxin-related secondary acute myeloid leukemia

Translocations at chromosomal band 11q23 characterize most de novo acute lymphoblastic leukemias (ALL) of infants, acute myeloid leukemias (AML) of infants and young children, and secondary AMLs following epipodophyllotoxin exposure. The chromosomal breakpoints at 11q23 have been cloned from isolated cases of de novo ALL and AML. Using an 859-base pair BamHI fragment of human ALL-1 complementary DNA that recognizes the genomic breakpoint region for de novo ALL and AML, we investigated two cases of secondary AML that followed etoposide-treated primary B-lineage ALL. In the first case, the translocation occurred between chromosomes 9 and 11 and the breakpoint at 11q23 localized to the same 9-kilobase region of the ALL-1 gene that is disrupted in most of the de novo leukemias. In the second case the translocation was between chromosomes 11 and 19. The breakpoint occurred outside of the ALL-1 breakpoint cluster region.