Generation of constitutively active recombinant caspases-3 and -6 by rearrangement of their subunits

Caspases play a major role in the transduction of the apoptotic signal and execution of apoptosis in mammalian cells. Ectopic overexpression of the short prodomain caspases-3 and -6 precursors in mammalian cells does not induce apoptosis. This is due to their inability to undergo autocatalytic processing/activation and suggests that they depend on the long prodomain caspases for activation. To investigate directly the apoptotic activity of these two caspases in vivo, we engineered constitutively active recombinant caspases-3 and -6 precursors. This was achieved by making contiguous precursor caspases-3 and -6 molecules, which have their small subunits preceding their large subunits. Unlike their wild type counterparts, these recombinant molecules were capable of autocatalytic processing in an in vitro translation reaction, suggesting that they are catalytically active. They were also capable of autoprocessing and inducing apoptosis in vivo independent of the upstream caspases. Furthermore, their autocatalytic and apoptotic activities were inhibited by the pancaspase inhibitor z-VAD-fluoromethylketone, but not by CrmA or Bcl-2, thus directly demonstrating that the targets of inhibition of apoptosis by CrmA and Bcl-2 are upstream of caspases-3 and -6. Since caspases-3 and -6 are the most downstream executioners of apoptosis, the constitutively active versions of these caspases could be used at very low concentrations in gene therapy model systems to induce apoptosis in target tissues or tumors.     

Adenosine acts by A1 receptors to stimulate release of prolactin from anterior-pituitaries in vitro

Adenosine has been identified in the anterior pituitary gland and is secreted from cultured folliculostellate (FS) cells. To determine whether adenosine controls the secretion of anterior pituitary hormones in vitro, adenosine was incubated with anterior pituitaries. It stimulated prolactin (PRL) release at the lowest concentration used (10(-10) M); the stimulation peaked at 10(-8) M with a threefold increase in release and declined to minimal stimulation at 10(-4) and 10(-3) M. Follicle-stimulating hormone release was maximally inhibited at 10(-8) M, whereas luteinizing hormone release was not significantly inhibited. Two selective A1 adenosine receptor antagonists (10(-7) or 10(-5) M) had no effect on basal PRL release, but either antagonist completely blocked the response to the most effective concentration of adenosine (10(-8) M). In contrast, a highly specific A2 receptor antagonist (10(-7) or 10(-5) M) had no effect on basal PRL release or the stimulation of PRL release induced by adenosine (10(-8) M). We conclude that adenosine acts to stimulate PRL release in vitro by activating A1 receptors. Since the A1 receptors decrease intracellular-free calcium, this would decrease the activation of nitric oxide synthase in the FS cells, resulting in decreased release of nitric oxide (NO). NO inhibits PRL release by activating guanylate cyclase that synthesizes cGMP from GTP; cGMP concentrations increase in the lactotrophs leading to inhibition of PRL release. In the case of adenosine, NO release from the FS cells decreases, resulting in decreased concentrations of NO in the lactotrophs, consequent decreased cGMP formation, and resultant increased PRL release.     

Prenatal diagnosis of a fetal head and neck neoplasm: differential diagnosis, management, and histology

A fetal head and neck malignancy was prenatally diagnosed. The parents allowed the fetus to die during labour, due to the poor prognosis. We discuss the corresponding pathology findings, differential diagnosis, and management of this rare entity. Prenatal diagnosis of fetal neoplasms theoretically improves outcome, although this was not true in our case.     

Internal validity of attention deficit hyperactivity disorder, oppositional defiant disorder, and overt conduct disorder symptoms in young children: implications from teacher ratings for a dimensional approach to symptom validity

A software system for interactive manipulation of three-dimensional data has been developed, based on the Open Inventor tool kit. The primary use of this software system is in the segmentation of tomographic reconstructions of subcellular structures. To this end, the reconstruction is represented by volume rendering and displayed in stereo. A three-dimensional cursor with adjustable shape and size is used to define and isolate regions of interest inside the volume, based on the user's expert knowledge. Once isolated, the region of interest can be conveniently analyzed and displayed.     

Anaesthesia and the broken hearted. 170th annual oration: Royal Victoria Hospital, Belfast 2nd October 1997

Endoscopic evaluation of the patient with lung metastases takes on many forms depending upon the extent of disease and the intent of treatment, be that curative or palliative. Thorascopy, and occasionally mediastinoscopy, may be helpful in assessing operability in patients with extensive disease on a preoperative computed tomography scan. However, when in doubt, exploration is always indicated in the young, good risk patient. Palliative efforts usually concern airway obstruction and malignant effects. A variety of technologies, including laser, brachytherapy, and endoluminal stents, helps manage symptomatic bronchial or tracheal lesions.     

Lysophosphatidic acid-induced proliferation-related signals in astrocytes

Lysophosphatidic acid (LPA) is a potent lipid biomediator that is likely to have diverse roles in the brain. Thus, LPA-induced events in astrocytes were defined. As little as 1 nM LPA induced a rapid increase in the concentration of intracellular free calcium ([Ca2+]i) in astrocytes from neonatal rat brains. This increase was followed by a slow return to the basal level. Intracellular calcium stores were important for the initial rise in [Ca2+]i, whereas the influx of extracellular calcium contributed significantly to the extended elevation of [Ca2+]i. LPA treatment also resulted in increases in lipid peroxidation and DNA synthesis. These increases in [Ca2+]i, lipid peroxidation, and DNA synthesis were inhibited by pretreatment of cells with pertussis toxin or H7, a serine/threonine protein kinase inhibitor. Moreover, the LPA-induced increase in [Ca2+]i was inhibited by a protein kinase C inhibitor, Ro 31-8220, and a calcium-dependent protein kinase C inhibitor, G? 6976. The increase in [Ca2+]i was important for the LPA-induced increase in lipid peroxidation, whereas the antioxidant, propyl gallate, inhibited the LPA-stimulated increases in lipid peroxidation and DNA synthesis. In contrast, pertussis toxin, H7, and propyl gallate had no effect on LPA-induced inhibition of glutamate uptake. Thus, LPA appears to signal via at least two distinctive mechanisms in astrocytes. One is a novel pathway, namely, activation of a pertussis toxin-sensitive G protein and participation of a protein kinase, leading to sequential increases in [Ca2+]i, lipid peroxidation, and DNA synthesis.     

Phenotype of the obese Koletsky (f) rat due to Tyr763Stop mutation in the extracellular domain of the leptin receptor (Lepr): evidence for deficient plasma-to-CSF transport of leptin in both the Zucker and Koletsky obese rat

The obese phenotypes of the diabetes (db) mouse and fatty fa) rat are due to functional null mutations of the leptin receptor (Lepr). The recessive mutation in the Koletsky (f) obese rat maps to the same genetic intervals as db and fa and fails to complement the fa mutation. Comparison of the sequence of brain Lepr cDNA from +/+ and f/f animals reveals a T2349A transversion resulting in a Tyr763Stop nonsense mutation in the gene just before the transmembrane domain. Virtual absence of Lepr mRNA in whole brain from f/f animals is consistent with the presence of a null mutation. The predicted reduced cerebrospinal fluid (CSF) transport of leptin in both f/f and fa/fa mutants is reflected in the approximately 10-fold lower ratio of CSF/plasma leptin concentration in the obese versus lean animals. However, equivalent CSF leptin concentration between lean and obese rats (fa/fa, f/f) indicates that leptin can enter the CSF through a non-Lepr-mediated mechanism, which may be saturated at normal physiological plasma leptin concentration.     

The prevalence of risk factors among women in the United States by race and age, 1992-1994: opportunities for primary and secondary prevention

OBJECTIVE: To analyze the prevalence of 11 modifiable behavioral risk factors, including multiple risk factors, among white, black, Asian and Pacific Islander, American Indian, and Hispanic women in the United States. DESIGN: We used Behavioral Risk Factor Surveillance System (BRFSS) data for 1992 to 1994 to examine risk factors (smoking; obesity; diabetes; heavy alcohol consumption; sedentary lifestyle; and inadequate use of seat belts, pap smears, consumption of fruits or vegetables, mammography and colorectal screening, and immunization), among women age 18 to 49, 50 to 64, and 65 and older. We also conducted a multiple regression analysis, comparing the odds of having either 1-2 versus 0 or 3 or more versus 0 risk factors among racial/ethnic groups, controlling for education and family income, to see if racial/ethnic differences can be attributed to socioeconomic differences. RESULTS: US women engage in a variety of behaviors that place them at risk for many causes of morbidity and mortality. Risk profiles vary substantially among racial/ethnic populations: Pacific Islanders have relatively low prevalences of most major risk factors, while blacks and American Indians have relatively high prevalences of many major risk factors. Prevalence differences among racial/ethnic populations are diminished but not eliminated when socioeconomic factors are accounted for. CONCLUSIONS: Appropriately designed programs to help women reduce their behavioral risk factors are needed. Action by health care providers, communities, and policy makers can substantially improve the health of women in the United States.     

Effects of phencyclidine on immediate early gene expression in the brain

An enzyme-linked immunosorbent assay (ELISA) was developed for sensitive and specific determination of pravastatin (PS) sodium, a HMG-CoA reductase inhibitor. Preparation of immunogens to obtain antisera was carried out using chemically modified PS; beta-alanine derivative of PS (for ELISA-1) and 5-deoxy- PS (for ELISA-2) were linked to bovine serum albumin via its terminal carboxylic acid by the N-succinimidyl ester method, to avoid intramolecular lactonization of PS. Enzyme-labeled antigens were prepared similarly by coupling with horseradish peroxidase, and were used by homogeneous combination of antisera. The enzymic activity was determined using a microtiter plate coated with second antibody and tetramethylbenzidine as a chromogenic substrate. Both of the ELISA systems enabled the determination of PS in a range of 5 to 500 pg/well, with an IC50 of 36 to 130 pg/well. Cross-reactivties with main metabolites in plasma, which differed from PS in decaline moiety, were less than a few percent. When ELISA-1 was applied to the determination of PS in human plasma directly after dilution with the ELISA buffer, the detection limit and the intra-assay coefficient (5 ng/ml of PS) were 500 pg/ml and 4.5%, respectively. Further, ELISA-1 was validated by gas chromatography-mass spectrometry with the determination of PS in human plasma after oral administration at a dose of 10 mg/body.