Development of a protein phosphatase-based assay for the detection of phosphatase inhibitors in crude whole cell and animal extracts

Diarrhetic shellfish poisoning (DSP) is a serious and globally widespread phytoplankton-related seafood illness. Although DSP is rarely life-threatening, it causes incapacitating diarrhea and vomiting with no known medical treatments. In addition, phytoplankton producing DSP toxins have been identified in temperate coastal waters worldwide, and their numbers may be increasing as a result of coastal eutrophication. The toxic effects of the major DSP toxins, okadaic acid and dinophysistoxin-1 (35-methylokadaic acid), appear to originate from their inhibitory activity against a family of structurally related serine/threonine protein phosphatases (PSPases). In particular, the inhibition of essential PSPases (e.g. PP1 and PP2A) has catastrophic consequences in most eukaryotic cells. Exploiting the potent inhibitory property of the DSP toxins, we have developed an enzyme-based assay (PP2A assay) capable of detecting both okadaic acid and dinophysis- toxin-1 in nanogram amounts. The assay employs purified PP2A, which has an extremely high affinity for both DSP toxins. This provides the PP2A assay with a level of sensitivity comparable to, or surpassing, that of most monoclonal antibody probes. To evaluate the PP2A assay as a means of detecting contaminated shellfish, a series of spike recovery experiments was conducted. The findings from these studies suggest that the PP2A assay has the potential for development into a rapid and relatively simple method for detecting PSPase inhibitors in crude extracts produced from shellfish.     

Development of tyrosine hydroxylase-, dopamine- and dopamine β-hydroxylase-immunoreactive neurons in a teleost, the three-spined stickleback

The development of catecholaminergic neuronal systems in the brain of a teleost, the three-spined stickleback, was studied through embryonic to early larval stages by immunocytochemistry using specific antibodies against dopamine, tyrosine hydroxylase and dopamine β-hydroxylase. By analysing the spatiotemporal patterns of development for the catecholaminergic nuclei, possible homologies with nuclei in amniote brains have been identified.

The noradrenergic neurons in the isthmus region of the rostral rhombencephalon originate in the same manner as the A4–A7 + subcoeruleus group in mammals. Their developmental characteristics show the largest similarities with the subcoeruleus group of birds and mammals, although some features are shared with developing A6 (locus coeruleus) neurons.

Catecholaminergic neurons never appear during development in the ventral mesencephalon of the three-spined stickleback. A group of large dopaminergic neurons that accompany the cerebrospinal fluid (CSF)-contacting neurons follows the border between the hypothalamus and the ventral thalamus into the caudal hypothalamus, where they are continuous with the dopaminergic neurons in the posterior tuberculum. They are thus topologically comparable with the dopaminergic neurons of the zona incerta in mammals.

The dopaminergic CSF-contacting neurons that line the median, lateral and posterior recesses of the third ventricle do not contain tyrosine hydroxylase-immunoreactivity at any developmental stage. This indicates that they take up and accumulate exogenous dopamine or -dihydroxyphenylalanine, and do not synthesize dopamine from tyrosine at any developmental stage. Tyrosine hydroxylase-immunoreactive neurons appear in the pineal organ on the day of hatching (120 h post-fertilization). They were still observed in 240-h-old larvae, but are absent in the pineal organ of adult sticklebacks.

The initial appearance and subsequent differentiation of catecholaminergic neurons in the stickle-back embryo follow essentially the same spatial and temporal pattern as in amphibian, avian and mammalian embryos. This observation supports the hypothesis that morphologically, topologically and chemically similar monoaminergic neurons in different vertebrate classes are homologous.     

Reliability of causal indicators for nonfatal injuries

The applicability of the ICD E code as a causal indicator of nonfatal injuries has been criticized. New codes have been developed to replace the ICD codes. We compared the coding reliability of the ICD E and place vs. the Nordic mechanism (M) and place codes. The mean accuracy (76 vs. 70%) (p less than 0.002) and the intercoder reliability (84 vs. 69%) (p less than 0.001) were better for the E than M code. The accuracy of the place codes was the same (83%). A short training improved (p less than 0.001) the accuracy of all four codes. The replacement of the E code with the M code would not improve the reliability of data on causes of injuries.     

Males as a high-risk group for trauma: the Finnish experience

The high incidence of trauma among young men has been observed in many countries. To better understand this risk, we analyzed male injuries and their causes in Finland. The incidence peak of medically attended trauma in men aged 15-24 years was caused by occupational and sports injuries, whereas that of hospitalizations was due to motor vehicle injuries. Age patterns of nonfatal trauma resembled those recorded in the U.S.A. However, patterns and causes of fatal trauma were very different in the two countries: early adulthood in the U.S.A. and late middle-age in Finland were the ages of highest injury mortality. The majority of deaths in young men were caused by motor vehicle injuries in the U.S.A., whereas suicides and the extremely high rate of fatal non-motor vehicle accidents, largely due to alcohol poisoning and drownings, formed the main bulk of injury mortality in Finland. Similarities in early adulthood suggest general biological factors, whereas the deviance of the middle-aged male Finns from the general pattern may indicate more specific psychosocial factors.     

Multicenter Pilot Study of a Serpentine Balloon-Expandable Stent (beStentTM): Acute Angiographic and Clinical Results

The beStent is a new stainless steel, balloon-expandable mesh stent which has a unique serpentine design. Rotation of the unique low stress junctions upon expansion leads to orthogonal locking of the wires, maximizing radial strength and assuring zero shortening. The stent has delineating gold markers which assure precise positioning. We aim to present the initial acute results in a pilot registry for stent evaluation. Two hundred eighty-four stents were used in a total of 217 patients (age 57.9 ± 3.10 years; 178 males; 39 females) in seven centers, for variable indications. Stents of 15-, 25-, and 35-mm length were used. The arteries treated were the left anterior descending (n = 112, 42%), circumflex (n = 54, 20.2%), right coronary (n = 95, 35.5%), left main (n = 1, 0.4%), and vein graft (n = 5, 1.9%). Lesion types were: A in 42 patients (16.5%); B1 in 53 patients (20.7%); B2 in 81 patients (31.8%); and C in 79 patients (31%). One hundred fifty-nine patients required one stent, 40 patients required two stents, and 18 patients required three or more stents. Anticoagulation protocol included procedural heparin with aspirin with/without ticlopidine. Smooth angiographie results were obtained in all cases with no plaque herniation. Acute angiographic success was obtained in 97% of the patients, and acute clinical success in 95% of the patients. Complications within 30 days were: 3 deaths (1.4%) (2 noncardiac); 2 (0.9%) myocardial infarctions; and 2 (0.9%) stent thromboses. Therefore, the beStent is useful in treatment of complex lesions of variable length and complexity, providing excellent acute results with a low complication rate, in spite of unfavorable basic clinical and angiographie characteristics.     

Binding of serotonergic ligands to brain membranes of alcohol-preferring AA and alcohol-avoiding ANA rats.

The alcohol-preferring AA rats have higher concentration of 5-hydroxytryptamine (5-HT) in the brain than the alcohol-avoiding ANA rats. In the present study, the 5-HT1, 5-HT2, and 5-HT3 receptors were studied with [3H]5-HT, [3H]ketanserin, and [3H]LY278584, respectively, in membrane homogenates from different brain regions of both rat lines using in vitro binding assays. No differences in the 5-HT1 and 5-HT2 receptor binding in the brainstem, hippocampus, frontal cortex, and hypothalamus or in the 5-HT3 receptor binding in the nucleus accumbens, amygdala, hippocampus, and frontal cortex were observed between the ethanol-naive animals of the rat lines. In rats given the opportunity to voluntarily consume alcohol, there was a tendency to increase 5-HT1 binding in the ANA rats, which tendency was, however, also found in their ethanol-naive controls subjected to the same handling and behavioral tests as the ethanol-experienced animals. The results do not, however, indicate that any genetic modifications of the 5-HT receptor-binding sites have occurred in the process of the selective breeding of AA and ANA rats for alcohol preference and avoidance, respectively.     

Associations of Early Premenopausal Fractures with Subsequent Fractures Vary by Sites and Mechanisms of Fractures

In a retrospective population-based study we assessed whether and how self-reported former fractures sustained at the ages of 20–34 are associated with subsequent fractures sustained at the ages of 35–57. The 12,162 women who responded to fracture questions of the baseline postal enquiry (in 1989) of the Kuopio Osteoporosis Study, Finland formed the study population. They reported 589 former and 2092 subsequent fractures. The hazard ratio (HR), with 95% confidence interval (CI), of a subsequent fracture was 1.9 (1.6–2.3) in women with the history of a former fracture compared with women without such a history. A former low-energy wrist fracture was related to subsequent low-energy wrist [HR = 3.7 (2.0–6.8)] and high-energy nonwrist [HR = 2.4 (1.3–4.4)] fractures, whereas former high-energy nonwrist fractures were related only to subsequent high-energy nonwrist [HR = 2.8 (1.9–4.1)] but not to low-energy wrist [HR = 0.7 (0.3–1.8)] fractures. The analysis of bone mineral density (BMD) data of a subsample of premenopausal women who underwent dual x-ray absorptiometry (DXA) during 1989–91 revealed that those with a wrist fracture due to a fall on the same level at the age of 20–34 recorded 6.5% lower spinal (P= 0.140) and 10.5% lower femoral (P= 0.026) BMD than nonfractured women, whereas the corresponding differences for women with a former nonwrist fracture due to high-energy trauma were −1.8% (P= 0.721) and −2.4% (P= 0.616), respectively. Our results suggest that an early premenopausal, low-energy wrist fracture is an indicator of low peak BMD which predisposes to subsequent fractures in general, whereas early high-energy fractures are mainly indicators of other and more specific extraskeletal factors which mainly predispose to same types of subsequent fractures only. Received: 21 February 1996 / Accepted: 24 September 1996     

Perfluorochemicals as US contrast agents for tumor imaging and hepatosplenography: preliminary clinical results

In animals, perfluorochemicals (PFCs) are effective ultrasound (US) contrast agents that produce hepatic, splenic, and tumor enhancement. The use of Fluosol-DA 20%, an emulsion of perfluorodecalin and perfluorotripropylamine, was studied in nine non-critically ill patients with cancer who had liver lesions. US studies without Fluosol were compared with studies obtained 24, 48, and 72 hours after Fluosol infusion. Vital signs and extensive laboratory analyses are performed before and after Fluosol infusion. Liver metastases from colonic, pancreatic, and gastric carcinoma exhibited rim or diffuse enhancement after a Fluosol dose of 1.6 g/kg or greater. Fluosol produced echogenic enhancement of the liver and spleen relative to kidney at a dose of 2.4 g/kg, allowing the detection of nonenhancing lesions. In addition, Fluosol at a dose of 1.6 g/kg or greater allowed detection of lesions not seen before contrast medium was administered in three of the seven patients studied. There was a mild increase in the level of serum glutamic oxaloacetic transaminase in two patients, one given 2.4 and the other 3.2 g/kg of Fluosol. Mild and transient allergic reactions without change in vital signs were experienced by two patients.