Multiple widely spaced elements determine the efficiency with which a distal cistron is expressed from the polycistronic pregenomic RNA of figwort mosaic caulimovirus

The polycistronic expression mechanism of the plant pararetrovirus figwort mosaic caulimovirus (FMV) depends upon cis-acting elements present in its pregenomic RNA and a trans-acting protein (P6) which is expressed from a monocistronic subgenomic RNA. Using transient expression of FMV-derived polycistronic reporter constructs in Nicotiana edwardsonii cell suspension protoplasts, we further analyzed the cis-acting elements involved in polycistronic expression. A cis-acting element located within the first 74 nucleotides of the 7,954-nucleotide pregenomic RNA appears to be essential for P6 to transactivate expression of an internal cistron. Expression of this internal cistron, in the presence of P6, is greatly enhanced by the combined presence of two cis-acting elements located at the 3' end of the polycistronic RNA. Surprisingly, deletion of the most upstream of these two 3' cis-acting elements exposed a negative-acting element located internally on the polycistronic RNA, at the 3' end of open reading frame I. The action of both this negative-acting internal element and the positive-acting 3' elements is more pronounced when the large 5' untranslated leader region is present. This indicates that the 5' untranslated leader region is central to regulation of the FMV gene expression mechanism. Although a limited set of elements suffices to direct polycistronic expression in this eukaryotic system, a complex interplay between elements is involved in the spatial regulation of the genes present on the pregenomic RNA of FMV.     

Children referred for specialist care: a nationwide study in Dutch general practice

BACKGROUND: Insight into referral patterns provides general practitioners (GPs) and specialists with a frame of reference for their own work and enables assessment of the need for secondary care. Only approximate information is available. AIM: To determine how often, to which specialties and for what conditions children in different age groups are referred, as well as how often a condition is referred given the incidence in general practice. METHOD: From data of the Dutch National Survey of Morbidity and Interventions in General Practice, 63,753 new referrals (acute and non-acute) were analysed for children (0-14 years) from 103 participating practices (161 GPs) who registered. Practices were divided into four groups. Each group of practices participated for three consecutive months covering a whole year altogether. We calculated referral rates per 1000 children per year and referability rates per 100 episodes, which quantifies the a priori chance of a condition being referred for specialist care. RESULTS: The referral rate varied by age from 231 for children under 1 year old to 119 for those aged 10-14 years (mean 159). The specialties mainly involved were ENT, paediatrics, surgery, ophthalmology, dermatology and orthopaedics. Referrals in the first year of life were most frequently to paediatricians (123); among older children the referral rate to paediatricians decreased (mean 36). Referrals to ENT specialists were seen particularly in the age groups 1-4 (71) and 5-9 (53). For surgery, the referral rate increased by age from 19 to 34. Differences between boys and girls were small, except for surgery. The highest referral rates were for problems in the International Classification of Primary Care (ICPC) chapters: respiratory (28); musculoskeletal (25); ear (24) and eye (21). Referability rates were, in general, low for conditions referred to paediatrics and dermatology and high for surgery and ophthalmology. The variation in problems presented to each specialty is indicated by the proportion of all referrals constituted by the 10 most frequently referred diagnoses: from 35% for paediatrics to 81% for ENT; for ophthalmology, five diagnoses accounted for 83% of all referrals. CONCLUSIONS: The need for specialist care in childhood is clarified with detailed information for different age categories, specialties involved and variation in morbidity presented to specialists, as well as the proneness of conditions to be referred.     

The insulin resistance syndrome in native Hawaiians. Native Hawaiian Health Research (NHHR) Project

OBJECTIVE: To investigate whether fasting hyperinsulinemia is associated with a clustering of cardiovascular disease (CVD) risk factors, manifesting as the insulin resistance syndrome (IRS), in a population of native Hawaiians. RESEARCH DESIGN AND METHODS: A total of 574 native Hawaiians > or = 30 years of age were examined for blood pressure, waist-to-hip ratio (WHR), BMI, oral glucose tolerance, and fasting lipid, insulin, and C-peptide concentrations. All statistical analyses (n = 384) excluded 190 individuals who had NIDDM or who were taking hypertension medication. Using logistic regression analysis, fasting insulin and C-peptide levels were compared with CVD risk factors (glucose intolerance, hypertension, central adiposity, elevated triglyceride levels, and low HDL cholesterol levels) after adjusting for age and obesity. RESULTS: Sixty-six percent of native Hawaiians were overweight or obese, and 70% were found to have central adiposity. Fasting insulin concentrations were correlated with BMI, WHR, blood pressure, and triglyceride, HDL cholesterol, and glucose concentrations. Fasting insulin was also significantly associated with an increasing number of CVD risk factors in each participant (P < 0.001). Fasting insulin and C-peptide concentrations were independently associated with glucose intolerance, high triglyceride levels, and low HDL cholesterol levels. However, only fasting C-peptide concentrations were independently associated with hypertension and central adiposity. Apparent differences in the correlates of fasting insulin and C-peptide may be related to multiple factors and warrant further evaluation. CONCLUSIONS: This study provides cross-sectional data confirming the existence of the IRS in native Hawaiians. However, further longitudinal studies are needed to examine the relationship of insulin resistance and/or surrogate markers to increased rates of NIDDM and CVD mortality in native Hawaiians.     

Failure of the nonselective beta-blocker propranolol to affect lipoprotein lipase gene expression in the rat

Treatment with beta-blockers has been reported to be associated with the development of hypertriglyceridemia. The etiology, even the existence, of this phenomenon is controversial. The purpose of our study was to examine whether the nonselective beta-blocker propranolol causes hypertriglyceridemia in the rat and whether its action is mediated by the modulation of lipoprotein lipase (LPL) messenger RNA (mRNA) accumulation or activity. LPL activity was assayed in fresh tissue by incubation with tritiated triglycerides. LPL mRNA was quantified in total RNA by slot-blot analysis using a mouse LPL complementary DNA probe. We have conducted three series of experiments in unanaesthetized rats in order to study the effects of different single doses of propranolol (1.5 to 6 mg i.p.) and different durations of treatment (15 min to 4 wk). We measured triglyceride and cholesterol levels in plasma as well as the LPL activity and mRNA levels in the heart and adipose tissue before and after propranolol administration. In these experiments we did not find any significant decrease in either the activity or the amount of mRNA of lipoprotein lipase nor was there any change in plasma lipids following treatment. Our results lead us to the conclusion that the nonselective beta-blocker propranolol affects neither the activity nor the mRNA level of LPL in the rat.     

Ischio-pubic-patellar hypoplasia: is it a new syndrome?

Aplasia/hypoplasia of the patella has been described as an isolated finding or, more commonly, as a part of congenital syndromes. We describe here bilateral absence of the patella in an 11-year-old girl with absence of the ischial and inferior pubic rami bilaterally. Other associated skeletal and soft-tissue deformities are also reported. To our knowledge, the constellation of these findings has not been described previously and represents a unique syndrome.     

Arthritic temporomandibular joint: correlation of macromolecular contrast-enhanced MR imaging parameters and histopathologic findings

PURPOSE: To assess the utility of macromolecular contrast material-enhanced magnetic resonance (MR) imaging parameters for determining the histopathologic severity of temporomandibular joint (TMJ) arthritis. MATERIALS AND METHODS: Ovalbumin was used to induce arthritis in the TMJs of 10 previously sensitized adult white rabbits. Five rabbits composed the sham-treated control group. Dynamic spin-echo imaging was performed immediately before and for 30 minutes after injection of macromolecular contrast medium. Histologic specimens of TMJ were assessed quantitatively for arthritis. Changes in MR signal intensity were derived from the synovial and subsynovial tissues of the TMJ, and plasma volume (PV) and permeability surface area product (PS) were calculated. These MR parameters and the arthritic scores were compared between sham-treated and antigen-challenged TMJs. The relationships between MR parameters and histopathologic indexes were also determined. RESULTS: Arthritic TMJs showed marked enhancement of the synovial and subsynovial tissues over the imaging period. PS and all histopathologic indexes of arthritis were significantly greater (P < .005) in antigen-challenged than in sham-treated TMJs. PS demonstrated strong positive relationships with all histologic parameters of arthritis, indicating its utility for assessing the severity of joint inflammation. CONCLUSION: Macromolecular contrast-enhanced MR imaging enables quantification of PS and PV in inflamed joints. This technique may provide insights into the pathogenesis of joint inflammation and noninvasive monitoring of disease severity and treatment response in arthritis.     

Transmitter-induced calcium responses differ in astrocytes acutely isolated from rat brain and in culture

Glial fibrillary acid protein (GFAP)-positive astrocytes isolated from the cerebral cortices of 3-10-day-old rats frequently showed increased intracellular Ca2+ concentration responses to L-glutamate and glutamate analogues. However, few of the acutely isolated cells responded to ATP, and no such cells responded to serotonin [5-hydroxytryptamine (5-HT)]. The same cell that failed to respond to ATP or 5-HT often responded to glutamate. Culturing acutely isolated cells in media containing horse serum decreased Ca2+ responses to glutamate but increased the responses to ATP and induced responses to 5-HT. In primary cultures prepared from the cerebral cortices of 1-day-old rats and cultured in horse serum, fewer of the cells responded to glutamate, but almost all cells responded to ATP and 5-HT. The lack of or limited response to, 5-HT or ATP in the acutely isolated cells seems unlikely to be due to selective damage to the respective receptors because acutely isolated GFAP-negative cells showed responses to ATP, several different proteases and mechanical dissociation yielded cells that also responded to glutamate but not to ATP, and exposure of primary cultures to papain did not abolish Ca2+ responses to several transmitters. The responses of the acutely isolated cells to glutamate but limited or lack of responses to ATP and 5-HT also correspond to what has been seen so far for astrocytes in situ. Thus, the present studies provide direct evidence that some of the receptors seen in primary astrocyte cultures may reflect a response to culture conditions and that, in the context of the relevant information so far available, acutely isolated astrocytes seem to reflect better the in vivo state.     

Influenza vaccination in 22 developed countries: an update to 1995

This study expands and updates through 1995 our earlier report on influenza vaccine use in 18 developed countries. Five of the six countries with high levels of vaccine use in 1992 (> or = 130 doses/1000 population) showed little change or slight declines over the subsequent 3 years. The exception was the United States, where a new federal program for vaccination reimbursement for the elderly helped to increase vaccine distribution from 144 to 239 doses/1000 population. The six countries with medium levels of vaccine use in 1992 (76-96 doses/1000 population) increased to > or = 100 doses/1000 population by 1995. Among the six low-use countries in 1992 (< or = 65 doses/1000 population), only Finland showed substantial improvement (96 doses/1000 population) in 1995. Four new countries were added to the study. In Germany, vaccine use increased to 80 doses/1000 population in 1995, but in Ireland it remained at a low level (48 doses/1000 population). In Korea, vaccine use increased from 17 to 95 doses/ 1000 population during the period 1987-1995. In Japan, very high levels of vaccine use (approximately 280 doses/1000 population) in the early 1980s were associated with vaccination programs for school children. However, vaccine use fell precipitously when these programs were discontinued, and only 2 and 8 doses/1000 population were used in 1994 and 1995, respectively. In all 22 countries, higher levels of vaccine use were associated with vaccination reimbursement programs under national or social health insurance and were not correlated with different levels of economic development. Excluding Japan, in 1995 there was still a greater than fourfold difference between the highest and lowest levels of vaccine use among the other 21 countries in the study. Given its well established clinical effectiveness and cost-effectiveness, none of these countries has yet achieved the full benefits of its programs for influenza vaccination.     

Hereditary defect in biosynthesis of aldosterone: aldosterone synthase deficiency 1964-1997

We studied two of the three patients with a hereditary defect in the biosynthesis of aldosterone originally described by Visser and Cost in 1964. All three presented as newborns with salt-losing syndrome and failure to thrive. The original biochemical studies showed a defect in the 18-hydroxylation of corticosterone. According to the nomenclature proposed by Ulick, this defect would be termed corticosterone methyl oxidase deficiency type I. We measured plasma steroids in the untreated adult patients and performed molecular genetic studies. Aldosterone and 18-OH-corticosterone were decreased, whereas corticosterone and 11-deoxycorticosterone were elevated, thus confirming the diagnosis of corticosterone methyl oxidase deficiency type I. Cortisol and its precursors were in the normal range. Genetic defects in the gene CYP11B2 encoding aldosterone synthase (P450c11Aldo) have been described in a few cases. We identified a homozygous single base exchange (G to T) in codon 255 (GAG) causing a premature stop codon E255X (TAG). This mutation destroys a Aoc II restriction site. Digestion of a PCR fragment containing exon 4 of CYP11B2 (261 bp) with this restriction enzyme revealed in the two patients homozygous for the E255X mutation only a 261-bp fragment, whereas the heterozygous parents had three fragments (261 bp from the mutant allele and 194 and 67 bp from the wild-type allele). The mutant enzyme had lost the five terminal exons containing the heme binding site, and thus there was a loss of function enzyme. We conclude that the biochemical phenotype of these prismatic cases of congenital hypoaldosteronism can be explained by the patients genotype.     

Physical status and expression of HPV genes in cervical cancers

Entamoeba histolytica and Entamoeba dispar have only recently been defined as two separate species. E. histolytica, the pathogenic species, is the microorganism causing invasive intestinal amoebiasis and/or liver abscess, while the morphologically similar E. dispar is nonpathogenic and noninvasive. The gold standard for the distinction of the two species has been the isoenzyme electrophoresis of phosphoglucomutases (EC 5.4.2.2) and hexokinases (EC 2.7.1.1), but there had also been a controversy about the possibility of a conversion of isoenzyme patterns. In this study, we cloned the phosphoglucomutase (PGM) cDNAs from the pathogenic and the nonpathogenic species. The deduced amino acid sequences were only 2.4% different. The cDNAs were expressed in Escherichia coli under the control of a T7 RNA polymerase promoter. The recombinant polypeptides displayed strong phosphoglucomutase activity, each of the recombinant enzymes comigrated with its natural counterpart from E. histolytica and E. dispar in the starch gel electrophoresis. Our results give a biochemical interpretation of the PGM isoenzyme pattern and support the clear distinction between the two species.