Importing genetically altered animals: ensuring quality.

The reproducibility of research using laboratory animals requires reliable management of their quality, in particular of their genetics, health and environment, all of which contribute to their phenotypes. The point at which these biological materials are transferred between researchers is particularly sensitive, as it may result in a loss of integrity of the animals and/or their documentation. Here, we describe the various aspects of laboratory animal quality that should be confirmed when sharing rodent research models. We also discuss how repositories of biological materials support the scientific community to ensure the continuity of the quality of laboratory animals. Both the concept of quality and the role of repositories themselves extend to all exchanges of biological materials and all networks that support the sharing of these reagents.

The Flannotator--a gene and protein expression annotation tool for Drosophila melanogaster.

SUMMARY: Understanding developmental processes and building towards integrative systems biology require detailed knowledge of the spatio-temporal expression of genes and proteins. We have developed a software package for collecting, storing and searching the annotation of protein or gene expression patterns in Drosophila melanogaster. Using standard Drosophila anatomy and Gene Ontologies, the system can readily capture expression patterns at any stage of development and in all recognized tissue types as well as details of sub-cellular localization. The web-based system allows multiple groups to work in collaboration and share images and annotation. AVAILABILITY: http://www.flannotator.org.uk/.

Migration patterns of clonally related granule cells and their progenitors in the developing chick cerebellum.

During cerebellar development, granule neurons and their progenitors undergo complex migrations. To define these migratory paths better, we used replication-incompetent retroviruses to label dividing cells early in cerebellar development. Clonally related granule cells were widely dispersed in both rostrocaudal and mediolateral planes; clones often spanned the midline. The data suggest that granule cell progenitors originate from the ventricular zone along the entire mediolateral extent of the caudal edge of the cerebellum. After reaching the cerebellar surface, progenitors move primarily rostrally and proliferate in the superficial external granule layer. Postmitotic granule cells then migrate long distances medially and laterally in the transverse plane in the deep external granule layer, where previously they had been thought simply to extend transverse processes.

Chemosensory control of surface antigen switching in the nematode Caenorhabditis elegans.

Nematodes change their surface compositions in response to environmental signals, which may allow them to survive attacks from microbial pathogens or host immune systems. In the free-living species Caenorhabditis elegans, wild-type worms are induced to display an L1 (first larval stage) surface epitope at later larval stages when grown on an extract of spent culture medium (Inducible Larval Display or ILD). Before this study, it was not known whether ILD was regulated by the well-characterized, neurologically based chemical senses of C. elegans, which mediate other behavioural and developmental responses to environmental signals such as chemotaxis and formation of the facultatively arrested dauer larva stage. We show here that ILD requires the activities of three genes that are essential for the function of the C. elegans chemosensory neurons. ILD was abolished in chemotaxis-defective che-3, osm-3 and tax-4 mutants. In contrast, chemotaxis-defective mutants altered in a different gene, srf-6, show constitutive display of the L1 epitope on all four larval stages. The ILD-defective che-3, osm-3 and tax-4 mutations blocked the constitutive larval display of an srf-6 mutant. Combining srf-6 and certain dauer-constitutive mutations in double mutants enhanced constitutive dauer formation, consistent with the idea that srf-6 acts in parallel with specific components of the dauer formation pathway. These results taken together are consistent with the hypothesis that ILD is triggered by environmental signals detected by the nematode's chemosensory neurons.

Distribution of mitochondrial enzymes between the perikaryal and synaptic fractions of immature and adult rat brain.

The subcellular distribution of mitochondrial enzymes was studied in cerebral hemispheres of 15-day-old and adult rats. At both ages the synaptosomal fraction contained very little glutamate dehydrogenase (EC 1.4.1.2) but significant amounts of succinate dehydrogenase (EC 1.3.99.1), glutaminase (EC 3.5.1.2), hexokinase (EC 2.7.1.1), malate NADP dehydrogenase (EC 1.1.1.40) and beta-hydroxybutyrate dehydrogenase (EC 1.1.1.30). In immature brain, in the fraction enriched with free (perikaryal) mitochondria, the concentrations of these enzymes were 9.5, 1.8, 2.0, 0.92, 1.5, and 2.1 times higher, respectively, than in the synaptosomes. The increase with age in succinate dehydrogenase and glutaminase was restricted to free mitochondria while hexokinase and malate NADP dehydrogenase accumulated and beta-hydroxybutyrate dehydrogenase diminished in both fractions. In adult brain, too, where the above ratios became 7.5, 5.2, 3.5, 0.84, 1.4, and 2.0, respectively, the concentrations of enzymes relative to each other distinguished clearly between free and synaptic mitochondria. The results substantiate previously noted signs of mitochondrial heteroeneity in adult brain, and extend them to immature brain. The chemical composition, the quantitative pattern of enzymes, of free and synaptic mitochondria is clearly different, and undergoes separate changes during postnatal differentiation.

Plasma fibrinogen level and the risk of major cardiovascular diseases and nonvascular mortality: an individual participant meta-analysis.

CONTEXT: Plasma fibrinogen levels may be associated with the risk of coronary heart disease (CHD) and stroke. OBJECTIVE: To assess the relationships of fibrinogen levels with risk of major vascular and with risk of nonvascular outcomes based on individual participant data. DATA SOURCES: Relevant studies were identified by computer-assisted searches, hand searches of reference lists, and personal communication with relevant investigators. STUDY SELECTION: All identified prospective studies were included with information available on baseline fibrinogen levels and details of subsequent major vascular morbidity and/or cause-specific mortality during at least 1 year of follow-up. Studies were excluded if they recruited participants on the basis of having had a previous history of cardiovascular disease; participants with known preexisting CHD or stroke were excluded. DATA EXTRACTION: Individual records were provided on each of 154,211 participants in 31 prospective studies. During 1.38 million person-years of follow-up, there were 6944 first nonfatal myocardial infarctions or stroke events and 13,210 deaths. Cause-specific mortality was generally available. Analyses involved proportional hazards modeling with adjustment for confounding by known cardiovascular risk factors and for regression dilution bias. DATA SYNTHESIS: Within each age group considered (40-59, 60-69, and > or =70 years), there was an approximately log-linear association with usual fibrinogen level for the risk of any CHD, any stroke, other vascular (eg, non-CHD, nonstroke) mortality, and nonvascular mortality. There was no evidence of a threshold within the range of usual fibrinogen level studied at any age. The age- and sex- adjusted hazard ratio per 1-g/L increase in usual fibrinogen level for CHD was 2.42 (95% confidence interval [CI], 2.24-2.60); stroke, 2.06 (95% CI, 1.83-2.33); other vascular mortality, 2.76 (95% CI, 2.28-3.35); and nonvascular mortality, 2.03 (95% CI, 1.90-2.18). The hazard ratios for CHD and stroke were reduced to about 1.8 after further adjustment for measured values of several established vascular risk factors. In a subset of 7011 participants with available C-reactive protein values, the findings for CHD were essentially unchanged following additional adjustment for C-reactive protein. The associations of fibrinogen level with CHD or stroke did not differ substantially according to sex, smoking, blood pressure, blood lipid levels, or several features of study design. CONCLUSIONS: In this large individual participant meta-analysis, moderately strong associations were found between usual plasma fibrinogen level and the risks of CHD, stroke, other vascular mortality, and nonvascular mortality in a wide range of circumstances in healthy middle-aged adults. Assessment of any causal relevance of elevated fibrinogen levels to disease requires additional research.

Characterization of Distinct Tombusviruses that Cause Diseases of Lettuce and Tomato in the Western United States.

A soilborne disease of lettuce, associated with necrosis and dieback, has been found with increasing frequency in California and Arizona over the last 10 years. An isometric virus, serologically related to Tomato bushy stunt virus (TBSV), was consistently isolated from lettuce plants with these disease symptoms. Back-inoculation to healthy lettuce plants and subsequent reisolation of the virus from symptomatic lettuce leaves suggested that this virus was the causal agent of this disease. A tombusvirus was also associated with a necrosis disease of greenhouse-grown tomatoes in Colorado and New Mexico. Complementary DNA representing the 3' end of viral genomic RNAs recovered from diseased lettuce and tomato plants had identical nucleotide sequences. However, these sequences were divergent (12.2 to 17.1%) from sequences of the previously described strains of TBSV, Petunia asteroid mosaic virus (PAMV), Artichoke mottled crinkle virus, and Carnation Italian ringspot virus. Additional tombusvirus isolates were recovered from diseased lettuce and tomato plants and these were most closely related to the TBSV-cherry strain (synonymous with PAMV) and to Cucumber necrosis virus based on comparison of 3'-end sequences (0.1 to 0.6% and 4.8 to 5.1% divergence, respectively). Western blot analysis revealed that the new tombusvirus isolated from diseased lettuce and tomato plants in the western United States is serologically distinct from previously described tombusvirus species and strains. Based on genomic and serological properties, we propose to classify this virus as a new tombusvirus species and name it Lettuce necrotic stunt virus.

First Report of Tomato Bushy Stunt Virus Isolated from Lettuce.

In recent years a disease causing dieback and necrosis of Romaine and leaf lettuce has become increasingly important in California and incidence is becoming more widespread. This disease has been primarily found in areas where soil has been dredged from a river or in flooded land. Tomato bushy stunt virus (TBSV) isolates have been isolated from roots and leaves of symptomatic lettuce. The particles are isometric with a diameter of 30 nm. Double-stranded RNA (dsRNA) profiles are identical to the tomato and Prunus isolates of TBSV. However, spurs are formed in agar double diffusion tests when antisera to the tomato and Prunus isolates were used. A similar dieback disease of lettuce was observed in several counties of California during the mid-1980s. Symptoms of this disease are very similar to those described for the "brown blight" disease of lettuce reported in the 1920s (1), including severe stunting of plants and extensive chlorosis, mottling, and necrosis of older leaves. Plants infected early in their development may die. Although inoculation under greenhouse conditions has not reproduced the dieback disease in lettuce, TBSV has been consistently isolated from field-grown, symptomatic lettuce. The question of whether this new dieback disease of lettuce is caused only by lettuce isolates of TBSV or if some other viruses are also involved needs further studies. Reference: (1) I. C. Jagger. Phytopathology 30:53, 1940.

Pets and the elderly. A social work perspective.

Pets can provide companionship, affection, and psychosocial stimulus for elderly people in a program that is planned and supervised by professionals who are knowledgeable about both the humans and the animals and who are clear about the goals to be achieved. Ineffective programs, or programs with negative effects, have developed when the proponents assumed that any pet would be good for elderly persons either living alone or in a residential center. A much-needed aspect of any people-pet program is the collection, analysis, and reporting of social data to provide a better information base for planning the use of pets in a therapeutic setting.

A program which determines mutagenic concentrations of chemical carcinogens via a diffusion bioassay.

A computer program implementing a mathematical model for determining mutagenic concentrations of chemical carcinogens was developed. The mathematical model describes the experiment in which a droplet of a suspected carcinogen is put at the center of a petri dish containing a bacterial lawn in an agar gel. After a period of incubation during which the chemical diffuses outward, one observes a concentric ring of mutants around the center. The largest radius at which mutation occurs, r mut, corresponds to the lowest (threshold) concentration of the chemical sufficient to produce bacterial mutation. Given a series of initial concentrations of a chemical and the resulting r mut's, the program computes and reports the threshold concentration and the decay time of the chemical. The program is also used as a method to determine the lowest mutagenic concentration for a particular time of exposure.

Graphical simulation of early development of the cerebral cortex.

Much experimental data exists concerning the development of the cerebral cortex. There is a need for a common vehicle to integrate this data and to allow the testing of hypotheses concerning development. Computer simulation and visualization are powerful mechanisms for hypothesis testing. Our long-term goal is to create a robust, extensible, portable tool for simulation and visualization of cortical development to serve both research and educational purposes. This paper describes a simulation program, SimCortex, which models the early stages of development of the cerebral cortex of the mouse. Version 1.0 of SimCortex models the proliferation of progenitor cells in the pseudostratified ventricular epithelium (PVE), the generation of young neurons and their migration into the cortical plate, which is the forerunner of cell layers II through VI of the adult cortex. We present an overview of the design and implementation of SimCortex, sample output of the system and a comparison of our results with experimental data. We conclude with a brief overview of proposed future enhancements of the system.

DNA marker C04107 for copper toxicosis in a population of Bedlington terriers in the United Kingdom.

The DNA microsatellite marker C04107, linked to the Bedlington terrier copper toxicosis locus, is used diagnostically in the USA to detect the disease allele. This marker has been typed in Bedlington terriers of known disease status in the United Kingdom, and it is concluded that it should be useful in eradicating the disease from the breed in the UK. The marker also identified a dog which had been diagnosed on the basis of a liver biopsy as having the disease, as being unaffected; a second liver biopsy confirmed that the dog did not have copper toxicosis.

Von Wille-brand's disease in UK dobermanns: possible correlation of a polymorphic DNA marker with disease status.

Ninety-one dobermanns have been typed for a polymorphic microsatellite DNA marker situated within an intron of the von Willebrand factor gene and the alleles correlated with von Wille-brand's disease status. Two alleles were identified, one associated only with the normal gene and the other with both normal and disease genes.

[Effect of low doses of estrogens in menopausal women]. / Efecto de bajas dosis de estrógenos en mujeres menopáusicas.

The effect of hormonal therapy in serum glucose, lipoproteins and the hemostatic system was studied in menopausal women, who were distributed in three groups: Group I (15 patients) received placebo in cycles of 21 days, followed by 7 days of rest; Group II (16 patients), received natural estrogens conjugated with progesterone in a sequential way, also in cycles of 21 days: Group III (16 patients), was treated with triphasic oral contraceptives in the same cyclical fashion. The patients completed 6, 12 or 18 cycles of the treatment. The results were as follows: The glucose levels were decreased by the 18th cycle in group II (P < 0.01). The HDL-cholesterol levels and the cholesterol/HDL cholesterol index were unchanged, however the basal levels of LDL-cholesterol in group III were high, but normal after 12 cycles of treatment. The coagulation factors did not changed significantly with the hormonal treatment, however fibrinogen was increased after 18 cycles under placebo. The von Willebrand factor and antithrombin III remained stable. Platelet aggregation with ADP increased after 12 cycles in group III (p < 0.05), and no changes were found with collagen. The above results suggest that the hormonal treatment in menopausal women, either with natural estrogens and progesterone or with triphasic oral contraceptives, has little effect in the lipidic concentrations of sera in these patients, however it can increase platelet activity, specially with prolonged treatment with semisynthetic estrogens.

[Role of environmental factors in the development of insulin-dependent diabetes mellitus (IDDM) in Venezuelan children]. / Papel de los factores ambientales en el desarrollo de la Diabetes Mellitus Insulino Dependiente (DMID) en niños venezolanos.

In view of the controversy surrounding the role of environmental factors, such as the presence of bovine albumin in milk, or viral infections, in the etiology of IDDM, a study was undertaken to determine the relationship between these events and the subsequent risk of developing IDDM. On 40 venezuelan diabetic children (< 18 y) and forty, age, sex and race-matched controls were studied at the same time. Parents of children completed a questionnaire on the infant's feeding habits, its environment and family history. The X2 method and the Fischer's exact test were used to analyze the results. We found that 20% of the controls, and 10% of IDDM (NS), were never breast-fed. In 95% of controls vs 65% of IDDM (p < 0.001), cow's milk was given exclusively from birth, or combined with breast-feeding, 65% of IDDM and 60% of controls (NS) were breast-fed (alone or combined with milk substitutes) for more than three months. These results do not support the hypothesis that early exposure to breast milk substitutes increases the risk of IDDM in venezuelan children. The study revealed, however, that a family history of diabetes mellitus was present in 55% of IDDM vs 30% of controls (p < 0.05) and mumps infection before the onset of diabetes was recorded in 42.5% of IDDM in comparison with 12.5% of controls (p = 0.005). Other viral infections (rubella, chicken pox) had no statistical significance. The latter results suggest an association between a family history of diabetes mellitus and viral infections with the development of IDDM among this group of children.

[Hyperinsulinism and lipid changes in the early stages of essential arterial hypertension]. / Hiperinsulinemia y alteraciones lipídicas en etapas tempranas de la hipertensión arterial esencial.

The purpose of this study was to evaluate the presence of insulin-resistance in non-obese, non-diabetic patients with mild to moderate essential arterial hypertension of recent diagnosis and without prior pharmacological treatment and its relationship with the lipid alteration found in those patients. Twenty-one controls (9 M/12F) and twenty-nine patients (19 M/10 F) were studied. The control group presented mean age: 29 +/- 1.5 years, BMI: 23.9 +/- 0.46 Kg/m2, SBP: 112.6 +/- 2.9 mm Hg, DBP: 68.0 +/- 2.9 mm Hg. The patient group presented mean age: 35 +/- 1.4 years, BMI: 27.3 +/- 0.45 Kg/m2, SBP: 140 +/- 26 mm Hg, DBP: 95.1 +/- 1.4 mm Hg. The fasting levels of glucose, insulin and lipids were measured in each individual. Both controls and patients were subjected to an oral glucose tolerance test (OGTT) with determination of glucose and insulin at 30, 60, and 120 minutes. The patients had significantly (p < 0.05) increased plasma glucose at 0, 30, 60 and 120 min. and increased (p < 0.05) plasma insulin levels at O and 120 min compared to controls. The G/I ratio was significantly lower (p < 0.005) in the hypertensive group, at 0 h and 120 min. Abnormalities in fasting lipid profile were also observed in the patients including a significant increase in TG, Cholesterol, VLDL-C and LDL-C. These increases were especially evident in men and those with higher BMI. There was a positive and significant association between basal-insulin and TG, VLDL-C and DBP in hypertensive patients. We conclude that hyperinsulinemia is present in the majority of hypertensive patients and that hyperinsulinemia is associated with lipid abnormalities specially in men and the most individuals with higher BMI.

[Glycemic index and insulin response to the ingestion of precooked corn flour in the form of "arepa" in healthy individuals]. / Indice glicémico y respuesta insulínica a la ingestión de harina precocida de maíz en forma de arepa en individuos sanos.

With the purpose of exploring the glucose and insulin responses to a breakfast composed of a complex carbohydrate (CC) in the form of a "arepa" prepared with precooked corn flour, with or without the addition of protein and fat (CC + P + F), we studied 6 healthy volunteers, ages ranging from 26-50 years and body mass index of 24.5 +/- 1.32. Three tests were performed on each individual: 1) 75 g OGTT, 2) Ingestion of 75 g of CC ("arepa") and 3) Ingestion of 75 g of CC + 6.7 g protein (low fat cheese) and 4 g fat (margarine). Glycemic values (glucose - oxidase method) and insulinemia (radioimmunoassay) were determined at basal, 30, 60, 120, 180 and 240 min. Glucose (GA) and insulin (IA) areas, glycemic index (GI) and insulin/glucose ratio (I/G) were calculated. We found that the "arepa" has a high GI (71.5%) that it is increased, although not significatively to 140% with the addition of protein and fat. Total GA as well as IA obtained for CC and for CC + P + F were similar to OGTT, however the profiles of the glucose and insulin responses during CC and CC + P + F were less abrupt but more prolonged, resulting in a greater I/G ratio for OGTT in comparison with CC or CC + P + F during the initial steps. We conclude that GI of this corn bread ("arepa") is high in comparison to other complex carbohydrates and it is not altered by the addition of protein and fat. This is possibly due to glucose and insulin responses similar to that produced by OGTT.

[Serum concentration of calcium and calcitonin in hyperthyroidism caused by Graves' disease]. / Concentraciones séricas de calcio y calcitonina en hipertiroidismo por enfermedad de Graves.

Serum calcium and calcitonin were determined in 13 patients (12 women and 1 man), ages ranging from 30 to 40 years, with clinical diagnosis of hyperthyroidism due to Graves' Disease, confirmed by serum determinations of T3 and T4, with the purpose of establishing the relationship that these two substances may have in this pathology. The results obtained showed a decrease in seric calcium concentration in relation to a control group (10.02 +/-) 0.48 vs 11.49 +/- 0.28 mg/dl; p less than 0.005) and an increase in calcitonin concentration (193.6 +/- 8.62 vs 116.7 +/- 7.61 pg/ml; p less than 0.0001). We also found a significative negative association (r = -0.69; p less than 0.01) between these two compounds in the group of patients with hyperthyroidism, not being found in the control group.

[Qualitative and quantitiative differences in the plasma lipoproteins of obese, hyperlipidemic or normolipidemic men and women]. / Diferencias cualitativas y cuantitativas en las lipoproteínas del plasma entre hombres y mujeres obesos, hiperlipidémicos o normolipidémicos.

The aim of this study was to evaluate the alterations of the lipoprotein composition and their relation with the insulin-resistance and/or hyperinsulinemia in non diabetic obese patients. Twenty-two no obese(13 women and 9 men) and 30 obese patients (BMI > 30) were studied, who were divided into two groups according to the total lipid levels. The first group was formed by 18 obese patients (10 women and 8 men) with normal serum cholesterol (Chol) concentration < 200 mg/dL and triglycerides (TG) < 150mg/dL (NO), while the second group were formed by 12 obese patients (3 women and 9 men) with elevated Chol level > 200mg/dL and/or TG > 150 mg/dL (HO). A clinical and anthropometric examination was performed to each patient, as well as a glucose tolerance test, including serum glucose and insulin determinations. Likewise, the plasma lipoproteins (VLDL, LDL, HDL2 and HDL3) were isolated by ultracentrifugation and their cholesterol and triglycerides content were determined by enzymatic methods. In this report, we demonstrate the existence of compensatory basal hyperinsulinemia in men and women on both obese patients populations as well as alterations in the lipoprotein composition, mostly a TG overload even on NO. On the other hand, the presence of lipids and lipoproteins modification were obvious in those patients with abdominal obesity, on whom the hyperinsulinemia was more evident, which could be related with the high risk of cardiovascular disease in this kind of patients.

[Glucose/insulin response to a glucose overload in subjects at risk for type 2 diabetes]. / Respuesta de la Glucosa/insulina a una sobrecarga glucosada en sujetos con riesgo a diabetes tipo 2.

With the purpose of determining how certain risk factors for type 2 diabetes such as family history of diabetes, obesity and dyslipidemia, affect the glucose-insulin response to a glucose challenge, 135 individuals (77 women and 58) men were studied. Their ages ranged from 20-68 years, their basal glycemic values were less than 110 mg/dL but they were considered at risk for diabetes due to the presence of one or more of those factors. We found that the presence of those risk factors did not affect the glycemic response in any case. However, the basal insulin levels as well as the post-challenge values were increased significantly (p < 0.0001) by the presence of obesity in men as well as in women. Dyslipidemia increased the basal and post challenge glucose insulin values only in men (p < 0.002). The coexistence of obesity and family history of diabetes provoked a decrease in the basal insulin levels as well as in the insulin response to glucose. We conclude that, without alteration of the glycemic response, the presence of risk factors as obesity, dyslipidemia or family history of diabetes leads to basal hyperinsulinemia, as well as glucose stimulated hyperinsulinemia, however the coexistence of obesity and family history of diabetes, is responsible for a deficit in the insulin secretion by the pancreas.