Cloning and expression of the cDNA encoding rat granulocyte colony-stimulating factor

Granulocyte colony-stimulating factor (G-CSF) acts on precursor hematopoietic cells to control the production and maintenance of neutrophils. Recombinant G-CSF (re-G-CSF)is used clinically to treat patients with neutropenia and has greatly reduced the infection risk associated with bone marrow transplantation. Cyclic hematopoiesis, a stem cell defect characterized by severe recurrent neutropenia, occurs in man and grey collie dogs, and can be treated by administration of re-G-CSF. Availability of the rat G-CSF cDNA would benefit the use of rats as models of gene therapy for the treatment of cyclic hematopoiesis. In preliminary rat experiments, retroviral-mediated expression of canine G-CSF caused neutralizing antibody formation which precluded long-term increases in neutrophil counts. To overcome this problem we cloned the rat G-CSF cDNA from RNA isolated from skin fibroblasts. The rat G-CSF sequence shared a high degree of identity in both the coding and non-coding regions with both the murine G-CSF (85%) and human G-CSF (74%). The signal peptides of murine and human G-CSF both contained 30 amino acids (aa), whereas the deduced signal sequence for rat G-CSF possessed 21 aa. A retrovirus encoding the rat G-CSF cDNA synthesized bioactive G-CSF from transduced vascular smooth muscle cells.

Ultrastructural and morphometric aspects of ageing in the retinal capillaries of rats

Age-related morphological, ultrastructural and morphometric changes in the capillaries of the superficial and deep plexuses of the rat retina were studied in animals aged from 3 to 15 months. Our results suggest that age-related morphological alterations start occurring in the retina of rats at about 12 months of age. Increased glycogen deposits, pinocytotic vesicles, residual bodies and cell debris were observed in both the endothelial and pericytic cells of 12- and 15-month-old animals. In addition, heterogeneous osmiophilic accumulations, electron-transparent spaces were observed in the basement membrane as well as projections of the basement membrane towards the neighboring cells. Morphometric examination of the two vascular plexuses studied did not show differences in the area of the endothelial or pericytic cells, basement membrane or vascular lumen between rats of different ages.

Efeito do Nível de Energia da Dieta e da Exposição Prévia ao Calor sobre a Respiração Mitocondrial do Músculo Cardíaco de Frangos de Corte

This work was conducted aiming to evaluate the effect of dietary energy level and the previous heat exposure on the respiratory activity of the mitochondria from the cardiac muscle of broiler chickens. It was used broiler chickens (Hubbard) from both sexes and fed with diet containing 2900 kcal ME/kg or 3200 kcal ME/kg. The birds were heat stressed (35°C/4h) in the 1 st, 21 st and 42 nd days of age. The respiratory activity of mitochondria from cardiac muscle was evaluated in a Gilson oxygraph, model 5/6, by using alpha-ceto-glutaric as substrate and the ADP (adenosine-di-phosphate) to stimulate the respiratory activity. There was not effect of dietary energy level and previous heat exposure on the respiratory activity of the mitochondria from cardiac muscle. However, the females presented higher respiratory activity than males. By reason of the low oxidative capability of the heart muscle fiber of the male broiler chickens, these could be more sensitive to cardiac disease than females.

Atherosclerosis morphology and pathogenesis

Atherosclerosis is a very common and important disease being the most important cause of mortality in Brazil. Indeed, in 1995, 23.3% of deaths, all ages, in our country, were the consequence of atherosclerosis. This percentage grows to 26.3% for S. Paulo and 32.7% for Rio Grande do Sul. Morphologically, there are 3 main types of lesions: fatty streaks, fibrous plaques, and complicated lesions. Fatty streaks are inocuous and occur early in life. In some persons, with age, they change into fibrous plaques that may lead to stenosis. They also may become complicated by erosion, calcification, hemorrhage and thrombosis. Atherosclerosis is initiated by endothelial functional alterations responsible for increase in permeability to macromolecules, adhesion, and migration of monocytes-macrophages and lymphocytes plus recruitment of platelets and smooth-muscle medial cells. Adhesion molecules, cytokines, growth factors, and free radicals are locally synthesized, favoring proliferation of extracellular matrix and progression of the lesion. Experimental, clinical, and epidemiological evidence point to the importance of lipids, mainly cholesterol-rich low-density lipoprotein (LDL), as one of the most important molecules involved in the genesis and progression of atherosclerosis. Patients with a genetic disorder of cholesterol metabolism (familial hyperlipidemia), caused by a decrease in the availability of receptors for LDL, develop severe atherosclerosis early in life. A series of other factors, such as age, diabetes melitus, diet, hypertension, lack of exercise, elevated hemocysteinemia, immunological disorders, and coagulation instability, are related to the progression of atherosclerosis. All of them are capable of altering the endothelium or increasing the offer of LDL. All the above-mentioned factors are systemic; but atherosclerosic lesions are focal, located at preferential sites such as the emergence of colaterals, bifurcations, and curvatures of arteries, all areas in which the laminar flow is disturbed. In these areas shear stress is diminished favoring the prolongation of permanence time of lipid particles, cells, cytokines, growth factors, etc., in the vicinity of the endothelium. Moreover, the endothelium has sensors that act as transducers of mechanical forces in biological responses. Experimental data demonstrate that the number and quality of adhesion molecules, cytokines, and growth factors synthetized, as well as the local production of radicals, and pro and anticoagulation factors may change with shear stress favoring or not the local establishment and progression of atherosclerotic lesions.

Mouse extensor digitorum longus and soleus show distinctive ultrastructural changes induced by veratrine

We investigated whether veratrine (5 μl, 10 ng/kg) injected into the mouse extensor digitorum longus (EDL) (fast-twitch) and soleus (SOL) (slow-twitch) muscles provokes distinctive ultrastructural disturbances 15, 30 and 60 min later. The mitochondria in SOL were affected earlier (within 15 min) than in EDL. Swelling of the sarcoplasmic reticulum terminal cisternae was more marked in EDL than in SOL and caused distortion of sarcomeres so that fragmentation of myofilaments was more pronounced in EDL. Hypercontracted sarcomeres were seen mainly in SOL and veratrine caused infoldings of the sarcolemma only in this muscle. In both muscles, the T-tubules remained unaffected and by 60 min after veratrine most of the above alterations had reverted to normal. Pretreatment with tetrodotoxin prevented the alterations induced by veratrine. This suggests that most of the alterations resulted from the enhanced influx of Na+ into muscle fibers. These results emphasize the importance of considering the type of muscle when studying the action of myotoxic agents.

Histoenzymology and morphometry of the masticatory muscles of tufted capuchin monkey (Cebus apella Linnaeus, 1758)

Samples of the anterior and posterior regions of the masseter and temporal muscles and of the anterior belly of the digastric muscle of 4 adult male tufted capuchin monkeys (Cebus apella) were removed and stained with HE and submitted to the m-ATPase reaction (with alkaline and acid preincubation) and to the NADH-TR and SDH reactions. The results of the histoenzymologic reactions were similar, except for acid reversal which did not occur in fibers of the fast glycolytic (FG) type in the mandibular locomotor muscles. FG fibers had a larger area and were more frequent in all regions studied. No significant differences in frequency or area of each fiber type were detected, considering the anterior and posterior regions of the masseter and temporal muscles. The frequency of fibers of the fast oxidative glycolytic (FOG) and slow oxidative (SO) types and of FOG area differed significantly between the anterior belly of the digastric muscle and the mandibular locomotor muscle. The predominance of fast twitch (FG and FOG) fibers and the multipenniform and bipenniform internal architecture of the masseter and temporal muscles, respectively, are characteristics that permit the powerful bite typical of tufted capuchin monkeys.

Static lung compliance and body pressures in Tupinambis merianae with and without post-hepatic septum

The surgical removal of the post-hepatic septum (PHS) in the tegu lizard, Tupinambis merianae, significantly reduces resting lung volume (VLr) and maximal lung volume (VLm) when compared with tegus with intact PHS. Standardised for body mass (MB), static lung compliance was significantly less in tegus without PHS. Pleural and abdominal pressures followed, like ventilation, a biphasic pattern. In general, pressures increased during expiration and decreased during inspiration. However, during expiration pressure changes showed a marked intra- and interindividual variation. The removal of the PHS resulted in a lower cranio-caudal intracoelomic pressure differential, but had no effect on the general pattern of pressure changes accompanying ventilation. These results show that a perforated PHS that lacks striated muscle has significant influence on static breathing mechanics in Tupinambis and by analogy provides valuable insight into similar processes that led to the evolution of the mammalian diaphragm. © 2003 Elsevier Science B.V. All rights reserved.

Morphology of oesophagus and crop of the partrigde Rhynchotus rufescens (Tiramidae)

Twenty adult partridges Rhynchotus rufescens were used to study the morphology of oesophagus and crop. Materials to the morphologic study were collected and lengths of the oesophagus and of the crop were measured. For histological study, fragments of the oesophagus and of the crop were stained routinely with Masson's trichrome stain. Total oesophagus was larger in females. In the entrance of the thorax, its ventral wall is enlarged broadly, forming the crop, which is larger in males. Oesophagus mucosa is constituted by stratified squamous epithelium, with mucous glands and lymphatic tissues. There are three layers of smooth muscle, involved in serosa. The histological structure of the crop is similar to the oesophagus. The mucous glands are reduced in size and the lymphatic tissues are dispersed in the connective tissue. There is a thin stratum of smooth muscle and other two wider layers of longitudinal and circular musculature.

Molecular hybridization: A useful tool in the design of new drug prototypes

Molecular hybridization is a new concept in drug design and development based on the combination of pharmacophoric moieties of different bioactive substances to produce a new hyrid compound with improved affinity and efficacy, when compared to the parent drugs. Additionally, this strategy can results in compounds presenting modified selectivity profile, different and/or dual modes of action and reduced undesired side effects. So, in this described several example of different strategies for drug design, discovery and pharmacomodulation focused on new innovative hybrid compounds presenting analgesic, anti-inflammatory, platelet anti-aggregating, anti-infections, anticancer, cardio- and neuroactive properties.

Características ultraestruturais do segmento abdominal da aorta de rato albino

The objective of the present research was to investigate the ultrastructural peculiarities of the aortic wall of the rat. Seven young adult rats were used, from which fragments of the infrarenal abdominal aorta were collected. After collection, the vascular segments were fixed and sent for analysis by scanning electron microscope. The elastic lamellae appear interposed with smooth muscular fibers; this pattern was verified mainly at the medial layer structure. Among the mural elements a well defined interrelationship was established through connective lamellae of the arterial wall. The collagen lamellae mainly provided anchoring among the elastic and smooth muscular constituents. The intimal layer showed special ultrastructural features, such as a non-continuous inner elastic lamina presented in certain sites of the vascular wall, followed by endothelial pores. This mural pattern of the abdominal aorta provided support to vascular functions such as shrinkage among the laminar composition of the arterial layers, also acting in mechanical properties of the vascular wall, such as viscoelasticity and contractility - essential actions to blood vessel hemodynamics.

Morfologia e morfometria dos tipos de fibras do músculo reto abdominal de cadelas (canis familiaris) durante a gestação

The present paper deals with the evaluation of morphological and morphometric alterations of slow-twist (I) and fast-twitch (II) fibers of rectus abdominis muscle of adult female dog during pregestational phase, at 30 and 60 days of pregnancy and at 30 days after the parturition. At the every phases, using the open biopsy technique, muscle samples were collected. The samples were plunged. Histological sections were cut in a microtome. For general morphology, some sections were stained with HE. Subsequent sections were reacted for myofibrillar ATPase (m-ATPase), after alkaline (pH 10.4) and acid (pH 4.4) pre-incubations, in order to identificate type I and II fibers. In the pre-gestational phase, muscle tissue revealed to be composed by fibers with different diameters, presenting polygonal outlines and one or more periphery nuclei. At 30 days of pregnancy, muscle fiber characteristics were similar. At 60 days, in addition to the existence of normal fibers, polymorphic and small diameter fibers were frequent. At 30 days after the parturation, the morphology of muscle fiber were similar to that observed in the pre-gestational phase. In the four phases, type II fiber diameters were lager than type I. The diameters of both fiber types showed a significant reduction in the 30 days phase and a significative increasing at 60 days. The expansion of the abdominal wall during the pregnancy represents a chronic stimulus, induced changes in the morphology and in the fiber type diameters.

Exercise training improves relaxation response and SOD-1 expression in aortic and mesenteric rings from high caloric diet-fed rats

Background. Obesity has been associated with a variety of disease such as type II diabetes mellitus, arterial hypertension and atherosclerosis. Evidences have shown that exercise training promotes beneficial effects on these disorders, but the underlying mechanisms are not fully understood. The aim of this study was to investigate whether physical preconditioning prevents the deleterious effect of high caloric diet in vascular reactivity of rat aortic and mesenteric rings. Methods. Male Wistar rats were divided into sedentary (SD); trained (TR); sedentary diet (SDD) and trained diet (TRD) groups. Run training (RT) was performed in sessions of 60 min, 5 days/week for 12 weeks (70-80% VO2max). Triglycerides, glucose, insulin and nitrite/nitrate concentrations (NOx -) were measured. Concentration- response curves to acetylcholine (ACh) and sodium nitroprusside (SNP) were obtained. Expression of Cu/Zn superoxide dismutase (SOD-1) was assessed by Western blotting. Results. High caloric diet increased triglycerides concentration (SDD: 216 ± 25 mg/dl) and exercise training restored to the baseline value (TRD: 89 ± 9 mg/dl). Physical preconditioning significantly reduced insulin levels in both groups (TR: 0.54 ± 0.1 and TRD: 1.24 ± 0.3 ng/ml) as compared to sedentary animals (SD: 0.87 ± 0.1 and SDD: 2.57 ± 0.3 ng/ml). On the other hand, glucose concentration was slightly increased by high caloric diet, and RT did not modify this parameter (SD: 126 ± 6; TR: 140 ± 8; SDD: 156 ± 8 and TRD 153 ± 9 mg/dl). Neither high caloric diet nor RT modified NO x - levels (SD: 27 ± 4; TR: 28 ± 6; SDD: 27 ± 3 and TRD: 30 ± 2 μM). Functional assays showed that high caloric diet impaired the relaxing response to ACh in mesenteric (about 13%), but not in aortic rings. RT improved the relaxing responses to ACh either in aortic (28%, for TR and 16%, to TRD groups) or mesenteric rings (10%, for TR and 17%, to TRD groups) that was accompanied by up-regulation of SOD-1 expression and reduction in triglycerides levels. Conclusion. The improvement in endothelial function by physical preconditioning in mesenteric and aortic arteries from high caloric fed-rats was directly related to an increase in NO bioavailability to the smooth muscle mostly due to SOD-1 up regulation. © 2008 de Moraes et al; licensee BioMed Central Ltd.

The possible involvement of hyperpolarizing mechanisms in histamine-induced relaxation of the rat portal vein

The present study evaluated the effects of histamine 10 -2 M on longitudinal preparations of rat portal vein. It was observed that histamine 10 -2 M induced relaxation of rat portal vein preparations pre-contracted with phenylephrine 10 -4 M. On the other hand, no pharmacological effects were observed in preparations not pre-contracted. The observed histamine-induced relaxing effect was absent in preparations pre-contracted with KCl (120 mM) or in the presence of depolarizing nutritive solution. However, the histamine-induced relaxation was still present in the endothelium-removed preparations. The histamine-induced relaxation also was not prevented by astemizole (10 -6 M, 10 -5 M and 10 -4 M), cimetidine (10 -5 M, 10 -4 M and 10 -3 M) or thioperamide (10 -6 M, 10 -5 M and 10 -4 M), selective antagonists H 1, H 2 and H 3, respectively. The presence of L-NAME 10 -4 M or L-NAME 10 -4 M plus indomethacin 10 -5 M also did not prevent the histamine-induced relaxation observed in rat portal vein. Thus, the histamine-induced relaxation observed in rat portal vein appears to involve a non-endothelial hyperpolarizing mechanism independent of H 1, H 2 and H 3 receptors.

Plasma lipid profile of experimentally induced hyperlipidemic New Zealand white rabbits is not affected by resveratrol

Hyperlipidemia is well recognized as an important risk factor in the development of atherosclerosis. Low-density lipoproteins (LDL) are components of cholesterol that are highly associated to an increased risk of cardiovascular diseases. Hypercholesterolemia induces proteolytic and oxidative changes in vasculature, leading to a local inflammatory response. Since dietary antioxidants have attracted considerable attention as preventive and therapeutic agents, the polyphenolic compound resveratrol seems to play an important role in prevention of human atherosclerosis. Researches show that resveratrol inhibits LDL oxidation and platelet aggregation, as well as vascular prolifer ation of smooth muscle cells. However, recent findings in animal models reveal conflicting results regarding its effects on plasma lipid levels. The aim of the present study was to evaluate the effect of resveratrol on plasma biochemistry profile in New Zealand white rabbits submitted to a hypercholesterolemic diet. Twenty healthy, male, adult New Zealand white rabbits were fed with ordinary diet for one week before being divided into four treatment groups, containing five animals each. Group CT received maintenance diet; group R received maintenance diet and resveratrol (3mg/kg/day) given orally; group CL received maintenance diet enriched with 1.5% cholesterol; and group CR received maintenance diet enriched with 1.5% cholesterol and resveratrol (3mg/kg/day) given orally. During the experiment, from each animal, samples of 3mL venous blood were collected in heparin twice monthly for measurements of total cholesterol, triglycerides, and low- and high-density lipoproteins. The data analysis revealed that resveratrol did not have a hypolipidemic effect in experimentally induced hypercholesterolemic New Zealand white rabbits.

Arquitetura da parede vascular de segmentos torácico e abdominais da aorta de macaco prego (Cebus apella)

The intimal layer of the thoracic and abdominal aortic segments of Cebus apella monkey presented an evident inner elastic lamina. The medial layer of the thoracic aorta segment showed a basic lamellar elastic pattern having a medium average of 23.12 lamellae per analyzed cut sections, which appeared disposed circular and oblique relatively to the aortic lumen. The aortic adventitial layer, at this level, was formed mainly by collagen fibers, irregularly disposed between elastic fibers and smooth muscle cells. The aortic abdominal portion, in this monkey, at the suprarenal level presented a medium number of 19.12 elastic lamellae which showed a relatively disorganized and broken up pattern. At the infrarenal level the aortic medium layer showed a medium average of 11.75 elastic lamellae and next to the emission of the internal iliac arteries it was found only a middle average of 8.37 elastic lamellae intermingled with smooth muscle cells and collagen fibers whose concentration seemed to be increased next to the adventitial layer. The external elastic lamina was only present in the more distal aortic segment near to the emission of the internal iliac arteries.