Characterization of the DNA binding features of Saccharomyces castellii Cdc13p.

The principal function of Saccharomyces cerevisiae Cdc13p is to provide a loading platform to recruit complexes that provide end protection and telomere replication. We isolated the Saccharomyces castellii Cdc13p homolog (scasCdc13p) and characterized the in vitro DNA binding features of the purified recombinant scasCdc13p. The full-length scasCdc13p binds specifically to G-rich single-stranded telomeric DNA, and not to double-stranded DNA or the C-rich strand.

Progestins initiate adverse events of menopausal estrogen therapy.

Objective: Recent report, particularly the Women's Health Initiative, demonstrated that hormone therapy with combined estrogen plus progestin increased the incidence of heart attacks, stroke, blood clots, breast cancer and dementia in women over 65 years old. We investigated the role of synthetic progestins in initiating the adverse events associated with estrogen therapy.

Methods: We used a fluorescence imaging technique, which allows video microscopic recordings of blood flow, blood vessel morphology and activities of various blood cells in a live animal.

Extraction of plasmid DNA from Escherichia coli cell lysate in a thermoseparating aqueous two-phase system.

The primary purification of a 6.1 kilo base pair (kbp) plasmid from a desalted alkaline lysate has been accomplished by a thermoseparating aqueous two-phase system [(50% ethylene oxide-50% propylene oxide)-Dextran T 500]. The partitioning of the different nucleic acids (plasmid DNA, RNA, genomic DNA) in the thermoseparating aqueous two-phase system was followed both qualitatively by agarose gel electrophoresis and quantitatively by analytical chromatography (size exclusion- and anion-exchange mode) and PicoGreen fluorescence analysis.

A comparison of the anti-inflammatory activities of conjugated estrogens and 17-beta estradiol.

Objective And Design: Unregulated chronic inflammatory process partly due to an estrogen deficiency may render postmenopausal women vulnerable to degenerative conditions such as arthritis, osteoporosis, atherosclerosis, and Alzheimer's disease. Current confusion regarding therapeutic efficacy of estrogen replacement therapy may be due to different estrogen formulations used, short term therapy, as well as advanced stage of the disease.

Materials And Methods: We compared anti-inflammatory activities of two major estrogen preparations, conjugated equine estrogen (CEE) and 17-beta estradiol, using an animal model (rat mesentery) of in vivo inflammatory reaction to intravenously infused amyloid-beta, examined by video recording and subsequently analyzed by transmission electron microscopy.

In vivo cerebrovascular actions of amyloid beta-peptides and the protective effect of conjugated estrogens.

Vascular dysfunction and inflammatory processes may be early events in the pathology of Alzheimer's disease (AD). Even though amyloid beta-peptides (Abeta) play a prominent role in the initiation and progression of cellular dysfunction in AD, the precise in vivo actions of various Abeta-peptides has not been established. The cerebrovascular actions of the major Abeta-peptides (1-40) and (1-42) in live animals were investigated using an open cranial window technique.

A vascular connection to Alzheimer's disease.

Alzheimer"s disease (AD) is characterized by a progressive and debilitating dementia in elderly people. The causes of this disease are not known, but major risk factors include old age and a family history of dementia, Down"s syndrome, female gender, low level of education, and head injury. There is no known cure for Alzheimer"s disease.

Estrogen and raloxifene activities on amyloid-beta-induced inflammatory reaction.

The prevalence of Alzheimer's disease (AD) in women is double that of men. Several studies indicate that use of estrogen after menopause by women may reduce the risk of developing AD. The risk of estrogen-dependent tumors associated with estrogen replacement therapy has prompted the use of alternatives, like the SERM raloxifene, which exert estrogen agonist effects on selected target tissues.

Vascular actions of estrogen and Alzheimer's disease.

Women are two to three times more likely to develop late-onset Alzheimer's disease (AD) than age-matched men. A large number of observational reports and a few randomized clinical trials have indicated that estrogen replacement therapy (ERT) may retard the development and severity of dementia in postmenopausal women. A chronic inflammatory reaction mediated by abnormal deposition of proteins such as amyloid-beta (A beta) is central to the pathology of AD.

Animal model of Alzheimer-like vascular pathology and inflammatory reaction.

This in vivo animal model of vascular inflammatory reaction facilitates morphologic and hemodynamic analyses of leukocyte-endothelial interaction and can be monitored by video microscopy and electron microscopy. The model has served as a rapid means to explore the deleterious vascular actions and inflammatory response to the cytokines tumor necrosis factor, interleukin-1 and amyloid-beta, as well as the protective effects of superoxide dismutase, estrogen, and cytokine antagonists.

Estrogen protects peripheral and cerebral blood vessels from toxicity of Alzheimer peptide amyloid-beta and inflammatory reaction.

Due to increases in life expectancy, women are living 30 years or more beyond menopause. This has led to an increasing interest in the association between postmenopausal estrogen deficiency and degenerative diseases associated with aging such as cardiovascular disease, osteoporosis and dementia. Women are two times more likely to develop late-onset Alzheimer's disease (AD) than age-matched men.

Amyloid-beta peptide induced inflammatory reaction is mediated by the cytokines tumor necrosis factor and interleukin-1.

A chronic inflammatory response possibly mediated by amyloid-beta (A beta) is believed to be a major factor in the pathology of Alzheimer's disease (AD). Recently, we demonstrated that in vivo administration of A beta produces an inflammatory response and vascular disruption as seen in the brains of AD patients. In an inflammatory response, leukocyte activation and extravasation involves cytokine production.

Animal model of vascular inflammation.

Inflammatory mechanisms play a central role in the pathology of a variety of conditions ranging from atherosclerosis, arthritis, cancer and Alzheimer's disease. Under normal conditions the inflammatory response initiates protective actions, but triggers tissue damage under pathological conditions. Acute or chronic inflammation is mediated by nascent expression of a host of proteins such as the cytokines interleukins (IL), tumor necrosis factor (TNF), and interferons.

In vivo vascular damage, leukocyte activation and inflammatory response induced by beta-amyloid.

beta-amyloid toxicity is central to the pathology of Alzheimer's disease. Recent evidence implicates vascular dysfunction as a contributing factor to the dementia of Alzheimer type. Using intravital microscopy we demonstrate that in vivo administration of beta-amyloid produces extensive vascular disruption including endothelial and smooth muscle damage, adhesion and migration of leukocytes across arteries and venules.

Ultrastructure changes associated with brain death in the human donor heart.

Electromicroscopic examinations were carried out on 30 myocardial biopsies taken from 22 human donor hearts immediately after excision (prestorage) or immediately before transplantation (poststorage). All electron micrographs were independently examined by two morphologists. Eleven structures were examined in each micrograph, and each structure was scored according to the degree of injury.

Differences in microcirculatory bed arrangement in some skeletal muscles of the rat and golden hamster.

The architectural features of the most peripheral parts of the microcirculatory bed were studied in selected skeletal muscles (cremaster, retractor, spinotrapezius, and ventral stripe of spinotrapezius) in rats and golden hamsters ranging in weight between 130 and 330 and 130 and 180 g, respectively. In the cremaster muscle of both species, a very complicated, irregular and heterogenous arrangement of terminal arterioles, as well as of postcapillary and collecting venules was found. In the retractor, the most regular arrangement was along the margins of the middle part of muscle, but an irregular brush-like branching of the terminal arterioles and collecting venules limits the use of these areas.

Early postnatal growth of skeletal muscle blood vessels of the rat.

The development of blood vessels during the first three postnatal weeks was studied in the ventral stripe of the spinotrapezius muscle of the rat by use of India ink-gelatine injections, and electron microscopy. The number of terminal arterioles and collecting venules remained unchanged postnatally in the observed area. A remarkable proximodistal gradient of vascular development was apparent: while the basic structure of the hilar vessels remained unchanged in the time studied, the intramuscular arteries and veins matured gradually.

Circulatory pattern and structure in the tail and tail fins of Xenopus laevis tadpoles.

This investigation was initiated with the intent of study capillary sprouting in the tadpole tail fin microcirculation of the African clawed frog, Xenopus laevis, using a combination of intravital video recordings and electron microscopy. The tadpoles were observed daily for periods up to one hour during one to two weeks. The capillary sprouts originated mostly from postcapillary venules, and within 24-48 h merged with other capillary sprouts, subsequently establishing a capillary loop with blood flow.

Capillary growth in the mesentery of normal young rats. Intravital video and electron microscope analyses.

Capillary sprouting was studied by a combination of intravital video recording and subsequent electron microscopy in the mesentery of young female rats without previous experimental manipulation. Selected segments of the mesenteric microvascular bed with capillary sprouts were carefully surveyed and mapped at a monitor magnification of 255 times and submitted to detailed in vivo analysis concerning flow pattern and cells at 2000 times magnification. The mesentery was preserved for light and electron microscopy by a superfusion of glutaraldehyde while observed and recorded on video, confirming earlier investigations that this type of fixation does preserve exceptionally well vascular topography and diameters of the mesenteric microvascular bed.

Perfusion and superfusion fixation effects on rat mesentery microvascular beds. Intravital and electron microscope analyses.

The effects of glutaraldehyde on dimensions and ultrastructure of microvascular beds in rat mesentery were studied in two kinds of experiment, administering the fixative by intra-arterial perfusion at a pressure of 80 mm Hg and by superfusion of the exteriorized mesenteric membrane. The microvascular segments were observed by means of intravital microscopy and recorded on videotape before, during, and after glutaraldehyde reached the microvascular segment being observed. Vascular outer diameters were measured at exactly the same points before and after fixation; in Epon embedded whole-mounts; and in sections analyzed by light and transmission electron microscopy, confirming positively the various segments of the microvascular bed and yielding information concerning the preservation of cellular components.

An electron microscopic study on the type I pneumocyte in the cat: postnatal morphogenesis.

This investigation describes the morphogenesis of the type I pneumocyte from the neonatal stage to the age of 3 months. Cells lining subpleural air spaces were photographed from electron microscopic serial sections and a three-dimensional representation of each cell was obtained by transferring the contours of the cell membranes from micrographs to transparent plastic sheets which were then spaced to scale and stacked. The portion of the reconstructed cell surface taking part in the formation of the blood-air barrier increased extensively in postnatal stages when compared with reconstructed cells of prenatal stages.

[Intravital and electron microscopic analysis of the arterioles by immersion and perfusion technics of fixation of the rat mesentery].

The technique of the intravital microscopic control, videotaping, cinemoregistration and per single-frame analysis made it possible to demonstrate for the first time that a double fixation in glutaraldehyde and osmium has no effect on the arteriolar lumen. Comparing the data on the intravital and a detailed electron microscopic study, it was possible to reveal certain peculiarities and advantages of the fixative perfusion and immersion techniques applied. The investigation establishes a basis for extended morphological studies of small blood vessels and morpho-physiological comparisons.

Combined intravital microscopy and electron microscopy of the blind beginnings of the mesenteric lymphatic capillaries of the rat mesentery. A preliminary report.

The blind beginnings (so-called terminal lymphatics) of the mesenteric lymphatic capillaries have been studied in the rat mesentery by means of intravital microscopy and electron microscopy. Intraluminal objects were observed to move slowly downstream, probably carried by the movement of the lymphatic fluid. No simultaneous changes in capillary lumen could be observed, and the subsequent electron microscope analysis of the same lymphatic capillary demonstrated the absence of smooth muscle cells in the wall of the lymphatic vessel.

An electron microscopic study on the type I pneumocyte in the cat: pre-natal morphogenesis.

This investigation describes the pre-natal morphogenesis of the type I pneumocyte subsequent to its differentiation from pulmonary epithelium. Cells lining subpleural alveolar septa were photographed from serial sections with the electron microscope, and a three-dimensional representation of each cell was obtained by transferring the contours of the cell membranes from montages to transparent plastic sheets which were then spaced to scale and stacked. The results of this study indicate that: The nascent blood-air barrier of a 50-day reconstructed cell was twice as thick as the average definitive barrier; definitive barrier thickness was observed in some areas in a 63-day reconstructed cell; the amorphous component of elastic tissue which appears peripherally in septal connective tissue during pre-natal morphogenesis may be directly juxtaposed to the basal lamina of the alveolar epithelium; the orientation of the cell junction between a pneumocyte and its neighboring cells, as observed in sections of alveolar septa, changes as the contour of the pneumocyte changes from simple abutment to overlapping patterns.