Weak-field H3O+ ion cyclotron resonance alters water refractive index.

Heretofore only observed in living systems, we report that weak-field ion cyclotron resonance (ICR) also occurs in inanimate matter. Weak magnetic field (50 nT) hydronium ICR at the field combination (7.84 Hz, 7.5 µT) markedly changes water structure, as evidenced by finding an altered index of refraction exactly at this combined field. This observation utilizes a novel technique which measures the scattering of a He-Ne laser beam as the sample is exposed to a ramped magnetic field frequency. In addition to the hydronium resonance, we find evidence of ICR coupling to a more massive structure, possibly a tetrahedral combination of three waters and a single hydronium ion. To check our observations, we extended this technique to D2O, successfully predicting the specific ICR charge-to-mass ratio for D3O+ that alters the index of refraction.

Lorentz force in water: evidence that hydronium cyclotron resonance enhances polymorphism.

There is an ongoing question regarding the structure forming capabilities of water at ambient temperatures. To probe for different structures, we studied effects in pure water following magnetic field exposures corresponding to the ion cyclotron resonance of H3O(+). Included were measurements of conductivity and pH. We find that under ion cyclotron resonance (ICR) stimulation, water undergoes a transition to a form that is hydroxonium-like, with the subsequent emission of a transient 48.5 Hz magnetic signal, in the absence of any other measurable field. Our results indicate that hydronium resonance stimulation alters the structure of water, enhancing the concentration of EZ-water. These results are not only consistent with Del Giudice's model of electromagnetically coherent domains, but they can also be interpreted to show that these domains exist in quantized spin states.

Lesions of the prepuce and penis in rams: A retrospective study.

In the ovine reproductive management, a thorough breeding soundness evaluation including specific inspection of the male genital tract represents a valuable tool for assessing the reproductive potential of a selected subject and for diagnosing genital disorders. During examination, accurate inspection on penis and prepuce is necessary, since conditions affecting these structures may hamper regular coitus. Records from 1270 males undergoing breeding soundness evaluation (n = 1232) or admitted for genital disorders to the Section of Obstetrics and Gynaecology (n = 38) of the Department of Veterinary Medicine, were collected, and lesions of penis and prepuce were therefore classified. The data collected revealed that 47/1270 rams examined presented lesions of the penis and prepuce. The most frequent condition was urolithiasis accounting for over 2% of the cases, followed by lack of the urethral process (0.39% incidence), lack of the glans penis and hypospadias (0.23% cases). Moreover, most of the conditions (40%) were observed in animals less than 2 years old, suggesting the importance of a careful breeding soundness evaluation in animals at young age.

Suppression of SPARC expression by antisense RNA abrogates the tumorigenicity of human melanoma cells.

Acquisition of invasive/metastatic potential is a key event in tumor progression. Cell surface glycoproteins and their respective matrix ligands have been implicated in this process. Recent evidence reveals that the secreted glycoprotein SPARC (secreted protein, acidic and rich in cysteine) is highly expressed in different malignant tissues. The present study reports that the suppression of SPARC expression by human melanoma cells using a SPARC antisense expression vector results in a significant decrease in the in vitro adhesive and invasive capacities of tumor cells, completely abolishing their in vivo tumorigenicity. This is the first evidence that SPARC plays a key role in human melanoma invasive-metastatic phenotype development.

The role of extracellular conditions during CaCo-2 cells growth: a preliminary study for numerical model validation.

OBJECTIVES: One important limitation in cell therapy protocols, and regenerative medicine (an innovative and promising strategy for different pathologies treatment), is the lack of knowledge about cells engraftment, proliferation and differentiation. In order to allow an efficient and successful cell transplant, it is necessary to predict the logistics, economic and timing issues during cellular injection. It has been reported that several parameters, such as cells number, temperature and extracellular pH (pH0) value can influence metabolic pathways and cellular growth. Numerical analysis and model can help to reduce and understand the effects of the above environmental conditions on cell survival. The aim of this paper is to develop the first step of cells transplantation in order to identify "in vitro", which parameters can be useful to develop and validate a numerical model, able to evaluate "in vivo" cells engraftment and proliferation. MATERIAL AND METHODS: We studied the variation of extracellular parameters--such as medium volume, buffer system, nutrient concentrations and temperature on human colon carcinoma cells (CaCo-2) "in vitro culture"--pursuing the goal of understanding in deeper details cellular processes such as growth, metabolic activity, survival and pH0. RESULTS: Results showed that CaCo-2 cells growth and mortality increase after two days in culture when cells were suspended in 3.5 ml volume to respect of 10 ml volume. Different temperature values influenced CaCo-2 cells growth and metabolic activity showing a direct relationship with the volume of the medium. CONCLUSIONS: Our results describe as CaCo-2 cell growth, metabolic activity, mortality and extracellular pH were influenced by extracellular parameters, enabling us to develop and validate a numerical model to be use to predict cells engraftment and proliferation.

Human muscle-derived stem cells. Effectiveness in animal models of faecal incontinence. Research scheduling.

Researchers believe that human muscle-derived cells are able to restore leak-point pressure to normal levels by differentiating into new muscle fibres that prevent anal sphincter muscle atrophy. Laboratory data are needed to identify exactly how these cells work to regenerate muscle. The objective of this study is to test whether stem cells can be employed to treat internal anal sphincter (IAS) injuries in humans; to this end, this work will use a two-step process to study: first, the effectiveness of the treatment in a sample of animals with artificial injuries to the IAS and then to verify the results in a population of selected humans affected by pathology.

Aging is associated with an increase in dye coupling and in gap junction number in satellite glial cells of murine dorsal root ganglia.

Glial cells in both central and peripheral nervous systems are connected by gap junctions, which allow electrical and metabolic coupling between them. In spite of the great current interest in aging of the nervous system, the effect of aging on glial cell coupling received little attention. We examined coupling between satellite glial cells in murine dorsal root ganglia using the dye coupling technique and electron microscopy. We studied mice at ages of postnatal 90-730 days. Dye coupling incidence between satellite glial cells associated with a single neuron increased from 24.2% at postnatal day 90 to 50.5% at postnatal day 730. Dye coupling between satellite glial cells that are in contact with two or more neurons increased from 2.7% at postnatal day 90 to 18.6% at postnatal day 730 (P<0.05). Examination of the ganglia with the electron microscope showed that the number of gap junctions per 100 microm2 of surface area of satellite glial cells increased from 0.22 at postnatal day 90 to 1.56 at postnatal day 730 (P<0.01). The mean length of individual gap junctions did not change with age. These results provide strong evidence for an increase of functional coupling between satellite glial cells during life. This increase is apparently due to an increase in the total area of the system of gap junctions connecting these cells.

The perineuronal glial tissue of spinal ganglia. Quantitative changes in the rabbit from youth to extremely advanced age.

The volumes of the nerve cell bodies and those of the enveloping satellite cell sheaths from spinal ganglia were determined by morphometric methods applied to electron micrographs in young, adult, old and very old rabbits. The mean volume of the nerve cell bodies increased progressively with age; this is probably related to the increase with age of the body size of the rabbits studied. The mean volume of the satellite cell sheaths did not differ significantly in young, adult and old animals, but was significantly smaller in very old animals. It is extremely unlikely that this marked reduction in the volume of the satellite cell sheath is the result of a pathological process. The mean value of the volume ratio between the satellite cell sheaths and the related nerve cell bodies did not differ significantly in young and adult animals, but was significantly smaller in old and very old animals. This ratio was particularly low in very old animals. Our analysis showed that in each age group the volume of the satellite cell sheath is linearly related to the volume of the related nerve cell body. This result suggests that in rabbit spinal ganglia the quantitative relations between glial and nervous tissue are tightly controlled throughout life. It is suggested that ganglionic neurons release signals to influence and control the volume of their associated glial tissue. Since satellite cells have important support roles for the neurons they surround, it is likely that the marked reduction in the volume of perineuronal sheaths in the extremely advanced age is accompanied by a reduction of those roles, with negative consequences for neuronal activity.

A study of mitochondria in spinal ganglion neurons during life: quantitative changes from youth to extremely advanced age.

In view of the central role that mitochondria are thought to play in the ageing process, we investigated changes in mitochondria of spinal ganglion neurons in rabbits aged 1, 3.6, 6.7, and 8.8 years (the latter extremely old). Mitochondrial size increased significantly with age, while mitochondrial structure did not change. The total volume of mitochondria within the perikaryon did not change significantly during life. This indicates that in these neurons mitochondrial degradation was completely compensated by the production of new mitochondria even in the extremely advanced age. We also found that the mean volume of neuronal perikaryon increased markedly with age, so that the mean percentage of perikaryal volume occupied by mitochondria decreased significantly with a difference of about 33% between the youngest and the oldest animals. This decrease is only in very small part due to lipofuscin accumulation, so that the ratio of the total volume of mitochondria to the volume of functionally active cytoplasm decreased with age. The mitochondria of the neurons studied seem therefore unable to adapt their total volume to the volume of functionally active cytoplasm, that increases with age. This result is consistent with the observation that the neurons of old animals have a reduced ability to respond to high energy demands.

The Golgi apparatus of spinal ganglion neurons: quantitative changes with aging.

The Golgi apparatus of spinal ganglion neurons was studied in 1, 3.6, 6.7, and 8.8-year-old rabbits. The structure of this organelle did not differ in the four age groups examined. While the mean volume of the neuronal perikaryon increased progressively with age, the total volume of the Golgi apparatus remained stable throughout life. As a consequence, the mean percentage of perikaryal volume occupied by this organelle decreased significantly with age. Since the percentage of perikaryal volume occupied by lipofuscin remained at low levels throughout life, the ratio of the total volume of the Golgi apparatus to the functionally active volume of cytoplasm decreased with age. It is possible that this decrease is related to the reduction in neuronal metabolism that occurs in senescence. The age-related quantitative changes in the Golgi apparatus were very similar in large light and in small dark neurons. Finally, neither fragmentation, nor peripheral displacement of the Golgi apparatus was observed with advancing age.

Increase in number of the gap junctions between satellite neuroglial cells during lifetime: an ultrastructural study in rabbit spinal ganglia from youth to extremely advanced age.

This study investigated quantitative aspects of the gap junctions between satellite neuroglial cells that envelope the spinal ganglion neurons in rabbits aged 1 year (young), 3.6 years (adult), 6.7 years (old), and 8.8 years (very old). Both the total number of gap junctions present in 30,000 microm2 of surface area occupied by perineuronal satellite cells, and the density of these junctions increased throughout life, including the extremely advanced age. By contrast, the mean length of individual gap junctions did not change with age. Thus, the junctional system which provides morphological support for the metabolic cooperation between satellite cells in rabbit spinal ganglia becomes more extensive as the age of the animal increases. These results support the hypothesis that the gap junctions between perineuronal satellite cells are involved in the spatial buffering of extracellular K+ and in neuroprotection.

Differences in amniotic amino acid concentrations between pregnancies obtained with transfer of vitrified thawed in vitro-produced embryos and with natural mating in sheep.

In vitro embryo production (IVP) and cryopreservation are associated with a high incidence of pregnancy complications and fetal abnormalities that may be linked with alterations of placental development. The amniotic fluid is partly derived from the transport of water and solutes across the placenta and provides the fetus with amino acids (AAs), which are the building blocks for biomolecules involved in physiological growth and development. To better understand the anomalies associated with IVP pregnancies, the present study was conducted to test the hypothesis that amniotic concentrations of AAs differ in pregnancies derived from vitrified/thawed (V/T) IVP embryos compared with gestations obtained with natural mating (NM) in sheep. Amniotic fluid was sampled in ewes that were pregnant after transfer of V/T IVP embryos and that had conceived with NM between Days 60 and 65 (V/T, n = 6; NM, n = 11) and between Days 80 and 85 (V/T, n = 5; NM, n = 14) of gestation via ultrasound-guided amniocentesis. Concentrations of 16 AAs in the amniotic fluid were measured using high-performance liquid chromatography. From Days 60 to 65 of gestation, concentrations of cystine, phenylalanine, and isoleucine were lower in V/T compared with NM ewes. From Days 80 to 85 of pregnancy, the mean concentrations of cystine and lysine were lower in the V/T versus NM groups. The total AA concentration per ewe was similar between the groups from Days 60 to 65 and 80 to 85 of gestation and decreased by 55% from Days 60 to 65 and 80 to 85 of gestation in all ewes. The most abundant AA from Days 60 to 65 of gestation was alanine in both groups, whereas from Days 80 to 85, the most abundant AAs were alanine in NM and glycine in V/T ewes; cystine was the less abundant detectable AA in all ewes at both stages of gestation. Results report that V/T IVP embryos have decreased concentrations of individual AAs in the amniotic fluid during the second trimester of gestation possibly because of an impaired placental vasculogenesis or because of a reduced placental transport. These novel findings are relevant to unravel the mechanisms responsible for the issues of pregnancies achieved with the transfer of IVP and cryopreserved embryos.

Expression of cathepsin D in primary and metastatic human melanoma and dysplastic nevi.

High levels of cytosolic cathepsin D expression have been associated with poor prognosis in breast cancer node-negative patients. In this work, we provide evidence that three cell lines established from human metastatic melanomas--IIB-MEL-J, IIB-MEL-LES, and IIB-MEL-IAN--express high levels of procathepsin D mRNA. IIB-MEL-J cells secreted into the conditioned media about 30% of the newly synthesized protein, which was active at acidic pH. Melanoma tumors arising in nude mice after injection of the three different cell lines expressed high levels of procathepsin D mRNA. Moreover, 13 human metastatic melanomas expressed variable levels of procathepsin D mRNA. To study the possible association between cathepsin D expression and melanoma development, samples corresponding to 10 primary tumors, 11 metastatic melanomas, 10 dysplastic nevi, 27 nevocellular nevi, and normal melanocytes were studied by immunohistochemistry for cathepsin D-specific staining. We found that cathepsin D was expressed in all of the dysplastic nevi and primary and metastatic melanomas tested but in only 18% of nevocellular nevi (five of 27), whereas normal melanocytes showed no cathepsin D expression. The overall data indicate that cathepsin D is expressed at a high level by melanoma cells, and because of its expression in preneoplastic lesions, it may be associated with melanoma development.

High levels of matrix metalloproteinases regulate proliferation and hormone secretion in pituitary cells.

Beside the digestion of the extracellular matrix during tumor invasion and metastasis, more recently, new functions for matrix metalloproteinases (MMPs) have been proposed. We studied the expression and function of these enzymes in pituitary cells. We observed the activities of MMP-2 and MMP-9 together with expression of membrane-type MMP and tissue inhibitor of metalloproteinase-1 in all types of human pituitary adenomas. We found surprisingly high levels of MMP activity and low levels of tissue inhibitor of metalloproteinases, indicating a high level of extracellular matrix-degrading activity in pituitary adenomas. To examine the function of metalloproteinase activity in pituitary cells we used the synthetic MMP inhibitor batimastat. These studies demonstrate that MMPs secreted by pituitary cells can release growth factors anchored to the extracellular matrix that, in turn, control pituitary cell proliferation and hormone secretion. These results define a new additional mechanism for the control of pituitary hormone secretion and indicate new potential therapeutic targets for pituitary adenomas.

Glial cell plasticity in sensory ganglia induced by nerve damage.

Numerous studies have been done on the effect of nerve injury on neurons of sensory ganglia but little is known about the contribution of satellite glial cells (SCs) in these ganglia to post-injury events. We investigated cell-to-cell coupling and ultrastructure of SCs in mouse dorsal root ganglia after nerve injury (axotomy). Under control conditions SCs were mutually coupled, but mainly to other SCs around a given neuron. After axotomy SCs became extensively coupled to SCs that enveloped other neurons, apparently by gap junctions. Serial section electron microscopy showed that after axotomy SC sheaths enveloping neighboring neurons formed connections with each other. Such connections were absent in control ganglia. The number of gap junctions between SCs increased 6.5-fold after axotomy. We propose that axotomy induces growth of perineuronal SC sheaths, leading to contacts between SCs enveloping adjacent neurons and to formation of new gap junctions between SCs. These changes may be an important mode of glial plasticity and can contribute to neuropathic pain.

Decrease in the nucleo-cytoplasmic volume ratio of rabbit spinal ganglion neurons with age.

Nuclear and perikaryal volumes, as well as the mean percentage of perikaryal volume occupied by lipofuscin, were estimated in spinal ganglion neurons of 12, 42 and 79 month-old rabbits. The volume ratio between nucleus and perikaryal cytoplasm (N/C volume ratio) decreased progressively and significantly with increasing age. This was not a consequence of the perikaryal enlargement due to the age-related lipofuscin accumulation since the same result was obtained when the volume occupied by lipofuscin was subtracted from the perikaryal volume. The decrease in the N/C volume ratio may depend on an age-related loss of DNA, a cytoplasmic influence on nuclear size, or other unknown causes.

Age-related decrease in the overall extent of perikaryal projections in rabbit spinal ganglion neurons.

The overall extent of the perikaryal projections of sensory neurons from spinal ganglia of young adult and aged rabbits was estimated by a stereological method using the electron microscope. The extent of perikaryal projections was significantly smaller in the aged animals. This age-related decrease did not seem to depend on factors intrinsic to the neuron, but on the absence of a satellite cell covering over extensive portions of the nerve cell body surface. This decrease may influence the organization of the subplasmalemmal cytoskeleton, metabolic exchange between the nerve cell body and its environment and perhaps also neuronal metabolism.

Quantitative changes in mitochondria of spinal ganglion neurons in aged rabbits.

Within the context of our research on the age-related structural changes in spinal ganglia, we studied the mitochondria of the neuronal perikaryon in the spinal ganglia of 12-, 42-, and 79-month-old rabbits. Both the volume of the perikaryon and the total mitochondrial mass within the perikaryon increased significantly passing from young adult to old animals. Hence, there is no net loss of mitochondria in these neurons with age. Since, however, the volume of the perikaryon increased by more than 63% while the total mitochondrial mass within the perikaryon increased by only 18%, the mean percentage of perikaryal volume occupied by mitochondria decreased with age. This decrease is only in very minor part a consequence of lipofuscin accumulation, so that the ratio between the total mitochondrial mass and the functionally active volume of cytoplasm decreased with age. Possible causes of this decrease are discussed briefly. Moreover, while the mitochondrial structure did not change, mitochondrial size increased with age. Finally, in each of the three age groups both the mean percentage volume of mitochondria and the mean mitochondrial size were very similar in large light and in small dark neurons.

Age-related quantitative changes in mitochondria of satellite cell sheaths enveloping spinal ganglion neurons in the rabbit.

We studied mitochondria in the satellite cell sheaths which envelope the spinal ganglion neurons of rabbits aged 12, 42, and 79 months. While the mean cytoplasmic volume of satellite cell sheaths did not change significantly with age, the mean percentage of cytoplasmic volume occupied by mitochondria decreased with age. This decrease is mainly due to a reduction in the total mitochondrial mass and only in minor part is a consequence of lipofuscin accumulation. Mitochondrial structure did not change, while mitochondrial size increased with age. Comparison between mitochondria in nerve cell bodies and those in satellite cell sheaths showed that: (1) the mean percentage of cytoplasmic volume occupied by mitochondria was greater in nerve cell bodies than satellite cell sheaths and the ratio between these two percentages remained constant with advancing age; (2) the total mitochondrial mass was much greater in nerve cell bodies than satellite cell sheaths and the ratio between these two values increased with age; (3) the extent of increase of mitochondrial size with age was similar in nerve cell bodies and satellite cell sheaths. The results of the present study suggest that: (1) the ability of satellite cell sheaths to produce energy decreases with age; (2) the decreased ability of sensory neurons in old animals to meet high energy demands may be partly due to the diminished contribution of their associated satellite cell sheaths.

Evaluation of the efficacy and toxicity of the cyclosporine A-flucortolone-methotrexate combination in the treatment of sarcoidosis.

Eleven patients with chronic sarcoidosis resistant to high-dose corticosteroids and other immunosuppressive treatments were treated with cyclosporine A at the initial daily dose of 5 mg per kg body weight (ideal weight in the case of overweight subjects) combined with flucortolone and methotrexate. A complete and lasting remission of the disease was obtained in all patients with total disappearance of pulmonary and extrapulmonary manifestations. In addition, the disease activity indexes normalized and remained normal for the rest of the follow-up period (24.82 +/- 8.22 months, range 12-33). No renal or hepatic toxicity was observed in any patient. Two of them presented hypertrichosis and one nausea.