[en] A grazing study in Area 13 of the Nevada Test Site (NTS) was initiated in May of 1973 and is proceeding on schedule. The accomplishments to date in this study include the quarterly collection of ingesta samples from fistulated steers, the quarterly sacrifice and sampling of a goat, the semiannual sacrifice and sampling of selected adult and young cattle, and the quarterly determination of digestibility of range plants. Dietary habits were tabulated with favored plant species being: two grasses, Indian ricegrass and galleta; two shrubs, winter fat and four-winged saltbush; and one forb, Russian thistle. Other analytical data are not yet available. (auth)

[en] The application of nuclear transfer technology is an interesting approach to investigate stem and progenitor cell transplantation therapy. If stem cells are used as a nuclear donor, donor cells can engraft into cloned animals without histocompatible problems. However, it is still uncertain whether donor cells can engraft to cloned animal and differentiate in vivo. To address this problem, we transplanted donor cells to dermal tissues of cloned pigs developed by using preadipocytes as donor cells. Preadipocytes are adipocytic progenitor which can differentiate to mature adipocytes in vitro. We showed that the donor preadipocytes were successfully transplanted into the cloned pigs without immune rejection and they differentiated into mature adipocytes in vivo 3 weeks after transplantation. In contrast, allogenic control preadipocytes, which can differentiate in vitro, did not differentiate in vivo. These results indicate that donor progenitor cells can differentiate in cloned animal

[en] Full text: Each cell of an animal's body contains full genetic code for the whole animal and nuclear transfer provides a way of converting cells to whole animal. Cells from endangered breeds collected by biopsy or from scrapings of soft skin or ear tissue or from hair follicle can be grown and multiplied in a laboratory and this would then be stored frozen indefinitely at 196 deg. C in liquid nitrogen. Mammary gland cells from sheep, mouse cumulus granulosa cells, bovine mural granulosa cells and fibroblast cells have all generated viable clones. The currently available methods of conservation, deep freezing of sperms (haploid genome) and storage of a large number of embryos are too expensive. In comparison, adult skin fibroblast cells are easy to obtain, hardy in culture and freezing, a good source of donor DNA without the limitations of age, sex and physiological state. Progenies were successfully obtained from nuclear transfer of serum-starved fibroblast cells from cattle, sheep and goat. Several other cell types successfully used for cloning are limited to female donors (cumulus and mammary epithelial, mural granulosa and oviductal cells) and are more difficult for long-term culture. Live progenies using skin fibroblasts have been produced in cattle. Sample collection and development of primary cultures: Samples were collected by biopsy of skin from ear pinna and transported in a complete medium (DMEM + HamsF12 with 10% FBS and penicillin and streptomycin) at 4 deg. C. Tissue samples were processed by removing hair form both sides, cut into small pieces and seeded in petridish containing fibroblast culture medium (DMEM + HamsF12, 10% FBS, penicillin and streptomycin and L-glutamine). The primary skin fibroblast cells started emerging out of tissues within 4-6 days and were allowed to grow up to 12-15 days till nearly 80% confluency was attained. Purification and sub-culturing of skin fibroblast cells: In isolated cases, there were contaminations of epithelial cells but these were removed mechanically as well as enzymatically to get pure fibroblasts. Sub-culturing or 'splitting cells' was done periodically removing growth media, washing the plate, dissociating the cells and diluting cell suspension in fresh media. Standard growth curve: Whenever, a new batch of culture media supplement was introduced, it was checked for its efficacy for growth of cells in culture and compared with standard growth curve. Goat skin fibroblasts remained in lag phase for initial two days when they settled on the solid surface of culture vessel and then came to log phase when maximum growth took place spanning from the third to the seventh day. As the confluencey level increased and media supplement was depleted, cells stopped dividing and a plateau was attained from the eighth day onwards and then showed decline due to contact inhibition. Cell proliferation index: Under standard culture conditions, skin fibroblast cells divide once in 24 hours but it is rarely achieved in normal culturing. The population doubling time and cell proliferation rate per day were checked at regular interval for quality assessment. For this, ELISA based MTT assay, incorporation of 5-bromo-de-oxyuridine method, and flow cytometer methods were used. Evaluation of cells for ploidy level: During long-term culturing the cells are likely to develop one or other type of chromosomal abnormalities. It must be ensured that the cells in different passages be checked for normal ploidy so that viable clones can be developed from them. Cultures showing increased frequency of aneuploidy or polyploidy must be terminated from further passaging. DNA from cultured somatic cells can be isolated using available DNA isolation kits and checked for its quality on 2% agarose. Cryo-freezing of cells: Cells are best frozen as cell suspension. Healthy culture were always employed to provide the stock to freeze cells. The cells were frozen at controlled freezing rate. The cells were kept at -80 deg. C as well as at -196 deg. C in Liquid nitrogen. Cells were found viable for subsequent growth after thawing at 38 deg. C even after 12 months. Applications: Skin fibroblast cells are widely used as nuclear donor and have produced viable clones in different livestock species. These cells after a series of passaging behave like totipotent cells and can be used as substitute of ES cells for gene targeting and production of transgenic animals. Cells can have wider application in understanding disease models and for cell therapy, and can be used for in vitro secretion of useful therapeutic protein in a culture medium. These can also be used for in vitro model studies on aging and cancer. (author)

[en] Laser surgery provides clean, fast and accurate modeling of tissue. However, the inability to determine what kind of tissue is being ablated at the bottom of the cut may lead to the iatrogenic damage of structures that were meant to be preserved. In this context, nerve preservation is one of the key challenges in any surgical procedure. One example is the treatment of parotid gland pathologies, where the facial nerve (N. VII) and its main branches run through and fan out inside the glands parenchyma. A feedback system that automatically stops the ablation to prevent nerve-tissue damage could greatly increase the applicability and safety of surgical laser systems. In the present study, Laser Induced Breakdown Spectroscopy (LIBS) is used to differentiate between nerve and gland tissue of an ex-vivo pig animal model. The LIBS results obtained in this preliminary experiment suggest that the measured spectra, containing atomic and molecular emissions, can be used to differentiate between the two tissue types. The measurements and differentiation were performed in open air and under normal stray light conditions. (paper)

[en] The articular-epiphyseal (A-E) cartilage complex of the distal humeral and femoral epiphyseal condyles of eight intact pigs (4 male & 4 female) of the same age were radiologically examined every 3 weeks, beginning at 42 days up to 147 days of age; to determine the age of onset, the course and trend of development of osteochondrosis (OCD). The earliest Dyschondroplasia (Osteochondrotic) lesions were demonstrated radiologically in the A-E complexes of the humeral condyles of 42-day-old pigs and in the femoral condyles at 63 days of age. Thus the radiographic examination of live animals to demonstrate subchondral radiolucency typical for OCD lesions in the condylar A-E complexes of pigs is not of limited value until the animals were > 100 days old as indicated in earlier reports. Also the course of further development of OCD lesions associated with A-E complexes was followed. The radiolucency was seen to develop to a certain extent, and then either to regress, and stabilize or even to progress as the animal grows. Some lesions regressed completely. Also some of the regressed lesions may become active again and become progressive. However, the course of development of femoral condyle A-E complexes OCD lesions was seen to be progressive continuously, or progressive then stable and then progressive again. The regressive course and trend of osteochondrotic lesions was not observed in the femoral condyle A-E complexes up to day 147 of age. Therefore, the course and trend of development of the A-E complexes OCD is not constantly the same

[en] Various European Union (EU) legislations request the use of in vitro tests for toxicological evaluations in order to increase the safety of consumer and patients but also to reduce the number vertebrates. The review provides a brief overview on EU legislations in place but without further interpretation. At present several ongoing EU projects address the need of developing predictive in vitro tests including projects assessing the potential of human embryonic stem cell (hESC) lines as basis for a range of toxicity tests. Tests based on human cells would avoid interspecies variations and as such predict more precisely adverse effects to the human body. However, the ethical situation on the use of toxicity tests based on hESCs is still under debate since no harmonization within Europe on the use of hESC lines has been achieved yet. A mutual acceptance of toxicity tests based on hESCs for regulatory applications is therefore challenging. Recent reports on the establishment of induced pluripotent stem cells (iPSC) are pointing to a way out of this dilemma, since these cells have apparently very similar characteristics as hESCs and could serve as basis for the development of toxicity tests. A careful scientific comparison between pluripotent cells of different origin is now needed in order to make final judgments. In any case, the development of reliable and relevant in vitro toxicity tests based on human pluripotent cells requires additional quality assessments of critical parameter that are also summarized within the review.

[en] One function ascribed to apoptosis is the suicidal destruction of potentially harmful cells, such as cancerous cells. Hence, their growth depends on evasion of apoptosis, which is considered as one of the hallmarks of cancer. Apoptosis is ultimately carried out by the sequential activation of initiator and executioner caspases, which constitute a family of intracellular proteases involved in dismantling the cell in an ordered fashion. In cancer, therefore, one would anticipate caspases to be frequently rendered inactive, either by gene silencing or by somatic mutations. From clinical data, however, there is little evidence that caspase genes are impaired in cancer. Executioner caspases have only rarely been found mutated or silenced, and also initiator caspases are only affected in particular types of cancer. There is experimental evidence from transgenic mice that certain initiator caspases, such as caspase-8 and -2, might act as tumor suppressors. Loss of the initiator caspase of the intrinsic apoptotic pathway, caspase-9, however, did not promote cellular transformation. These data seem to question a general tumor-suppressive role of caspases. We discuss several possible ways how tumor cells might evade the need for alterations of caspase genes. First, alternative splicing in tumor cells might generate caspase variants that counteract apoptosis. Second, in tumor cells caspases might be kept in check by cellular caspase inhibitors such as c-FLIP or XIAP. Third, pathways upstream of caspase activation might be disrupted in tumor cells. Finally, caspase-independent cell death mechanisms might abrogate the selection pressure for caspase inactivation during tumor development. These scenarios, however, are hardly compatible with the considerable frequency of spontaneous apoptosis occurring in several cancer types. Therefore, alternative concepts might come into play, such as compensatory proliferation. Herein, apoptosis and/or non-apoptotic functions of caspases may even promote tumor development. Moreover, experimental evidence suggests that caspases might play non-apoptotic roles in processes that are crucial for tumorigenesis, such as cell proliferation, migration, or invasion. We thus propose a model wherein caspases are preserved in tumor cells due to their functional contributions to development and progression of tumors

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