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Approximately 100 species become extinct a day. Despite increasing interest in using cloning to rescue endangered species, successful interspecies nuclear transfer has not been previously described, and only a few reports of in vitro embryo formation exist. Here we show that interspecies nuclear transfer can be used to clone an endangered species with normal karyotypic and phenotypic development through implantation and the late stages of fetal growth. Somatic cells from a gaur bull (Bos gaurus), a large wild ox on the verge of extinction, (Species Survival Plan < 100 animals) were electrofused with enucleated oocytes from domestic cows. Twelve percent of the reconstructed oocytes developed to the blastocyst stage, and 18% of these embryos developed to the fetal stage when transferred to surrogate mothers. Three of the fetuses were electively removed at days 46 to 54 of gestation, and two continued gestation longer than 180 (ongoing) and 200 days, respectively. Microsatellite marker and cytogenetic analyses confirmed that the nuclear genome of the cloned animals was gaurus in origin. The gaur nuclei were shown to direct normal fetal development, with differentiation into complex tissue and organs, even though the mitochondrial DNA (mtDNA) within all the tissue types evaluated was derived exclusively from the recipient bovine oocytes. These results suggest that somatic cell cloning methods could be used to restore endangered, or even extinct, species and populations.

Therapeutic cloning by somatic cell nuclear transfer offers potential for treatment of a wide range of degenerative disease. Nuclear transplantation with neo (r)-marked somatic nuclei from 10-13-year-old cows was used to generate cloned bovine fetuses. Clone fetal liver (FL) hematopoietic stem cells (HSC) were transplanted into two busulfan-treated and one untreated nuclear donor cows. Hematopoiesis was monitored over 13-16 months by in vitro progenitor and HSC assays. Chimerism was demonstrated by PCR in blood, marrow, lymph nodes, and endothelium, peaking at levels of 9-17% in blood granulocytes but at lower levels in lymphocyte subsets (0.1-0.01%). Circulating progenitors showed high levels of chimerism (up to 60% neo (r+)) with persisting fetal features. At sacrifice, the animal that had no pre-transplant myelosupression showed persisting donor cells in blood and lymph nodes, and in marrow 0.25% of progenitor cells and a detectable fraction of stem cells were neo (r+). The fetal HSC showed a 10-fold competition advantage over adult HSC. Cloning generated histocompatible HSC capable of long-term multilineage engraftment in a large animal model.

Human embryonic stem cells are likely to play an important role in the future of regenerative medicine. However, exposure of existing human embryonic stem-cell lines to live animal cells and serum risks contamination with pathogens that could lead to human health risks. We aimed to derive an embryonic stem-cell line without exposure to cells or serum.Frozen cleavage-stage embryos were thawed and cultured to the blastocyst stage. Inner cell masses were isolated by immunosurgery and plated onto extracellular-matrix-coated plates that can be easily sterilised. Six established human embryonic stem-cell lines were also maintained with this serum and feeder free culture system.A new stem-cell line was derived from human embryos under completely cell and serum free conditions. The cells maintained normal karyotype and markers of pluripotency, including octamer binding protein 4 (Oct-4), stage-specific embryonic antigen (SSEA)-3, SSEA-4, tumour-rejection antigen (TRA)-1-60, TRA-1-81, and alkaline phosphatase. After more than 6 months of undifferentiated proliferation, these cells retained the potential to form derivatives of all three embryonic germ layers both in vitro and in teratomas. These properties were also successfully maintained (for more than 30 passages) with the established stem-cell lines.This system eliminates exposure of human embryonic stem cells and their progeny to animal and human feeder layers, and thus the risk of contamination with pathogenic agents capable of transmitting diseases to patients.

Human stem-cell derivatives are likely to play an important role in the future of regenerative medicine. Evaluation and comparison to their in vivo counterparts is critical for assessment of their therapeutic potential. Transcriptomics was used to compare a new differentiation derivative of human embryonic stem (hES) cells--retinal pigment epithelium (RPE)--to human fetal RPE. Several hES cell lines were differentiated into putative RPE, which expressed RPEspecific molecular markers and was capable of phagocytosis, an important RPE function. Isolated hES cell-derived RPE was able to transdifferentiate into cells of neuronal lineage and redifferentiate into RPE-like cells through multiple passages (30 Population doublings). Gene expression profiling demonstrated their higher similarity to primary RPE tissue than of existing human RPE cell lines D407 and ARPE-19, which has been shown to attenuate loss of visual function in animals. This is the first report of the isolation and characterization of putative RPE cells from hES cells, as well as the first application of transcriptomics to assess embryonic stem-cell derivatives and their in vivo counterparts--a "differentiomics" outlook. We describe for the first time, a differentiation system that does not require coculture with animal cells or factors, thus allowing the production of zoonoses-free RPE cells suitable for subretinal transplantation in patients with retinal degenerative diseases. With the further development of therapeutic cloning, or the creation of the banks of homozygous human leucocyte antigen (HLA) hES cells using parthenogenesis, RPE lines could be generated to overcome the problem of immune rejection and could be one of the nearest term applications of stem-cell technology.

‡Polycarbonates are renowned for their tremendous mechanical properties and particularly their resistance to high energy impacts making them ideal for a variety of aerospace applications. Little is known about polycarbonates interaction with the space environment, with ultraviolet radiation being of specific concern. Results are presented from experiments conducted that exam ine the interaction of polycarbonate materials with ultraviolet radiation conditions similar to those experienced in the low earth orbit environment. Principally, the effect of ultraviolet radiation in the UV -C portion of the spectrum, i.e. 100 - 280 nm, i s examined via an array of mercury vapor discharge lamps ( � =254nm) . The optical properties, namely the percentage transmittance and reflectance, of polycarbonate samples were examined before irradiation and after certain periods of UV -C treatment allowing a relationship between UV -C exposure time and the extent of degradation to be determined. The effect of extended UV -C irradiation upon the mechanical properties of polycarbonate samples has also been evaluated. Tensile tests were conducted on untreated sa mples and the results compared to samples exposed to UV -C for 503 hours. Significant yellowing of polycarbonate was evident as a result of the UV -C induced photo Fries reaction. However, the discovery of an exponentially decreasing relationship between th e change in transmittance and the exposure time suggests that this macroscopic effect stabilizes with time. Minimal deviation of the mechanical properties of polycarbonate as a result of exposure to 503 hours of UV -C was found when compared with virgin pol ycarbonate. The results obtained and the conclusions drawn from this investigation suggest that polycarbonate is a viable candidate for use in low earth orbit applications.

Nuclear transfer techniques have been proposed as a strategy for generating an unlimited supply of rejuvenated and histocompatible stem cells for the treatment of cardiac diseases. For this purpose, c-kit–positive fetal liver stem cells obtained from cloned embryos were injected in the border zone of infarcted mice to induce tissue reconstitution. Cloned embryos were derived from somatic cell fusion between nuclei of cultured LacZ-positive fibroblasts and enucleated oocytes of a different mouse strain. We report that regenerating myocardium replaced 38% of the scar at 1 month. The rebuilt tissue expressed LacZ and was composed of myocytes and vessels connected with the coronary circulation. Myocytes were functionally competent and expressed contractile proteins, desmin, connexin43, and N-cadherin. These structural characteristics indicated that the new myocytes were electrically and mechanically coupled. Similarly, the formed coronary arterioles and capillary structures contained blood and contributed, therefore, to tissue oxygenation. Cardiac replacement resulted in an improvement of ventricular hemodynamics and in a reduction of diastolic wall stress. These beneficial effects were obtained by stem cell transdifferentiation and commitment to the cardiac cell lineages. Myocardial growth was independent from fusion of the injected stem cells with preexisting partner cells. In conclusion, c-kit–positive stem cells derived by nuclear transfer cloning restore infarcted myocardium. Although problems currently plague nuclear transplantation, including the potential for epigenetic and imprinting abnormalities, stem cells derived from cloned embryos are sufficiently normal to repair damaged tissue in vivo. Importantly, the magnitude of myocardial regeneration obtained in this study is significantly superior to that achieved with adult bone marrow cells.

Parthenogenesis is the biological phenomenon by which embryonic development is initiated without male contribution. Whereas parthenogenesis is a common mode of reproduction in lower organisms, the mammalian parthenote fails to produce a successful pregnancy. We herein describein vitroparthenogenetic development of monkey (Macaca fascicularis) eggs to the blastocyst stage, and their use to create a pluripotent line of stem cells. These monkey stem cells (Cyno-1 cells) are positive for telomerase activity and are immunoreactive for alkaline phosphatase, octamer-binding transcription factor 4 (Oct-4), stage-specific embryonic antigen 4 (SSEA-4), tumor rejection antigen 1-60 (TRA 1-60), and tumor rejection antigen 1-81 (TRA 1-81) (traditional markers of human embryonic stem cells). They have a normal chromosome karyotype (40 + 2) and can be maintainedin vitroin an undifferentiated state for extended periods of time. Cyno-1 cells can be differentiatedin vitrointo dopaminergic and serotonergic neurons, contractile cardiomyocyte-like cells, smooth muscle, ciliated epithelia, and adipocytes. When Cyno-1 cells were injected into severe combined immunodeficient mice, teratomas with derivatives from all three embryonic germ layers were obtained. When grown on fibronectin/laminin-coated plates and in neural progenitor medium, Cyno-1 cells assume a neural precursor phenotype (immunoreactive for nestin). However, these cells remain proliferative and express no functional ion channels. When transferred to differentiation conditions, the nestin-positive precursors assume neuronal and epithelial morphologies. Over time, these cells acquire electrophysiological characteristics of functional neurons (appearance of tetrodotoxin-sensitive, voltage-dependent sodium channels). These results suggest that stem cells derived from the parthenogenetically activated nonhuman primate egg provide a potential source for autologous cell therapy in the female and bypass the need for creating a competent embryo.

Nuclear transplantation (therapeutic cloning) could theoretically provide a limitless source of cells for regenerative therapy. Although the cloned cells would carry the nuclear genome of the patient, the presence of mitochondria inherited from the recipient oocyte raises questions about the histocompatibility of the resulting cells. In this study, we created bioengineered tissues from cardiac, skeletal muscle, and renal cells cloned from adult bovine fibroblasts. Long-term viability was demonstrated after transplantation of the grafts into the nuclear donor animals. Reverse transcription-PCR (RT-PCR) and western blot analysis confirmed that the cloned tissues expressed tissue-specific mRNA and proteins while expressing a different mitochondrial DNA (mtDNA) haplotype. In addition to creating skeletal muscle and cardiac "patches", nuclear transplantation was used to generate functioning renal units that produced urinelike fluid and demonstrated unidirectional secretion and concentration of urea nitrogen and creatinine. Examination of the explanted renal devices revealed formation of organized glomeruli- and tubule-like structures. Delayed-type hypersensitivity (DTH) testing in vivo and Elispot analysis in vitro suggested that there was no rejection response to the cloned renal cells. The ability to generate histocompatible cells using cloning techniques addresses one of the major challenges in transplantation medicine.

Somatic cell nuclear 'reprogramming' in livestock species is now routine in many laboratories. Here, Robert Lanza, Jose Cibelli and Michael West discuss how these techniques may soon be used to clone genetically matched cells and tissues for transplantation into patients suffering from a wide range of disorders that result from tissue loss or dysfunction.

LifeMap Discovery™ provides investigators with an integrated database of embryonic development, stem cell biology and regenerative medicine. The hand-curated reconstruction of cell ontology with stem cell biology; including molecular, cellular, anatomical and disease-related information, provides efficient and easy-to-use, searchable research tools. The database collates in vivo and in vitro gene expression and guides translation from in vitro data to the clinical utility, and thus can be utilized as a powerful tool for research and discovery in stem cell biology, developmental biology, disease mechanisms and therapeutic discovery. LifeMap Discovery is freely available to academic nonprofit institutions at http://discovery.lifemapsc.com.

e22083 Background: Limitations of current screening mammography, particularly in younger women, demonstrate the need for an alternative breast cancer screening strategy. A non-invasive, easily interpreted and low cost test should address this need. Methods: Gene expression microarray analysis was carried out on 128 individual tumor samples representing over 20 tumor types, 86 samples representing 31 diverse normal tissue types, 68 tumor cell lines and 97 diverse normal primary cell cultures. Genes were ranked for elevated expression in either: i) a large number and variety of tumors relative to normal tissues, or ii) in breast tumors. Elevated expression was verified for a subset of genes using qPCR in a set of independent RNA samples. Proteins coded by genes elevated in breast cancer samples were analyzed in a retrospective training set of breast cancer patient sera samples with cancer-free patient and benign pathology controls using ELISA or bead-based detection assay. Results: Based on availability of suitable reagents, 25 candidate biomarkers were assessed in patient sera samples (31-227 patient samples per biomarker) using ELISA or bead-based assays. Individually, the performance of individual markers varied (ROC AUC, 0.51 - 0.88); however, when expression levels of the best performing markers were combined, the multiplex test demonstrated high-sensitivity (>80%) and specificity (>90%) in identifying early-stage breast cancer patients. Conclusions: A multiplex, proteomic-based approach may provide for a high-performance, blood-based screening diagnostic for breast cancer.

Fibroblast senescence is associated with a loss of proliferative potential and an alteration in extracellular gene expression. Because the expression of extracellular gene products are frequently growth state dependent, we undertook a comparative study of the regulation of the components of the plasminogen activation system in young and senescent cells under controlled conditions of growth. Young and senescent cells were compared in quiescent and activated growth conditions for the secretion of tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), plasminogen activator inhibitor-1 (PAI-1) and plasminogen activator inhibitor-2 (PAI-2). Whereas young ceils showed decreased levels of PAI-1 in the secreted and extracellular matrix pools upon serum deprivation, senescent cells showed a more constitutive pattern of gene expression, with no noticeable decrease of the levels in a low concentration of serum. RNA analysis revealed that senescent lung and skin cells, independent of the growth state, constitutively express levels of u-PA and PAI-1 comparable to the expression levels in young mitotically growing cells. These expression levels are down-regulated in quiescent young cells. In contrast, both t-PA and PAI-2 were markedly overexpressed in senescent skin lung cells under all growth conditions. Total plasminogen activator activity in conditioned medium was 50-fold higher in senescent-cell medium compared to young when cultured in 0.5% fetal calf serum (FCS) for five days, with the majority of the activity co-migrating on zymograms with u-PA. Increases in PAI-1 was also observed in senescent human umbilical vein endothelial cells. In summary, cells of various types display alterations in plasminogen activator activity during replicative senescence. The inappropriate over-expression of plasminogen activator activity in vivo may be expected to lead to a progressive disruption of extracellular matrix maintenance. Thus, our observations suggest that cellular replicative senescence is associated with an altered expression of several genes regulating tissue maintenance which, in turn, could lead to degenerative changes in tissue in age-related disease(s).

Eukaryotic chromosomes are capped with repetitive telomere sequences that protect the ends from damage and rearrangements. Telomere repeats are synthesized by telomerase, a ribonucleic acid (RNA)-protein complex. Here, the cloning of the RNA component of human telomerase, termed hTR, is described. The template region of hTR encompasses 11 nucleotides (5'-CUAACCCUAAC) complementary to the human telomere sequence (TTAGGG)n. Germline tissues and tumor cell lines expressed more hTR than normal somatic cells and tissues, which have no detectable telomerase activity. Human cell lines that expressed hTR mutated in the template region generated the predicted mutant telomerase activity. HeLa cells transfected with an antisense hTR lost telomeric DNA and began to die after 23 to 26 doublings. Thus, human telomerase is a critical enzyme for the long-term proliferation of immortal tumor cells.

Synthesis of DNA at chromosome ends by telomerase may be necessary for indefinite proliferation of human cells. A highly sensitive assay for measuring telomerase activity was developed. In cultured cells representing 18 different human tissues, 98 of 100 immortal and none of 22 mortal populations were positive for telomerase. Similarly, 90 of 101 biopsies representing 12 human tumor types and none of 50 normal somatic tissues were positive. Normal ovaries and testes were positive, but benign tumors such as fibroids were negative. Thus, telomerase appears to be stringently repressed in normal human somatic tissues but reactivated in cancer, where immortal cells are likely required to maintain tumor growth.

The possibility of cloning humans has raised questions as to whether nuclear transfer can be used to reproducibly generate healthy adult animals. Reports in the popular and scientific press on genetic, immunological, and other developmental problems raise the question of whether there are “any

We have developed a simian virus 40 (SV40) T-antigen immortalized human cell line, 1MR90-D305.2H4 (IDH4), in which the expression of T-antigen is controlled by the mouse mammary tumor virus (MMTV) promoter and thus regulated by steroids such as dexamethasone. Studies on the regulation of proliferation by T-antigen led to the formulation of a two-stage model for human cell immortalization, in which a mortality stage 1 mechanism (M1) was the target of T-antigen action, and an independent mortality stage 2 mechanism (M2) produced crisis and prevented T-antigen from directly immortalizing cells. Rarely, a cell expressing T-antigen escaped crisis (e.g., M2) and was capable of indefinite proliferation. This model predicted that the deinduction of T-antigen in IDH4 cells would lead to the reexpression of the M1 mechanism, and thus a reexpression of the senescent phenotype. Our study confirms the prediction that, in the absence of steroids, IDH4 cells express a variety of morphological and biochemical markers characteristic of normal senescent human fibroblasts.

A high density 'blue' mode has been observed when operating the Helicon Double Layer Thruster (HDLT) with xenon. Using a Langmuir probe and a retarding field energy analyser (RFEA), the plasma source and exhaust have been characterized at various radio frequency (RF) powers and operating pressures. When operating at low RF powers, the HDLT prototype is shown to be in a capacitively coupled mode. As the RF power is increased, a discrete mode transition occurs over a small RF power range (at about 625 W at 0.45 mTorr) and the plasma inside the source increases in density significantly and changes to a bright white/blue colour. This high density mode exhibits hysteresis, and radial measurements inside the source reveal a centrally peaked profile that is indicative of a helicon wave-sustained discharge. The quality, or Q, factor of the matching box is determined as a function of RF power and is shown to decrease in the high density mode, consistent with the increase in plasma density observed. The xenon exhaust of the HDLT prototype is investigated axially with the Langmuir probe and the RFEA and is shown to follow a Boltzmann expansion with an electron temperature of about 6 eV.

A high sensitivity momentum flux measuring instrument based on a compound pendulum has been developed for use with electric propulsion devices and radio frequency driven plasmas. A laser displacement system, which builds upon techniques used by the materials science community for surface stress measurements, is used to measure with high sensitivity the displacement of a target plate placed in a plasma thruster exhaust. The instrument has been installed inside a vacuum chamber and calibrated via two different methods and is able to measure forces in the range of 0.02-0.5 mN with a resolution of 15 microN. Measurements have been made of the force produced from the cold gas flow and with a discharge ignited using argon propellant. The plasma is generated using a Helicon Double Layer Thruster prototype. The instrument target is placed about 1 mean free path for ion-neutral charge exchange collisions downstream of the thruster exit. At this position, the plasma consists of a low density ion beam (10%) and a much larger downstream component (90%). The results are in good agreement with those determined from the plasma parameters measured with diagnostic probes. Measurements at various flow rates show that variations in ion beam velocity and plasma density and the resulting momentum flux can be measured with this instrument. The instrument target is a simple, low cost device, and since the laser displacement system used is located outside the vacuum chamber, the measurement technique is free from radio frequency interference and thermal effects. It could be used to measure the thrust in the exhaust of other electric propulsion devices and the momentum flux of ion beams formed by expanding plasmas or fusion experiments.

Parthenogenesis is the process by which an egg can develop into an embryo in the absence of sperm. This process has been characterized to some extent in nonhuman primates ([1][1], [2][2]); however, to date, no primate parthenogenetic embryonic stem (ES) cell lines have been derived. Although

Human embryonic stem (hES) cells are unique in their demonstrated potential to differentiate into all cell lineages. Reports by T. Wakayama et al. (“Differentiation of embryonic stem cell lines generated from adult somatic cells by nuclear transfer,” 27 Apr., p. [740][1]) and N. Lumelsky et al. (“Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets, “ScienceExpress, [26 Apr., 10.1126][2]) testify to the enormous promise of ES cell research. The editorial “Disappearing stem cells, disappearing science,” by Irving L. Weissman and David Baltimore (27 Apr., p. [601][3]) emphasizes the implications of this research for human health. Weissman and Baltimore point out that hES cells are currently the most promising source of cells for tissue regeneration research. They also note that this area has enormous potential for shedding light on some of the greatest mysteries of early human development. During this same week, the U.S. Department of Health and Human Services suddenly asked the National Institutes of Health (NIH) to cancel a planned first meeting of a committee that was to review applications from scientists seeking federal funds for hES cell research. This announcement heightens concerns that the Bush administration may eventually block implementation of the NIH's guidelines supporting this research. We hope that these fears are groundless and that the Bush administration will use this additional time to move toward support of the guidelines. Whatever the outcome, however, these delays have a real cost in terms of human suffering. According to data from the Centers for Disease Control's National Center for Health Statistics, approximately 3,000 Americans die every day from diseases that in the future may be treatable with ES-derived cells and tissues. We believe that these urgent health needs provide strong moral grounds for pursuing ES cell research. In addition, at least three ethical considerations recommend federal funding for this research. First, withdrawal of support will slow this research, but not stop it from going forward. Private organizations and overseas researchers will fill the void. In some cases, they will do so without the kinds of comprehensive ethical oversight provided by U.S. human subjects regulations. Second, prohibiting such funding will not prevent the destruction of embryos. Each year, thousands of spare embryos created in infertility procedures are routinely destroyed at the request of their progenitors. A very small number of these embryos could be used to produce immortal stem cell lines that could be grown and used for research without ever using more embryos. The relevant ethical question is whether these spare embryos will simply be thrown away or used for human benefit. Third, and finally, we note that the United States is a religiously and ethically pluralistic nation. Many of those who oppose ES cell research base their position on religious views not shared by other citizens. As much as possible, the government should try to avoid taking sides in these debates and confine itself to policies that promote public health and welfare. ES cell research within the framework of the NIH guidelines is such a policy. [1]: /lookup/doi/10.1126/science.1059399 [2]: http://www.sciencemag.org/cgi/content/short/1058866v1 [3]: /lookup/doi/10.1126/science.292.5517.601

Last month, 70 members of the U.S. Congress, including Henry Hyde, Chairman of the House Judiciary Committee, and J. C. Watts Jr. Republican Conference Chairman, signed a letter urging the federal government to ban all research on stem cells obtained from human embryos and fetuses. The letter calls upon the U.S. Department of Health and Human Services (DHHS) to reverse National Institutes of Health (NIH) Director Harold Varmus's decision to allow funding of pluripotent stem cell research. The lawmakers object “in the strongest possible terms” to Varmus's decision, as well as to the memorandum issued in January by DHHS General Counsel Harriet Rabb, which served as the legal basis for Varmus's position. In their letter, the members of Congress state, “Any NIH action to initiate funding of such research would violate both the letter and spirit of the federal law banning federal support for research in which human embryos are harmed or destroyed.” Federal laws and regulations, they claim, have protected human embryos and fetuses “from harmful experimentation at the hands of the Federal government” for more than two decades. “This area of law has provided a bulwark against government's misuse and exploitation of human beings in the name of medical progress. It would he a travesty for this Administration to attempt to unravel this accepted ethical standard.”

Background: The dramatic alterations in the appearance of the integument with increasing age are due in part to a progressive destruction of the delicate architecture of the connective tissue components of the dermis. Both collagenous and elastic components display a degeneration consistent with the overexpression of proteolytic activity. Recent advances in the field of molecular gerontology, using in vitro models of cellular aging, are yielding clues as to the fundamental causes of dermal aging. Observations: Dermal fibroblasts possess a finite replicative capacity of 50 to 100 doublings, then cease replicating in response to growth factors. Cells cultivated to the end of their replicative lifespan in vitro display alterations consistent with their playing a role in aging in vivo. In particular, senescent dermal fibroblasts overexpress metalloproteinase activities that may explain the age-related atrophy of extracellular matrix architecture. Conclusions: The recent discovery of a structural change in the telomeric region of the genome with cellular aging and new insights into DNA damage checkpoint mechanisms offer new opportunities to uncover both the molecular mechanisms regulating cellular aging and possibly to devise new strategies to manipulate these molecular events for therapeutic effect. (Arch Dermatol. 1994;130:87-95)

The atrophy of extracellular matrix is a common event during the aging of connective tissues. In this study, we tested the hypothesis that the altered ability of senescent cells to be modulated by serum growth factors correlated with a loss of regulation of collagenase synthesis. We examined the levels of immunoreactive procollagenase and collagenase inhibitor (the tissue inhibitor of metalloproteinases, TIMP) associated with young and senescent fibroblasts cultured in vitro. Young fibroblasts cultured in low (0.5%) concentrations of fetal bovine serum respond to increased (10%) serum by increasing levels of procollagenase and TIMP beginning 4.0 h after serum stimulation. In contrast, senescent fibroblasts constitutively produce relatively high levels of procollagenase even when cultured in low levels of serum and do not respond to serum stimulation by increasing procollagenase synthesis. In addition, senescent fibroblasts constitutively express a relatively small amount of TIMP which is not induced upon serum stimulation. This altered expression of collagenase and TIMP appears unique to the senescent phenotype and not merely a result of growth inhibition, since young cells growth arrested by density-dependent growth inhibition displayed a temporal pattern of procollagenase and TIMP expression upon serum stimulation similar to that of subconfluent young cultures. An assay of net collagenase activity revealed a greater than 20-fold elevation of activity in trypsin-activated extracts from senescent versus young fibroblasts when cultured in a low concentration of fetal bovine serum. These results demonstrate for the first time a direct correlation between alterations in the molecular pathways regulating connective tissue homeostasis and those of replicative senescence. The increased collagenolytic activity of senescent compared to young fibroblasts cultured in the presence of a low serum concentration suggests that aging fibroblasts may become increasingly fibroclastic causing many of the age-associated alterations in dermal collagen observed during aging in vivo.

Lactic dehydrogenase (LD), an enzyme which can catalyze the conversion of lactate to pyruvate, has been demonstrated in the serum of animals1-3and humans.4-6Hill and Levi4studied 51 patients with neoplastic disease, only 2 of whom failed to exhibit elevated serum levels of this enzyme. Bierman and his associates5found elevated levels in 121 out of 132 patients with carcinoma. In an earlier report7from this laboratory, elevated LD levels were noted in half of 30 patients with carcinoma. Serum glutamic oxalacetic transaminase (GOT), an enzyme which catalyzes the reversible transfer of an amino group from aspartate to α-ketoglutarate has also been studied in neoplastic disease. Wroblewski and LaDue8reported elevated serum GOT levels in 23 of 25 patients with proved carcinomatosis involvement of the liver. Subsequently, these workers reported elevations of GOT in 75 out of 100 patients with primary hepatoma or

Keywords: longevity ; aging ; biomarkers ; geroprotectors ; epigenetics ; transcriptomics Reference EPFL-ARTICLE-227576doi:10.18632/aging.101163View record in Web of Science Record created on 2017-05-01, modified on 2017-05-26

• A 67-year-old woman with a history of thrombophlebitis had been taking warfarin sodium for 1½ years when she developed multiple skin lesions resembling warfarin-induced skin necrosis or purpura fulminans. Despite discontinuing the warfarin and administering prednisone, the lesions increased in size. Disseminated intravascular coagulation (DIC) was found and resolved with heparin sodium therapy, and her skin lesions healed. This patient was believed to have an atypical form of purpura fulminans rather than warfarin-induced skin necrosis because of the duration of warfarin therapy prior to the illness, the presence of DIC, and the dramatic resolution with heparin. A monoclonal (IgG) gammopathy was found, which stabilized as the skin lesions resolved, and fulfilled the criteria for a benign (asymptomatic) monoclonal gammopathy. ( Arch Dermatol 114:578-580, 1978)

Excerpt Several published studies1, 2, 3, 4have shown that patients with megaloblastic anemia have markedly elevated serum levels of lactic dehydrogenase. The sera of patients with sickle cell anem...

Nitrofurantoin (Furadantin), N-(5-nitro-2-furfurylidene)-1-aminohydantoin, a drug related to the antimicrobial nitrofurans, has been employed in the past few years in the treatment of acute and chronic urinary infections. Side-effects include nausea, occasionally accompanied by vomiting, diarrhea, pyrosis, headaches, and urticara. No ill-effects on the liver or kidney have been reported. There is one reported instance of leukopenia after the use of nitrofurantoin.1There has been no other report of hematological abnormality during its use. The case reported below is thought to represent the development of hemolytic anemia with the use of this drug. Report of a Case A 28-year-old Negro man was admitted to the hospital on Sept. 2, 1955, with the complaint of a swollen, tender penis for approximately two weeks. At the onset of this swelling he had received two injections of penicillin, but had used no other drugs prior to admission to the hospital. A history of