Viable offspring derived from fetal and adult mammalian cells

Fertilization of mammalian eggs is followed by successive cell divisions and progressive differentiation, first into the early embryo and subsequently into all of the cell types that make up the adult animal. Transfer of a single nucleus at a specific stage of development, to an enucleated unfertilized egg, provided an opportunity to investigate whether cellular differentiation to that stage involved irreversible genetic modification. The first offspring to develop from a differentiated cell were born after nuclear transfer from an embryo-derived cell line that had been induced to become quiescent. Using the same procedure, we now report the birth of live lambs from three new cell populations established from adult mammary gland, fetus and embryo. The fact that a lamb was derived from an adult cell confirms that differentiation of that cell did not involve the irreversible modification of genetic material required for development to term. The birth of lambs from differentiated fetal and adult cells also reinforces previous speculation that by inducing donor cells to become quiescent it will be possible to obtain normal development from a wide variety of differentiated cells.     

A study of the ion selectivity and the kinetic properties of the calcium dependent slow inward current in mammalian cardiac muscle

1. A voltage-clamp method combining a single surcose gap and two intracellular micro-electrodes was used to measure membrane currents in ventricullar myocardial fibres. 2. The adequacy of the voltage-clamp method is demonstrated by comparing the total current, It, across the gap with the voltage difference, delta V, between the two intracellular micro-electrodes, i.e. another independent way of measuring membrane currents. With both current measurements the slow inward current, Is, shows the same voltage- and time-dependences. 3. The sensitivity of the slow inward current to variation in external Ca and Na concentrations was investigated systematically. The reversal potential of the slow inward current was sensitive to variation of both ion species. 4. From the reversal potential measurements relative permeabilities of the conductance channels of the slow inward current were estimated as PCa/PNa approximately 1/0-01 and PCa/PK approximately 1/0-01 by means of the constant field equation. 5. The activation and inactivation kinetics of the slow inward current were explored in detail and related to the plateau of the action potential.     

Myosin light chains (MLCs) heterogeneity in mammalian smooth and cardiac muscle

Tuberous sclerosis (TS) is well known to be occasionally associated with subependymal giant cell astrocytoma (SGCA). SGCA is considered to be a benign tumor in its clinical course and morphology. However, this tumor is grown sometimes so rapid and caused hydrocephalus. To our knowledge, little is known about hemocirculation and metabolism, particularly in relation with proliferating activity of TS and SGCA. We measured hemocirculation and metabolism of SGCA developed in a case of TS using positron emission tomography (PET). A 13-year-old-boy who had frequently developed convulsions four months after birth. He was diagnosed as TS and had been medically treated with anticonvulsants, since multiple intraventricular calcifications were detected by CT, at the age of five months. The convulsions had been well controlled. In March 1993, he presented with syncopal attack and admitted to our hospital. CT showed multiple subependymal nodules. Among the nodules, one of the left anterior horn exceeded 2cm in size obliterated Monro's foramen. The tumor was homogeneously enhanced with contrast medium. The lesion detected by postcontrast T1-weighted MR imaging had almost the same status as that by CT. T2-weighted image revealed cortical tubers as high intensity area at the left frontal and parietooccipital regions. PET was performed with the Headtome IV. Hemocirculation of the tumor was lower than that of contralateral gray matter, which suggested poor blood supply. The oxygen and glucose metabolism of the tumor were decreased compared with contralateral gray matter, indicative of a low activity of proliferation and a clinically benign tumor in the present case.(ABSTRACT TRUNCATED AT 250 WORDS)     

Constitutive properties of hard-alpha titanium

The flow and fracture behavior of hard-alpha Ti was studied as a function of nitrogen content, stress state, strain rate, and temperature. Hard-alpha Ti specimens with nitrogen contents ranging from 2 to 11.6 wt pct were fabricated by powder-metallurgy techniques. Stress-strain curves were obtained under various states of stress by performing uniaxial compression, indirection tension, indentation, and plane-strain compression tests at two strain rates. By varying the test technique and the specimen geometry, deformation and fracture in hard-alpha Ti was characterized for mean pressures as high as 6 times the flow stress. Most of these tests were conducted at 954 °C, but some were performed at 25 °C, 927 °C, and 982 °C. The experimental results indicated that, during compression testing at 927 °C to 982 °C, hard-alpha Ti exhibited substantial plastic deformation for nitrogen contents less than 4 wt pct, but showed brittle fracture with little plastic flow for nitrogen contents of 5.5 to 11.6 wt pct. Both the yield and fracture strengths increase with increasing nitrogen content and pressure, but decrease with increasing temperature. The yield strength increases with strain rate, while the fracture strength shows little or no rate sensitivity. The fracture strength in tension is substantially lower than that in compression. These observed deformation and fracture characteristics are explained on the basis of microcrack formation during inelastic deformation.     

Constitutive chemokine production results in activation of leukocyte function-associated antigen-1 on adult T-cell leukemia cells

Adult T-cell leukemia (ATL) is characterized by massive infiltration of circulating ATL cells into a variety of tissues, a finding often associated with poor prognosis. Leukocyte migration from circulation into tissue depends on integrin-mediated adhesion to endothelium, and integrins are tightly regulated by several stimuli, such as inflammatory chemokines. However, the exact mechanisms that enhance adherence of leukemic cells to the endothelium and infiltration into tissues remain to be fully understood. We investigated the mechanisms of extravasation of leukemic cells using ATL cells and report the following novel features of endogenous chemokine-induced adhesion of ATL cells to the endothelium. ATL cells spontaneously adhered to endothelial cells without exogenous stimulation. Integrin leukocyte function-associated antigen-1 (LFA-1) on ATL cells was spontaneously activated. ATL cells produced high amounts of chemokines, macrophage inflammatory protein-1alpha (MIP-1alpha), and MIP-1beta. Adhesion of ATL cells to endothelial cells and the expression of activated form of LFA-1 were reduced by pretreatment with pertussis toxin, wortmannin, or anti-MIP-1alpha and MIP-1beta antibodies or transfection with antisense of MIP-1alpha or MIP-1beta. Spontaneous polymerization of cytoskeletal F-actin was observed in ATL cells, which was also inhibited by pertussis toxin and wortmannin. We propose that ATL cells adhere to endothelial cells through an adhesion cascade similar to normal leukocytes and that the chemokines produced by ATL cells are involved in triggering integrin LFA-1 through cytoskeletal rearrangement induced by G-protein-dependent activation of phosphoinositide 3-kinases in an autocrine manner. These events result in a strong adhesion of ATL cells to the endothelium and spontaneous transendothelial migration.     

The cytoskeleton in skeletal, cardiac and smooth muscle cells

The muscle cell cytoskeleton consists of proteins or structures whose primary function is to link, anchor or tether structural components inside the cell. Two important attributes of the cytoskeleton are strength of the various attachments and flexibility to accommodate the changes in cell geometry that occur during contraction. In striated muscle cells, extramyofibrillar and intramyofibrillar domains of the cytoskeleton have been identified. Evidence of the extramyofibrillar cytoskeleton is seen at the cytoplasmic face of the sarcolemma in striated muscle where vinculin- and dystrophin-rich costameres adjacent to sarcomeric Z lines anchor intermediate filaments that span from peripheral myofibrils to the sarcolemma. Intermediate filaments also link Z lines of adjacent myofibrils and may, in some muscles, link successive Z lines within a myofibril at the surface of the myofibril. The intramyofibrillar cytoskeletal domain includes elastic titin filaments from adjacent sarcomeres that are anchored in the Z line and continue through the M line at the center of the sarcomere; inelastic nebulin filaments also anchored in the Z line and co-extensible with thin filaments; the Z line, which also anchors thin filaments from adjacent sarcomeres; and the M line, which forms bridges between the centers of adjacent thick filaments. In smooth muscle, the cytoskeleton includes adherens junctions at the cytoplasmic face of the sarcolemma, which anchor beta-actin filaments and intermediate filaments of the cytoskeleton, and dense bodies in the cytoplasm, which also anchor actin filaments and intermediate filaments and which may be the interface between cytoskeletal and contractile elements.     

Functional development of intrinsic properties in ganglion cells of the mammalian retina

Senosory neurons manifest pronounced changes in excitability during maturation, but the factors contributing to this ubiquitous developmental phenomenon are not well understood. To assess the contribution of intrinsic membrane properties to such changes in excitability, in the present study whole cell patch-clamp recordings were made from developing ganglion cells in the intact retina of postnatal rats. During a relatively brief developmental period (postnatal days P7-P27) ganglion cells exhibited pronounced changes in the discharge patterns generated by depolarizing current injections. The youngest cells (P7-P17) typically responded to maintained depolarizations with only a single spike or a rapidly adapting discharge pattern. In contrast, the predominant response mode of more mature cells (P21-P27) was a series of repetitive discharges that lasted for the duration of the depolarization period, and by P25 all cells responded in this manner. These functional changes characterized all three morphologically defined cell classes identified by intracellular labeling with Lucifer yellow. To determine if expression of the potassium current (Ia) and the kinetics of the Na-channel related to the increased excitability of developing ganglion cells described above, current- and voltage-clamp recordings were made from individual neurons. The different firing patterns manifested by developing retinal ganglion cells did not reflect the presence or absence of the Ia conductance, although cells expressing Ia tended to generate spikes of shorter duration. With maturation the speed of recovery from inactivation of the Na current increased markedly and this related to the increased excitability of developing ganglion cells. Neurons yielding only a single spike to maintained depolarization were characterized by the slowest speed of recovery; cells with rapidly adapting discharges showed a faster recovery and those capable of repetitive firing recovered fastest from Na-channel inactivation. It is suggested that these changes in intrinsic membrane properties may relate to the different functional roles subserved by ganglion cells during development.     

Receptive-field properties of adult cat's retinal ganglion cells with regenerated axons

Receptive-field properties of retinal ganglion cells (RGCs) that had regenerated their axons were studied by recording single-unit activity from strands teased from peripheral nerve (PN) grafts apposed to the cut optic nerve in adult cats. Of the 286 visually responsive units recorded from PN grafts in 20 cats, 49.7% were classified, according to their receptive-field properties, as Y-cells, 39.5% as X-cells, 6.6% as W-cells, and 4.2% were unclassified. The predominant representation of Y-cells is consistent with a corresponding morphological study (Watanabe et al. 1993a), which identified alpha-cells as the RGC type with the largest proportion of regenerating axons. Among the X-cells, we only found ON-center types, whereas both ON-center and OFF-center Y-cells were found. As in intact retinas, the receptive-field center sizes of Y-cells and W-cells were larger than those of X-cells at corresponding displacements from the area centralis. Within the 10 degrees surrounding the area centralis, the receptive fields of X-cells with regenerated axons were larger than those in intact retinas, suggesting that some rearrangement of retinal circuitry occurred as a consequence of degeneration and regeneration. Receptive-field center responses of Y-, X-, and W-type units with regenerated axons were similar to those found in intact retinas, but the level of spontaneous activity of Y- and X-type units was, in general, less than that of intact RGCs. Receptive-field surrounds were weak or not detected in more than half of the visually responsive RGCs with regenerated axons.     

Heterogeneity among muscle precursor cells in adult skeletal muscles with differing regenerative capacities

Skeletal muscle has a remarkable capacity to regenerate after injury, although studies of muscle regeneration have heretofore been limited almost exclusively to limb musculature. Muscle precursor cells in skeletal muscle are responsible for the repair of damaged muscle. Heterogeneity exists in the growth and differentiation properties of muscle precursor cell (myoblast) populations throughout limb development but whether the muscle precursor cells differ among adult skeletal muscles is unknown. Such heterogeneity among myoblasts in the adult may give rise to skeletal muscles with different regenerative capacities. Here we compare the regenerative response of a masticatory muscle, the masseter, to that of limb muscles. After exogenous trauma (freeze or crush injuries), masseter muscle regenerated much less effectively than limb muscle. In limb muscle, normal architecture was restored 12 days after injury, whereas in masseter muscle, minimal regeneration occurred during the same time period. Indeed, at late time points, masseter muscles exhibited increased fibrous connective tissue in the region of damage, evidence of ineffective muscle regeneration. Similarly, in response to endogenous muscle injury due to a muscular dystrophy, widespread evidence of impaired regeneration was present in masseter muscle but not in limb muscle. To explore the cellular basis of these different regenerative capacities, we analyzed the myoblast populations of limb and masseter muscles both in vivo and in vitro. From in vivo analyses, the number of myoblasts in regenerating muscle was less in masseter compared with limb muscle. Assessment of population growth in vitro indicated that masseter myoblasts grow more slowly than limb myoblasts under identical conditions. We conclude that the impaired regeneration in masseter muscles is due to differences in the intrinsic myoblast populations compared to limb muscles.     

In vivo clonal analyses reveal the properties of endogenous neural stem cell proliferation in the adult mammalian forebrain

The adult mammalian forebrain contains a population of multipotential neural stem cells in the subependyma of the lateral ventricles whose progeny are the constitutively proliferating cells, which divide actively throughout life. The adult mammalian brain is ideal for examining the kinetics of the stem cells due to their strict spatial localization and the limited and discrete type of progeny generated (constitutively proliferating cells). Clonal lineage analyses 6 days after retrovirus infection revealed that under baseline conditions 60% of the constitutively proliferating cells undergo cell death, 25% migrate to the olfactory bulb and 15% remain confined to the lateral ventricle subependyma (where they reside for approximately 15 days). Analysis of single cell clones 31 days after retroviral infection revealed that the stem cell divides asymmetrically to self-renew and give rise to constitutively proliferating cells. Following repopulation of the depleted subependyma the average clone size is 2.8 times larger than control, yet the absolute number of cells migrating to the olfactory bulb is maintained and the stem cell retains its asymmetric mode of division. The number of neural stem cells in the adult forebrain 33 days after repopulation of the subependyma was estimated using bromodeoxyuridine labeling of subepenydmal cells. There were calculated to be 1200-1300 cells between the rostral corpus callosum and rostral anterior commissure; these data support a lineage model similar to those based on stem cell behavior in other tissue types.