Biomedical subjects
G E Davis
Publications and source records attributed to G E Davis.
Lowry protein assay using an automatic microtiter plate spectrophotometer.
The method of protein determination reported by Lowry et al. (1951, J. Biol. Chem. 193, 265-275) has been adapted for use with 96-well microtiter plates and an automatic microplate spectrophotometer. The spectrophotometer has been interfaced with a computer which plots the standard curve and calculates the protein content of each sample. The adapted method offers advantages over previously reported methods in that it is more rapid and uses a smaller sample volume (100 microliters) for samples containing 3-300 micrograms/ml (0.3-30 micrograms/assay) of protein. The method of Bensadoun and Weinstein (1976, Anal. Biochem. 70, 241-252) for precipitating microgram amounts of protein away from substances which interfere with the Lowry assay has also been adapted to this microplate procedure. These techniques should be particularly useful for laboratories where large numbers of samples containing a wide range of protein concentrations are assayed.
Mapping of domains in human laminin using monoclonal antibodies: localization of the neurite-promoting site.
Monoclonal antibodies were made against a truncated form of human laminin isolated from placenta. 12 antibodies were isolated and characterized. All antibodies stained basement membranes in placenta and immunoprecipitated laminin from media of cultured choriocarcinoma cells. Three antibodies, 3E5, 4C7, and 4E10, partially blocked the neurite-promoting activity of laminin. Addition of a second antibody, goat anti-mouse IgG, caused more complete blocking of the activity. Two of the blocking antibodies, 4C7 and 4E10, reacted with epitopes within the globular domain at the end of the long arm of laminin, and the third one, 3E5, reacted at the end of the rod-like portion of the long arm adjacent to the globular domain, as shown by electron microscopy after rotary shadowing. Five nonblocking antibodies used in the same test reacted with epitopes in other domains of the molecule. Blocking antibodies 3E5 and 4E10 could be used in immunoblotting and both antibodies reacted with the same polypeptides in pepsin fragments of human laminin, the predominant polypeptides being approximately 400 kD. When a crude extract of human amnion was used as a source of intact laminin, the 4E10 antibody detected a single polypeptide of approximately 400 kD. A nonblocking antibody, 2E8, which reacted at the center of the laminin cross, reacted predominantly with a 200-kD polypeptide in human laminin fragments and exclusively with a 200-kD polypeptide in amnion extract and in rat laminin. Our results with human laminin match the results by Edgar, D., R. Timpl, and H. Thoenen, 1984, EMBO (Eur. Mol. Biol. Organ.) J., 3:1463-1468, in which the neurite-promoting activity of mouse laminin resides at the end of the long arm, which is also the site for heparin binding. However, since the active fragments of human laminin did not bind to heparin, the neurite-promoting site should be different from the heparin-binding site. Our results further suggest that the neurite-promoting site may be contained in or close to the 400-kD component of laminin.
Characterization of a laminin-containing neurite-promoting factor and a neuronotrophic factor from peripheral nerve and related sources.
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Characteristics of life events during adolescence.
Two studies of the characteristics of life events during adolescence are reported. In the first, open-ended reports of major life events and daily events were obtained from early, middle, and late adolescents. Analyses of the frequency of past events perceived as positive versus negative revealed effects for type of event (major vs. daily), age, and gender. In contrast, anticipated events were perceived similarly across age and gender. The second study examined judgments by older adolescents of the valence, type, and domain of life events. Consensus regarding these characteristics was achieved on only a small portion of events. The implications of these findings for the measurement of life events during adolescence are highlighted.
Tumor-promoting phorbol diester mimics two distinct neuronotrophic factors.
The two purified neuronotrophic factor proteins, nerve growth factor (NGF) and ciliary neuronotrophic factor (CNTF), differ in molecular properties and in their action on certain neuronal cell types. The survival in culture of dissociated chick embryonic day 8 dorsal root ganglionic neurons is supported by NGF and day 8 ciliary ganglionic neurons by CNTF, but neither factor can be a substitute for the other. Here we report that a known tumor-promoting phorbol diester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), can support the survival of both neuronal types in the absence of either NGF or CNTF and does so with the same efficacy as the corresponding trophic factor. The inactive phorbol derivative, 4-alpha-phorbol-didecanoate, does not support either neuronal type. The combined provision of suboptimal doses of TPA and NGF or CNTF does not reveal synergistic effects. These results suggest that TPA, NGF and CNTF may exert their neuronal survival promoting influences through a common molecular mechanism.
Parameters of neuritic growth from ciliary ganglion neurons in vitro: influence of laminin, schwannoma polyornithine-binding neurite promoting factor and ciliary neuronotrophic factor.
Ciliary ganglion neurons extend neuritic processes when cultured for 24 h in medium containing ciliary neuronotrophic factor (CNTF) and on a polyornithine substratum precoated with either laminin or a Schwannoma-derived neurite promoting factor (PNPF). We have examined the roles of laminin, PNPF and CNTF for each of four parameters of neuritic growth, including: initiation time, neuronal polarity, neuritic branching and average neurite output (lengths) with time. Increasing laminin and PNPF levels were found to advance the time of neurite initiation as well as shift the majority (70-80%) of the neurons from a unipolar to multipolar neuritic morphology. The polarity imposed by any given concentration of either neurite promoting factor remained constant over the 24 h culture period examined. The average lengths from the longest neurites per neuron over a 10-28 h culture interval were not affected by increasing levels of laminin or PNPF, but total neuritic output per neuron was increased. This increased total neuritic output could be attributed to a combination of earlier neuritic initiation time and an increased neuronal polarity at high laminin or PNPF levels. CNTF at threshold survival levels did not promote initiation time, neuronal polarity or total neuritic output. However, cultures receiving less CNTF than that required for maximal neuronal survival displayed an increased neuronal polarity and a reduced neuritic output before any apparent loss of neurons. Neuritic branching was not affected by either the neurite promoting or trophic factors after 24 h of culture. Laminin and PNPF were found to be indistinguishable in their effects on the ciliary ganglion neurons in each of the four parameters studied.
Comparison of the effects of laminin and the polyornithine-binding neurite promoting factor from RN22 Schwannoma cells on neurite regeneration from cultured newborn and adult rat dorsal root ganglion neurons.
We have investigated the effects of two neurite promoting factors (NPFs)--laminin and the semipurified polyornithine-binding neurite promoting factor (PNPF-1) from RN 22 Schwannoma cells--on neurite regeneration from dissociated newborn and adult rat dorsal root ganglion (DRG) neurons during 24 and 48 h culture periods in the absence of exogenous neuronotrophic factors. Both laminin and PNPF, when used to pretreat the polyornithine substratum, significantly enhanced neurite recruitment from surviving newborn and adult DRG neurons as compared to an untreated polyornithine substratum. However, the responses of newborn neurons at saturating concentrations of laminin and PNPF were consistently greater (46% neurite-bearing cells at 24 h, 81% at 48 h) than those of adult neurons (14 and 45%, respectively). The responsive neurons of both newborn and adult DRG displayed extensive neuritic networks at 48 h. The ED50 of laminin, or PNPF was 0.15-0.2 micrograms/ml for both newborn and adult neurons. The similarities in the responses of newborn and adult DRG neurons to NPFs validate the use of neurons from embryonic and newborn animals for the in vitro assays of NPFs that can be collected from injured and regenerating adult peripheral nervous tissues.
Purified proteins acting on cultured chick embryo ciliary ganglion neurons.
Chick embryo ciliary ganglion neurons in dissociated monolayer culture have been used to examine molecular requirements for neuronal survival and neurite growth. These neurons will rapidly die in vitro unless supplied with an adequate level of ciliary neuronotrophic factor (CNTF), and even in the presence of CNTF they will not vigorously extend neurites on polyornithine substrata unless supplied with appropriate amounts of polyornithine-binding neurite-promoting factors (PNPFs). Recent work on the purification and partial characterization of embryonic chick eye CNTF and rat schwannoma PNPF is reviewed, and in vitro responses of ciliary ganglion neurons to other purified proteins such as laminin, fibronectin, insulin, and nerve growth factor are mentioned.
Isolation and characterization of rat schwannoma neurite-promoting factor: evidence that the factor contains laminin.
Rat RN22 schwannoma cells in vitro release into their growth medium a macromolecular factor that, when bound to polyornithine-coated culture substrata, will stimulate neuritic regeneration from axotomized peripheral and central neurons. During the purification of this factor, the neurite-promoting activity co-purifies with laminin immunoreactivity as measured by an enzyme-linked immunoadsorbent assay. The purified factor has an immunoreactivity per milligram of protein similar to that of purified rat yolk sac tumor laminin. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, the purified factor exhibits a major band at 200 kilodaltons (kD) and two minor ones at about 130 and 35 kD. The 200-kD band comigrates with the 200-kD band of purified rat laminin. After SDS-PAGE under non-reducing conditions, the rat schwannoma factor and rat laminin both exhibit a band in the 900-kD range with the schwannoma factor band migrating slightly faster than the laminin one. The 200-kD (reducing conditions) and 900-kD (non-reducing conditions) bands of both the schwannoma factor and laminin are stained by immunoblotting with antisera raised against rat and human laminin and against a partially purified preparation of the schwannoma factor. On immunoblots the 400-kD band of laminin (a band not seen in the schwannoma factor preparation) also stains with all three antisera. When the antibodies from each of the three antisera are immobilized on protein A-agarose beads, the beads will completely remove from solution the neurite-promoting activities of both the schwannoma factor and laminin. Antibodies raised against rat laminin fail to block the neurite-promoting activity of the purified schwannoma factor but totally block that of rat laminin. In contrast, antibodies raised against the schwannoma factor will block the neurite-promoting activities of both the schwannoma factor and laminin. By rotary shadowing electron microscopy the schwannoma factor preparation exhibits cross-shaped images similar but not identical to those previously reported for rat and mouse laminin. In addition, the schwannoma factor preparation contains images resembling proteoglycans.
Localized survival of ciliary ganglionic neurons identifies neuronotrophic factor bands on nitrocellulose blots.
A novel and sensitive method has been developed to identify ciliary neuronotrophic factors (CNTFs) from tissue extracts after blotting to nitrocellulose paper. The CNTF proteins are required for the in vitro survival of embryonic chick ciliary ganglionic neurons. Tissue extracts containing such CNTFs are electrophoresed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose paper. Purified ciliary ganglionic neurons are seeded on the surface of the nitrocellulose blot, and the culture is incubated for 24 hr in medium lacking CNTF. CNTF can be localized on the blot because it retains its ability to support the survival of the neurons cultured on the nitrocellulose. A band of viable neurons, easily visualized by staining with a vital dye, is supported by the blotted CNTF polypeptide. The number of neurons surviving on the blotted CNTF is related to the amount of CNTF originally loaded on the electrophoretic gel. As little as 2 ng (16 trophic units) of CNTF protein contained in crude tissue extracts can be loaded on the sodium dodecyl sulfate gel and still be recognized by the cultured neurons. This method was used to identify CNTF polypeptides from extracts of adult rat nerve (24,000 and 19,000 daltons) and from tissue found near experimentally induced adult rat brain lesions (24,000 daltons). The electrophoretic mobilities of these peptides are distinct from the previously purified chick eye CNTF polypeptide (20,400 daltons).
Neurite-promoting factors and extracellular matrix components accumulating in vivo within nerve regeneration chambers.
The outgrowth of neurites from cultured neurons can be induced by the extracellular matrix glycoproteins, fibronectin and laminin, and by polyornithine-binding neurite-promoting factors (NPFs) derived from culture media conditioned by Schwann, or other cultured cells. We have examined the occurrence of fibronectin, laminin and NPFs during peripheral nerve regeneration in vivo. A previously established model of peripheral nerve regeneration was used in which a transected rat sciatic nerve regenerates through a silicone chamber bridging a 10 mm interstump gap. The distribution of fibronectin and laminin during regeneration was assessed by indirect immunofluorescence. Seven days after nerve transection the regenerating structure within the chamber consisted primarily of a fibrous matrix which stained with anti-fibronectin but not anti-laminin. At 14 days, cellular outgrowths from the proximal and distal stumps (along which neurites grow) had entered the fibronectin-containing matrix, consistent with a role of fibronectin in promoting cell migration. Within these outgrowths non-vascular as well as vascular cells stained with anti-fibronectin and anti-laminin. Within the degenerated distal nerve segment, cell characteristic of Bungner bands (rows of Schwann cells along which regenerating neurites extend) stained with anti-fibronectin and laminin. The fluid surrounding the regenerating nerve was found to contain NPF activity for cultured ciliary ganglia neurons which markedly increased during the period of neurite growth into the chamber. In previous studies using this particular neurite-promoting assay, laminin but to a much lesser extent fibronectin also promoted neurite outgrowth.(ABSTRACT TRUNCATED AT 250 WORDS)
Fetal calf serum-mediated inhibition of neurite growth from ciliary ganglion neurons in vitro.
Embryonic chick ciliary ganglion (CG) neurons cultured in fetal calf serum-containing medium have been previously reported to extend neurites on polyornithine (PORN) substrata precoated with a neurite-promoting factor (PNPF) from rat schwannoma-conditioned medium. On PORN substrata alone, however, no neuritic growth occurred. This was interpreted as evidence that PORN was an incompetent substratum for ciliary neuritic growth. In this study, we now find that an untreated PORN substratum allows neuritic growth in serum-free defined medium. When PNPF was added to PORN, a more rapid and extensive neuritic response occurred. After 5 hr of culture, a 60% neuritic response occurred on PNPF/PORN, whereas no neurons initiated neurites until 10-12 hr on PORN. The inhibitory effect of fetal calf serum noted above on PORN could be obtained in part by pretreating the substratum with serum for 1 hr. Maximal inhibitory effects in the PORN pretreatment were achieved after 30 min and were not further improved by treatments up to 4 hr. Bovine serum albumin was also found to inhibit neurite growth on PORN to about 60% of the inhibition obtained by an equivalent amount of serum protein. Fetal calf serum was shown to cause a 15% reduction in the percentage of neurons bearing neurites after its addition to 18-hr serum-free PORN cultures and to cause statistically significant reductions in neurite lengths measured 2 hr later.
Laminin promotes neuritic regeneration from cultured peripheral and central neurons.
The ability of axons to grow through tissue in vivo during development or regeneration may be regulated by the availability of specific neurite-promoting macromolecules located within the extracellular matrix. We have used tissue culture methods to examine the relative ability of various extracellular matrix components to elicit neurite outgrowth from dissociated chick embryo parasympathetic (ciliary ganglion) neurons in serum-free monolayer culture. Purified laminin from both mouse and rat sources, as well as a partially purified polyornithine-binding neurite promoting factor (PNPF-1) from rat Schwannoma cells all stimulate neurite production from these neurons. Laminin and PNPF-1 are also potent stimulators of neurite growth from cultured neurons obtained from other peripheral as well as central neural tissues, specifically avian sympathetic and sensory ganglia and spinal cord, optic tectum, neural retina, and telencephalon, as well as from sensory ganglia of the neonatal mouse and hippocampal, septal, and striatal tissues of the fetal rat. A quantitative in vitro bioassay method using ciliary neurons was used to (a) measure and compare the specific neurite-promoting activities of these agents, (b) confirm that during the purification of laminin, the neurite-promoting activity co-purifies with the laminin protein, and (c) compare the influences of antilaminin antibodies on the neurite-promoting activity of laminin and PNPF-1. We conclude that laminin and PNPF-1 are distinct macromolecules capable of expressing their neurite-promoting activities even when presented in nanogram amounts. This neurite-promoting bioassay currently represents the most sensitive test for the biological activity of laminin.
"Minified" Goldmann applanating prism for tonometry in monkeys and humans.
The tip diameter of the standard Goldmann applanating prism was reduced from 7.0 mm to 4.0 or 4.5 mm, but the endpoint of intraocular pressure measurement-applanation of a circle of cornea having a diameter of 3.06 mm-was not changed. The "minified" tonometers exhibited standard mechanical calibration characteristics in three standard configurations. The 4.5-mm tonometer was calibrated for cynomolgus monkey eyes in vivo by open and closed stopcock manometry. By open stopcock manometry, between pressures of 4 and 70 mm Hg, IOP(tonometer) = 1.01 IOP(manometer)-0.72, with very little scatter or curvature. During closed stopcock manometry, between pressures of 5 and 55 mm Hg, IOP(tonometer)=1.07 IOP(manometer)-1.32, again with very little scatter or curvature. The tonometer elevated manometric IOP by an average of about 7.5%; the percentage pressure elevation decreased slightly as pressure increased. The 4.0-mm tonometer was calibrated against a standard tonometer in living human eyes. Over the pressure range from 0 to 75 mm Hg, IOP(minitonometer)=0.98 IOP(standard tonometer)+ 0.82, with very little scatter or curvature. Similar comparison of two standard tonometers showed a virtually identical relationship and scatter. We conclude that minified Goldmann tonometers provide accurate and reprodible measurements of IOP in cynomolgus monkey and human eyes.
Reducing the stress of high-density living: an architectural intervention.
The present study assesses the effects of an architectural intervention on residential crowding stress and poststressor effects. Residents of long-corridor, short-corridor, and long-corridor-intervention dormitory floors were surveyed and social behavior and space use patterns were systematically observed over a 3-month period. As predicted, although students living in the three environments were initially comparable, residents of the long-corridor floor (40 residents sharing space) reported more crowding and residential social problems over time, whereas short-corridor residents (20 residents sharing space) and modified long-corridor residents (20 residents sharing space) reported fewer of these problems. The results are interpreted in terms of a model of crowding in which architectural features of interior spaces are associated with space use patterns that facilitate or inhibit informal group development and regulation of the frequency of interaction and the amount of privacy. These conditions, in turn, are related to stress and stresslike symptoms.
Major and modified nucleosides in tRNA hydrolysates by high-performance liquid chromatography.
We describe a high-performance liquid chromatographic analytical method that can be readily placed in operation, and which is particularly well suited to scientists investigating tRNA structure, biosynthesis, and function, and for the determination of major and modified nucleosides of tRNA. The method is characterized by the following features: (1) Sensitivity at the nanogram level; (2) High chromatographic resolution and selectivity; (3) Direct measurement of nucleosides with accuracy and precision; (4) Analysis is non-destructive and the high capacity of this chromatographic system allows easy isolation of pure nucleosides for further characterization; (5) Rapid separation and measurement in ca. 1 h after hydrolysis to nucleosides; and (6) Quantitation without use of radiolabeled compounds; however, labeled compounds are readily isolated and measured.
Excretion of creatine and creatinine in feces of man.
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