Application of Raman spectroscopy to biological macromolecules.

The Raman spectra of biological macromolecules arise from molecular vibrations of either the backbone chains or the side chains. The frequencies of the Raman bands lie in a region between 200 cm-1 and 3000 cm-1. From certain frequencies of the vibrations of the backbone chains one can determine the conformation or secondary structure of a macromolecule. Thus for polypeptides and proteins the frequencies of the Amide I and Amide III vibrations allow one to determine the averge conformation of their backbone chain. In polynucleotides and nucleic acids, the frequency of the phosphate diester stretch of the phosphate furanose chain varies between 814 cm-1 for A conformation and 790 cm-1 for B conformation. Raman spectra of the bases in nucleic acids can be used to determine base stacking and hydrogen bonding interactions. Thus Raman spectroscopy is an important tool for determining the conformation structure of proteins and nucleic acids.     

The display of microtubules in transformed cells.

Monospecific tubulin antibodies have been used in indirect immunofluorescence microscopy on a variety of well characterized, transformed cell lines grown in tissue culture. Networks of colcemid-sensitive fibers are seen in SV40-transformed 3T3 cells, SV40-transformed rat embryo cells, HeLa cells and other transformed cell lines. In each case, greater than 90% of the cells contain visible microtubular networks, and where individual microtubules can be distinguished, they run for long distances. Documentation of these metworks is more difficult in transformed than in normal cells, because transformed cells are in general more rounded and have less well spread cytoplasm. In addition, the microtubular networks can be readily visualized in "cytoskeletons" of both normal and transformed cells, obtained by treatment of cells with nonionic detergents in a buffer which stabilizes microtubules in vitro. Addition of calcium to this buffer results in in situ fragmentation and destruction of the microtubular network. In view of these results, we conclude that transformed cells contain significant numbers of microtubules, and that in transformed cells, as in normal cells, microtubules are arranged in networks.     

Developmental uncoupling between blastoderm and yolk cell in the embryo of the teleost Fundulus

During cleavage and blastula stages of embryos of the teleost Fundulus heteroclitus all of the cells are both electotonically coupled and dye coupled to one another, as determined by microelectrode impalements and spread of Lucifer Yellow. At about the time that gastrulation begins we observed a specific loss of junctional coupling between the yolk cell and cells of the blastoderm. Passage of Lucifer Yellow between the yolk cell and blastoderm was reduced at stage 12 (late blastula), and not detected at stage 13 and thereafter, although cells of the blastoderm remain dye coupled to one another through gastrula stages. Also, junctional electrical coupling between the yolk cell and blastoderm became substantially reduced at stage 13 and thereafter. The loss of coupling at this specific cell apposition and time and the large size of the yolk cell may prove useful in analyzing the underlying cellular mechanisms.     

Effect of prostaglandins E2 and F2α on umbilical blood flow and fetal hemodynamics

The hemodynamic effects of PGF_2α, PGE₂, and norepinephrine injected into the umbilical arterial circulation were compared in nine fetal lambs in utero. Umbilical blood flow was measured with radioactive microspheres and an electromagnetic flow transducer implanted on the distal aorta of the fetus after ligation of external iliac arteries and other accessible distal aortic branches.PGF_2α and norepinephrine increased fetal arterial pressure and umbilical blood flow while umbilical vascular resistance increased slightly (PGF_2α) or not at all (norepinephrine). PGE₂ increased fetal arterial pressure, decreased umbilical blood flow, and exerted a profound active vasoconstrictor effect on the fetal placental bed. Our data taken together with the observations of others suggest that prostaglandins may play a role in the circulatory adaptations of the fetus at birth and that PGE₂ in high concentrations is likely to have deleterious hemodynamic consequences in the fetus in utero.     

Metastable secondary structures in ribosomal RNA molecular hysteresis in the acid-base titration of Escherichia coli ribosomal RNA

The metastable conformational states which underlie the hysteresis displayed by Escherichia coli ribosomal RNA in its pH titration in the acid range have been analyzed in terms of acid-stable RNA secondary structures. Sedimentation measurements show that the phenomenon is intramolecular, so that analysis of the hysteresis loops can, in principle, reveal details of molecular architecture. Hysteresis cycles obtained spectrophotometrically and potentiometrically were compared for RNA in solutions of different ionic strengths and ionic compositions. The effect is much smaller at lower ionic strength and disappears in the absence of magnesium ions. The curve followed upon addition of acid appears to reflect the equilibrium state of the system at each pH value. On the “base branch” of the loop, a slow absorbance change (complete in hours) was observed after the pH was raised by addition of a portion of base. This slow process is attributed to the annealing of “mismatched” multihelical regions of the ribosomal RNA. Certain regions, however, remain in metastable configurations for days and it is these long-lived non-equilibrium structures that underlie the hysteresis. Titration at 35 °C gave hysteresis loops of the same size and shape as at 20 °C; indeed, we found that the metastabilities are not removed even at 80 °C. Ultraviolet light absorbance difference spectra at 80 °C between solutions at the same pH, but on different branches of the cycle, give insight into the nature of the metastable conformation(s).Our experimental observations lead us to propose that the hysteresis is due to the formation at acidic pH of double-helical structures involving protonated guanine and adenine base pairs. The G.G pairs seem especially important to account for the very high thermal stability, as well as for the fact that the structures formed at a given pH value as acid is added dissociate only at higher pH values when the solution is titrated with base. Titrations of transfer RNA, along with literature data on 16 S rRNA primary structure, imply that the metastable regions in rRNA may consist of perhaps 10 to 15 base pairs.     

Biosynthesis of ergosta-4,6,8(14),22-tetraen-3-one. A novel oxygenative pathway

Specifically (tritium) labeled precursors (VIII, X, XIV, XV, and XVI), upon feeding to Penicillium rubrum, are incorporated into ergosta-4,6,8(14),22-tetraen-3-one (IV) to the extent of 14.2, 4.5, 11.4, 16.3, and 5.5% respectively. Proof that the ergostane skeleton was incorporated intact was afforded by a chemical-biosynthetic cycle, the latter stages of which entailed reduction of isolated (IV) to ergosterone (VIII), followed by removal of the label through base-catalyzed exchange. A search of the growth medium of P. rubrum revealed the presence of nonartefactual ergosterol epidioxide (XIII) and ergosta-6,22-dien-3β,5α,8α-triol (XVIII). The incorporation data are consistent with a set of multiple pathways with no unique biosynthetic sequence apparent.     

Involvement of the cystathionine pathway in the biosynthesis of glutathione by isolated rat hepatocytes

The ability of l-methionine to support glutathione biosynthesis has been investigated in isolated rat hepatocytes under conditions of normal and depleted glutathione status. The addition of l-[³⁵S]methionine or [l-[³⁵S]homocysteine to incubation media containing hepatocytes results in the incorporation of ³⁵S into intracellular glutathione. Additionally both l-methionine and l-homocysteine are capable of supporting the resynthesis of glutathione in isolated hepatocytes after prior depletion with diethyl maleate. The inclusion in the incubation medium of 1 mm propargylglycine, which is an irreversible inhibitor of the terminal enzyme of the cystathionine pathway, substantially blocks the incorporation of ³⁵S from methionine and l-homocysteine into cellular glutathione. Propargylglycine treatment of hepatocytes in the presence of [³⁵S]methionine is shown to result in the intracellular accumulation of [³⁵S]cystathionine. These results strongly support the conclusion that in rat hepatocytes the cystathionine pathway enables methionine to provide a significant source of l-cysteine for the support of glutathione biosynthesis, under both normal and glutathione-depleted conditions.     

Immunochemical studies on blood groups: Purification,chemical and immunochemical properties of blood group-active glycoproteins from horse gastric mucosae

Digestion of the gastric mucosae of 10 horses with pepsin or Pronase was followed by phenol/ethanol fractionation. Chemical and immunochemical examination of the fractions showed the mucosae to possess various combinations of A, B and H activities. Most were B-active, three had weak A activity, one had strong H activity and the remainder were weakly H-active; one mucosa possessed neither A, B nor H activity. Digestion with pepsin or Pronase of different portions of the same mucosa yielded products equivalent in serological and most chemical properties. Materials digested by Pronase tended to have less peptide nitrogen than those treated with pepsin. Fractions with the strongest serological activities contained significantly higher amounts of carbohydrate and lesser amounts of peptide nitrogen than those with weak A, B or H activity or with no activity. All mucosae, independent of their A, B or H activity, reacted with concanavalin A. The fractions precipitable by 10% ethanol from 90% phenol reacted most strongly.     

Evidence for selenocysteine in ovine tissue organelles

Subcellular fractions were prepared by differential centrifugation from heart and liver of a lamb labeled with 75Se-selenite. Crude fractions of nuclei, mitochondria, and microsomes from both tissues were solubilized with sodium dodecyl sulfate and chromatographed on columns of sephacryl S-200. A low molecular weight (MW) 75Se labeled cardiac cytosol protein (approximately 10,000 daltons) was partially purified by gel filtration chromatography. The major 75Se peaks from the sephacryl columns and the low MW cardiac protein were hydrolyzed in HCl under an inert atmosphere. When chromatographed on an amino acid analysis column, 75Se from each hydrolysate chromatographed in the identical position of 2,7-diamino-4-thia-5-selenaoctanedioic acid, the mixed oxidized dimer of cysteine and selenocysteine. The low MW cardiac protein was reacted with chloroacetate after reduction with borohydride. 75Se from a hydrolysate of this derivatized protein eluted in the same position as Se-carboxymethylselenocysteine on the amino acid analysis column. Thus, selenocysteine appears to be the predominant form of selenium in ovine heart and liver.     

Control of yeast cell type by the mating type locus: II. Genetic interactions between MATα and unlinked α-specific STE genes

The alleles of the yeast mating type locus, MATα and MATa, determine the yeast cell types, a,α, and a/α. It has been proposed that the MATα2 product negatively regulates expression of unlinked a-specific genes, and that the MATα1 product positively regulates expression of unlinked α-specific genes. The behavior of mutants defective in MATα2, which are deficient in mating and in production of α-factor, can thus be attributed to antagonism between a-specific and α-specific functions expressed simultaneously in matα2^? strains. If this view is correct, then elimination by mutation of the specific functions required to mate as α may allow matα2 mutants to mate as a. In order to test this possibility, we examined the interactions between matα2 mutations and various unlinked mutations that cause α cells but not a cells to be mating defective (α-specific STE mutations). Three α-specific mutations (ste3, ste13 and kex2) were found to be non-allelic. Furthermore, although matα2 mutants mate weakly as a, matα2, ste3 double mutants, but not matα2 ste13 or matα2 kex2 double mutants, mate efficiently as a. The ability of matα2 ste3 strains to mate as a supports the view that matα2 mutants express a-specific mating functions, and suggests that a mating functions are expressed constitutively in MATa cells. The mating behaviour of the matα2 ste3 double mutant is consistent with the proposal that STE3 is positively regulated by the MATα1 product.     

Control of yeast cell type by the mating type locus: I. Identification and control of expression of the a-specific gene BAR1

The MATα allele of the yeast mating type locus confers the α mating phenotype and contains two complementation groups, MATα1 and MATα2. The α1–α2 hypothesis proposes that MATα1 is a positive regulator of α-specific genes and that MATα2 is a negative regulator of a-specific genes. According to this hypothesis, matα2 mutants, which are defective in mating and in production of extracellular α-factor, express both a-specific functions (because they lack MATα2 product) and α-specific functions (because they contain MATα1 product). Failure to produce extracellular α-factor results from antagonism between these functions; in particular, because α-factor (an α-specific function) is degraded by an a-specific function. If this view is correct, matα2 mutants should acquire the ability to produce α-factor if they also carry a defect in the gene(s) responsible for α-factor degradation. We have isolated a derivative of a matα2 mutant that produces α-factor and have characterized the suppressor mutation in this strain. (1) This strain carries a mutation (bar1-1) tightly linked to HIS6 (on chromosome IX) that allows matα2 mutants to produce α-factor. (2) It does not allow matα1 mutants to produce α-factor. (3) Haploids of the a mating type bearing the bar1-1 mutation still mate, but are unable to act as a barrier to the diffusion of α-factor. MATa bar1-1 cells display increased sensitivity to α-factor. (4) A mutation (sst1?2) that causes increased sensitivity to α-factor is allelic to bar1-1 and also allows α-factor synthesis by matα2 mutants. The ability of matα2 bar1 double mutants to produce extracellular α-factor indicates that matα2 mutants do produce α-factor but that it is degraded by the Barrier function. These results suggest that BAR1 is normally expressed only in a cells, and is negatively regulated in α cells by the MATα2 product.     

Dental pathology of prehistoric residents of Oregon

Dentitions of 208 prehistoric skeletal specimens from five geographic regions of Oregon were studied to describe their dental status. Comparisons were made of the incidence of specific pathologies among regions and between jaws. Maxillary premortem tooth losses were significantly higher than mandibular tooth loss. A relatively high caries incidence occurs in samples from the Willamette Valley and Klamath Basin, where plant foods were used aboriginally to a significant extent. A high frequency of abscesses was recorded in the Klamath Basin and the Lower Columbia River sample, which also showed the highest levels of attrition. Each of the five subsamples shows patterns of dental pathology consistent with former use of natural resources.     

Selective protection of murine thymic helper T cells from glucocorticosteroid inhibition by macrophage-derived mediators

We investigated the in vitro effects of dexamethasone (DEX) on the functional capacities of virgin murine T cells cultured in the absence and presence of adjuvant-stimulated macrophages or factors derived from them. Immunologically mature thymocytes, isolated on the basis of their inability to bind peanut agglutinin (PNA^? thymocytes), were used as virgin T cells. Treatment of PNA^? thymocytes with DEX for 24 hr in vitro eliminated their subsequent capacity to function as helper cells for primary humoral responses, to proliferate when stimulated by plant mitogens or allogeneic cells, or to generate T-cell-mediated cytotoxic responses. However, when PNA^? thymocytes were pretreated with DEX in combination with either adjuvant-activated macrophages or their culture supernatants, which contained Interleukin 1, the capacity of the T cells to subsequently express helper activity was preserved. The macrophage products, however, did not prevent DEX from inhibiting the capacities of PNA^? thymocytes to proliferate in response to plant mitogens or alloantigens or to generate cytotoxic effector cells; thus, protection was selective. The data indicate that, prior to activation, helper T cells are distinguished by their capacity to become steroid resistant in response to macrophage products. Although T-cell proliferative and cytotoxic responses have been reported to be protected from DEX inhibition by Interleukin 2, our results suggest that macrophages prevent steroid effects on virgin helper T cells by Interleukin-1-dependent mechanisms that do not involve Interleukin 2. While we have not delineated the biochemical pathways of protection, we show that the acquisition of DEX resistance by helper T cells cannot be attributed to the polyclonal induction of helper activity by macrophage factors.     

Human Y-chromatin : I. Dispersion and condensation

The fluorescent Y-body was observed to manifest striking variations in morphology in interphase lymphocyte and fibroblast nuclei. These variations appeared to be largely independent of the specific fixation or hypotonic treatment. The Y-chromatin body ranged from a highly condensed mass to a constellation of smaller discrete particles. Dispersion of Y-chromatin was not seen in mitotically inactive cells from buccal mucosa and hair root sheaths. Comparison of uncultured and cultured lymphocytes confirmed a higher proportion of dispersed Y-bodies in cultured cells, particularly those with a relatively large mean nuclear diameter. In serially cultured lymphocytes, the frequency of Y-body dispersion showed a direct relationship to the time in culture. Mean nuclear diameter also increased directly with time in culture. In cultured fibroblasts dispersion of the Y-body increased during the logarithmic phase of growth and declined during the post-logarithmic phase.     

Airway constrictor effects of leukotriene D4 in dogs with hyperreactive airways

Leukotriene D₄ (5 μg/ml) aerosol constricts airways of dogs with nonspecific airway hyperreactivity but not of mongrel dogs which lack nonspecific airway hyperreactivity. R_L increased 200 + 25% and C_dyn decreased to 77 ± 5% of the pre-challenge value. LTD₄ (10 μg/ml) produced no further increase. Atropine (0.2 mg/kg) prevented the increase in R_L and decrease in C_dyn, suggesting that part of the effect of LTD₄ on airways is neurally mediated.     

Fire and Litter Effects on Seedling Establishment in Western Oregon Upland Prairies

Prescribed burning is an important tool for managing and restoring prairies and other ecosystems. One effect of fire is plant litter removal, which can influence seedling establishment. Four experimental treatments (burned, clipped and raked to remove litter, burned with litter reapplied, and unmanipulated) were applied to 2 × 2.5–m plots in three western Oregon, United States, upland prairies to determine how burning affects seedling establishment. Seeds of common exotic and native prairie species were sowed into the experimental plots after treatments. Seedlings were censused the following spring. The experiment was repeated on each of the three sites, representing three common types of prairie vegetation: an Annual Exotic Grass site, a Perennial Exotic Grass site, and a Native Bunchgrass site. In both the Annual Exotic Grass and the Perennial Exotic Grass sites, burning significantly improved native, but not exotic, seedling establishment over those on unburned plots. Litter removal was a significant component of this burn effect, particularly on the Perennial Exotic Grass site. In these winter‐moist systems, the net effect of litter is to inhibit seedling establishment. Burning treatments on the Native Bunchgrass site significantly increased seedling establishment only of short‐lived exotic species. These results suggest that in prairie ecosystems similar to the Annual and Perennial Exotic Grass sites, prescribed burning followed by sowing native seeds can be an effective restoration technique. Burning alone or sowing alone would be counter‐productive, in the first case because increased establishment would come from exotic species and in the second case because establishment rates are low in unburned plots.     

Population dynamics of the threatened Oregon spotted frog before and after drought mitigation

Amphibians are among the most sensitive taxa to climate change, and species inhabiting arid and semiarid landscapes at the extremes of their range are especially vulnerable to drought. The Jack Creek, Oregon, USA, population of Oregon spotted frogs (Rana pretiosa) faces unique challenges because it occupies the highest elevation site in the species' extant range and one that has been transformed by loss of American beavers (Castor canadensis), which historically maintained open water. We evaluated the effects of drought mitigation (addition of excavated ponds) on relationships between local and regional water availability, inactive legacy beaver dams, and Oregon spotted frog population dynamics in the Jack Creek system. We conducted egg mass surveys and capture-mark-recapture sampling at a treatment reach with excavated ponds and 3 reference reaches over 13 years; surveys spanned a period before and after pond excavation at the treatment and 1 primary comparison reference reach. We analyzed data using a combination of robust design capture-mark-recapture estimators and generalized linear mixed models to characterize population dynamics. Adult Oregon spotted frog survival was approximately 19.5% higher at the treatment reach than the primary reference reach during the study period. Annual survival was most strongly associated with late summer vegetation greenness, a proxy for water availability, and males had higher survival than females. Among the 4 study reaches, the treatment reach consistently had higher late summer vegetation greenness, and the hydrology functioned more independently of regional precipitation patterns relative to the reference reaches; however, these dynamics were not linked to pond excavation. Breeding was concentrated in 2 legacy beaver ponds that were deepened by excavation during the study compared to an unexcavated beaver pond, 2 excavated ponds without legacy beaver dams, and 9 reference ponds. These results point to the benefit of enhancing existing beaver structures and indicate that management actions aimed at maintaining surface water for breeding in spring and saturated soils and ponded water for adults in late summer would benefit this unique population of Oregon spotted frogs in the face of drought.     

VERTICAL MIGRATION OF A MIXED‐SPECIES EUGLENA (EUGLENOPHYTA) ASSEMBLAGE INHABITING THE HIGH‐INTERTIDAL SANDS OF NYE BEACH,OREGON1

Comparatively little is known about the vertical migration of the microphytobenthic community forming visible patches on high‐energy beaches. We collected surface and cored samples to evaluate the timing and extent of downward migration of a multispecies Euglena assemblage inhabiting Nye Beach, Oregon. Euglena density at the surface was highly variable and was not correlated with the time of low tide or instantaneous irradiance measurements; however, triplicate cores collected at low and high tides revealed a tidal rhythm in mean depth. On average, 95% of the assemblage occurred within 1 cm of the surface during low tide, but 54% of the assemblage was collected between 1 and 8 cm below the surface during high tide. A midday shading experiment revealed that short‐term changes in irradiance levels altered the Euglena density at the sediment surface by inducing vertical migration. This response to short‐term fluctuations in light may explain the weak correlation between cell density at the surface and time of day. The high‐intertidal location of these patches prevented the removal of nonmigrating cells by daily high tides, which increased the variability in surface samples and obscured the tidal migration rhythm detected in the core samples. Due in part to the semidiurnal nature of Oregon tides, this study provides in situ confirmation of past mesocosm research indicating that sediment disturbance during daily submersed periods is an important process in maintaining the quasi‐tidal rhythm in the appearance and disappearance of Euglena spp. from the surface of beaches and intertidal sandflats.     

THE EARLIEST SIRENIAN (MAMMALIA: DUGONGIDAE) FROM THE EASTERN PACIFIC OCEAN

The partial skull and mandible of an unidentified halitheriine dugongid, collected from the Early Miocene Nye Mudstone in Lincoln County, Oregon, USA, is the earliest record of the Sirenia in the eastern Pacific Ocean. It is probably earlier than Early or Middle Miocene sirenians recently found in Peru, and is definitely earlier than any known from California or Baja California. However, it appears to be slightly younger than fossil sirenian remains recently reported from Late Oligocene rocks in Japan. The Oregon specimen is also the most northern record of the Sirenia on the west coast of North America prior to the Pleistocene although other sirenians did evidently reach and surpass such latitudes by the Late Miocene when a dispersal took place from America to the North Pacific coasts of the Old World. The Oregon specimen probably represents sirenians that spread to the North Pacific from the Caribbean, quite possibly prior to the Miocene. The Nye Mudstone was deposited during the warmest period of the Neogene on the coast of Oregon, and it does not seem necessary to postulate a greater degree of cold-tolerance for the Oregon sirenian than is exhibited by living sea cows.     

Macroinvertebrate community response to natural and forest harvest gradients in western Oregon headwater streams

1. To examine the effects of forest harvest practices on headwater stream macroinvertebrates, we compiled a 167 site database with macroinvertebrate, fish, physical habitat and catchment land cover data from the three forested ecoregions in western Oregon. For our analysis, headwater streams were defined by catchment areas <10 km² and perennial water during summer low flows. Almost all sites in the database were selected using a randomised survey design, constituting a representative sample of headwater streams in these ecoregions. 2. Macroinvertebrate taxonomic and functional feeding group composition were very similar among the three ecoregions in the study area (Coast Range, Cascades and Klamath Mountains). On average, 55% of the individuals at each site were in the orders Ephemeroptera, Plecoptera or Trichoptera. Dipteran taxa (mostly chironomids) accounted for another 34%. At almost all sites, non‐insects made up <10% of the macroinvertebrate assemblage. Almost half (49%) of the assemblages were collectors; remaining individuals were about evenly divided among scrapers, shredders and predators. 3. There were 189 different macroinvertebrate taxa at the 167 sites with richness at individual sites ranging from 7 to 71 taxa. Ordination by non‐metric multidimensional scaling revealed a strong association between % Ephemeroptera, especially Baetis, and site scores along the first axis. This axis was also strongly related to % coarse substratum and fast water habitat. The second axis was strongly related to % intolerant individuals, site slope and altitude. No strong relationships were evident between any ordination axis and either logging activity, presence/absence of fish, catchment size or ecoregion. 4. Based on macroinvertebrate index of biotic integrity (IBI) scores, 62% of the sites had no impairment, 31% of the sites had slight impairment and only 6% of the sites had moderate or severe impairment. IBI scores were not strongly related to forest harvest history. All four severely impaired sites and five of the seven sites with moderate impairment were lower altitude, shallower slope stream reaches located in the Coast Range with evidence of agricultural activity in their catchment or riparian zone. % sand + fine substratum was the environmental variable most strongly related to macroinvertebrate IBI.