A microassay for lysyl oxidase activity.

Pinnell and Martin's [(1968) Proc. Nat. Acad. Sci. USA61, 708–716] standard assay for lysyl oxidase is modified to determine activity in small samples. Data collected by both methods are comparable, and the microassay has the advantages of being economical and rapid.     

Thymosin β4 in cultured mammalian cell lines

Thymosin β₄, originally isolated from calf thymus [Low et al., Proc. Nat. Acad. Sci. USA78, 1162–1166 (1981)] is present in a number of cell lines unrelated to the reticuloendothelium, including myoblasts and fibroblasts. It is also actively synthesized by these cell lines. Its content and rate of synthesis in the cell lines examined appear to be correlated with their ability to adhere and their motility.     

Radiosensitivity and time of origin of Mauthner neuron in the zebra fish

Regeneration was induced in the mature skeletal muscles of adult dystrophic mice ($\text{C57B1}\text{6J}$ dy/dy) by the im injection of hypertonic saline. Regenerating myogenic cells were isolated from the saline-treated muscles by cold trypsinization with the aid of Yaffe's selective plating procedure [Yaffe, D. (1968). Proc. Nat. Acad. Sci. USA61, 477–483]. The myogenic cells thus isolated readily developed into muscle fibers possessing cross striations when cultured in vitro.     

Carbonylated proteins are eliminated during reproduction in C. elegans

Oxidatively damaged proteins accumulate with age in many species (Stadtman (1992) Science 257 , 1220–1224). This means that damage must be reset at the time of reproduction. To visualize this resetting in the roundworm Caenorhabditis elegans, a novel immunofluorescence technique that allows the detection of carbonylated proteins in situ was developed. The application of this technique revealed that carbonylated proteins are eliminated during C. elegans reproduction. This purging occurs abruptly within the germline at the time of oocyte maturation. Surprisingly, the germline was markedly more oxidized than the surrounding somatic tissues. Because distinct mechanisms have been proposed to explain damage elimination in yeast and mice (Aguilaniu et al. (2003) Science 299 , 1751–1753; Hernebring et al. (2006) Proc Natl Acad Sci USA 103 , 7700–7705), possible common mechanisms between worms and one of these systems were tested. The results show that, unlike in yeast (Aguilaniu et al. (2003) Science 299 , 1751–1753; Erjavec et al. (2008) Proc Natl Acad Sci USA 105 , 18764–18769), the elimination of carbonylated proteins in worms does not require the presence of the longevity‐ensuring gene, SIR‐2.1. However, similar to findings in mice (Hernebring et al. (2006) Proc Natl Acad Sci USA 103 , 7700–7705), proteasome activity in the germline is required for the resetting of carbonylated proteins during reproduction in C. elegans. Thus, oxidatively damaged proteins are eliminated during reproduction in worms through the proteasome. This finding suggests that the resetting of damaged proteins during reproduction is conserved, therefore validating the use of C. elegans as a model to study the molecular basis of damage elimination.     

Size does matter: overcoming the adeno-associated virus packaging limit

Recombinant adeno-associated virus (rAAV) vectors mediate long-term gene transfer without any known toxicity. The primary limitation of rAAV has been the small size of the virion (20 nm), which only permits the packaging of 4.7 kilobases (kb) of exogenous DNA, including the promoter, the polyadenylation signal and any other enhancer elements that might be desired. Two recent reports (D Duan et al: Nat Med 2000, 6:595-598; Z Yan et al: Proc Natl Acad Sci USA 2000, 97:6716-6721) have exploited a unique feature of rAAV genomes, their ability to link together in doublets or strings, to bypass this size limitation. This technology could improve the chances for successful gene therapy of diseases like cystic fibrosis or Duchenne muscular dystrophy that lead to significant pulmonary morbidity.     

The dsRNA ofTrichomonas vaginalis is associated with virus-like particles and does not correlate with metornidazole resistance

Twelve metronidazole-resistant and twelve metronidazole-susceptible strains ofTrichomonas vaginalis were tested for the presence of dsRNA. Three resistant and five susceptible strains were found to contain dsRNA which indicated that metronidazole resistance does not correlate with the absence of dsRNA. Electron microscopy showed the homogenates of all dsRNA -positive strains to contain virus-like particles 32 –38 nm in diameter, while no such particles were found in the dsRNA-negative strains. A mutual relationship between the dsRNA and virus-like particles seems to exist. After this paper had been accepted for publication the occurrence of virus-like particles in dsRNA-positive trichomonads was reported by others (Wang A.L., Wang C.C.: The double stranded RNA inTrichomonas vaginalis may originate from virus-like particles.Proc. Nat. Acad. Sci. USA 83, 7956–7961, 1986).     

Excipients activate peroxidases in specific but not in non-specific reactions in organic solvents

Co-lyophilization with ligands and lyoprotectants markedly activated horseradish and soybean peroxidases in the oxidation of guaiacol in organic solvents (Dai & Klibanov (1999) Proc. Natl. Acad. Sci. USA 96: 9475–9478). However, such excipients gave little or no activation of these enzymes in non-specific reactions, sulfoxidation of thioanisole and epoxidation of styrene, presumably because they do not require a precise fine tuning of the enzyme active site.     

In vitro expression of Escherichia coli ribosomal protein L 10 gene: tripeptide synthesis as a measure of functional mRNA

The in vitro synthesis of the initial tripeptide, fMet-Ala-Leu, of Escherichia coli ribosomal protein L10 has been studied in a plasmid DNA-directed system using purified factors. In contrast to the synthesis of the dipeptide, fMet-Ala, described recently (N. Robakis L. Meza-Basso N. Brot, and H. Weissbach, 1981, Proc. Nat. Acad. Sci. USA78, 4261–4264), tripeptide formation leads to a stable peptidyl-tRNA:mRNA:70 S complex. Using mRNA for L10, a good correlation has been observed between the amount of tripeptide formed and the amount of fMet-tRNA bound to a mRNA:ribosome complex. These results indicate that tripeptide formation in this system can be used as a simple and specific assay for the amount of functional mRNA present.     

Cell differentiation in the absence of intracellular cyclic AMP pulses in Dictyostelium discoideum

Repeated additions of cyclic AMP to a morphogenetic mutant of Dictyostelium discoideum, agip 53, induced cell differentiation to the aggregation competent state as previously reported [Darmon, Brachet, and Pereira da Silva (1975). Proc. Nat. Acad. Sci. USA72, 3163–3166]. Cyclic AMP additions elicited transient increases of the intracellular cyclic GMP concentration but no significant increases of the intracellular cyclic AMP concentration. These results suggest that transient increases of the intracellular cyclic AMP concentration are not necessary for cell differentiation. Agip 53 seems to be unable to relay cyclic AMP signals. A defect in the receptor-mediated activation of adenylate cyclase could be the biochemical basis of the mutant phenotype of agip 53.     

Excitation energy flow at 77 K in the photosynthetic apparatus of overwintering evergreens

The flow of excitation energy from the antennae to photosynthetic reaction centre complexes at 77 K was studied in leaves of two evergreen species, namely, snow gum (Eucalyptus pauciflora Sieb. ex Spreng.) and a hemiparasitic mistletoe (Amyema miquelii, Lehm. ex Miq.). The leaves that were naturally acclimated to winter conditions of freezing temperatures and high irradiance displayed the recently discovered cold‐hard‐band or CHB feature of the chlorophyll a fluorescence spectra (Gilmore & Ball, Proc. Nat. Acad. Sci. USA 97:11098–11101, 2000). A streak‐camera‐spectrograph was used and the double convolution integral method for global analysis was applied to simultaneously acquire and simulate, respectively, the time‐ and wavelength‐dependence of all major chlorophyll a components (Gilmore et al. Phil. Trans. Roy. Soc. B‐London 355:1371–1384, 2000). The CHB coincided with changed amplitudes and decreased excited state lifetimes for the main F685 nm and F695 nm emission bands from the photosystem II (PSII) core‐inner‐antenna. The CHB dissipates energy as heat separate from PSII while also reducing the PSII quantum yield by competing for both photon absorption and antenna excitation. The CHB did not correlate with changes in the decay kinetics of the PSI antenna F740 nm band. The spectral‐kinetic features of the altered energy flow were similar in the unrelated evergreen species. These results are consistent with a functional association between the CHB, PSII energy dissipation and protective storage of chlorophyll in overwintering evergreens.     

Sniffing out cancer using “chemical nose” sensors

Comment on: Bajaj A, et al. Proc Nat Acad Sci 2009; 106:10912-16.     

Hydrogen-bonding preferences in 2,6-diaminopurine: uracil (thymine) and 8-methyl adenine:uracil (thymine) complexes

We present molecular mechanical calculations on 2,6-diaminopurine (2,6-DAP):uracil (thymine) and 8-methyladenine (8-methyl A): uracil (thymine) hydrogen-bonded complexes of various geometries, namely, Watson-Crick (normal and reverse), Hoogsteen (normal and reverse), and purine N3 type. In contrast to earlier calculations [Ornstein, R. L. & Fresco, J. R. (1983) Proc. Natl. Acad. Sci. USA 80 , 5171–5175], the 2,6-DAP:uracil (thymine) complexes are predicted to be Watson-Crick and the 8-methyladenine:uracil (thymine) to be Hoogsteen. The results presented here are more consistent with the observed crystallographic preferences.     

Terrestrial fluxes of carbon in GCP carbon budgets

The Global Carbon Project (GCP) has published global carbon budgets annually since 2007 (Canadell et al. [2007], Proc Natl Acad Sci USA, 104, 18866–18870; Raupach et al. [2007], Proc Natl Acad Sci USA, 104, 10288–10293). There are many scientists involved, but the terrestrial fluxes that appear in the budgets are not well understood by ecologists and biogeochemists outside of that community. The purpose of this paper is to make the terrestrial fluxes of carbon in those budgets more accessible to a broader community. The GCP budget is composed of annual perturbations from pre‐industrial conditions, driven by addition of carbon to the system from combustion of fossil fuels and by transfers of carbon from land to the atmosphere as a result of land use. The budget includes a term for each of the major fluxes of carbon (fossil fuels, oceans, land) as well as the rate of carbon accumulation in the atmosphere. Land is represented by two terms: one resulting from direct anthropogenic effects (Land Use, Land‐Use Change, and Forestry or land management) and one resulting from indirect anthropogenic (e.g., CO₂, climate change) and natural effects. Each of these two net terrestrial fluxes of carbon, in turn, is composed of opposing gross emissions and removals (e.g., deforestation and forest regrowth). Although the GCP budgets have focused on the two net terrestrial fluxes, they have paid little attention to the gross components, which are important for a number of reasons, including understanding the potential for land management to remove CO₂ from the atmosphere and understanding the processes responsible for the sink for carbon on land. In contrast to the net fluxes of carbon, which are constrained by the global carbon budget, the gross fluxes are largely unconstrained, suggesting that there is more uncertainty than commonly believed about how terrestrial carbon emissions will respond to future fossil fuel emissions and a changing climate.     

Large scale isolation and storage of Neurospora plasma membranes.

Functionally inverted plasma membrane vesieles isolated from the eukaryotic microorganism Neurospora crassa generate and maintain a transmembrane electrical potential via ATP hydrolysis catalyzed by a plasma membrane ATPase (G. A. Scarborough, 1976, Proc. Nat. Acad. Sci. USA73, 1485–1488). In order to facilitate investigation of the molecular mechanism of the electrogenic ATPase, and other transport systems, we have developed a method for the large scale isolation and storage of Neurospora plasma membranes in a stable form. Large quantities of open plasma membrane sheets (ghosts) are isolated by a scaled-up modification of the original method (G. A. Scarborough, 1975, J. Biol. Chem.250, 1106–1111) and stored at ?26°C in 60% glycerol ($\text{v}\text{v}$). As needed, the ghosts are washed free of glycerol and then converted to closed vesicles by a modification of the original method. With this technique, plasma membrane vesicles with normal electrogenic pump activity can be prepared daily in approximately 2.5 h.     

Construction and BamHL analysis of chimeric plasmids containing EcoRL DNA fragments of the F sex factor.

The construction of seven chimeric plasmids (pRS series) carrying EcoRI endonuclease-generated segments of the F sex factor cloned onto the vector pSC101 is described. BamHI endonuclease analysis of these seven plasmids, the six previously described pRS plasmids (Skurray, R. A., Nagaishi, H., and Clark, A. J. (1976) Proc. Nat. Acad. Sci. USA73, 64–68) and F plasmid DNA has enabled a partial BamHI map of F to be constructed; the orientation of insertion of F DNA segments into the pSC101 vector was also established for nine of the pRS plasmids. Results indicate that in the absence of their normal promoter, F cistrons cloned into the EcoRI site of pSC101 are expressed regardless of orientation of insertion although there is a preferred orientation for high levels of expression.     

Ontogeny of maternal and newly transcribed mRNA analyzed by in situ hybridization during development of Caenorhabditis elegans

We have reexamined conditions for release of labeled cell surface proteins from sea urchin eggs and the effects of exogenous cell surface protein on the status of protein synthesis in activated eggs. As reported earlier (J. D. Johnson and D. Epel, 1975, Proc. Nat. Acad. Sci. USA72, 4474–4478), we find insemination and the Ca-ionophore A23187 induce release of labeled cell surface polypeptides but in contrast to that report we do not observe increased release during incubation in ammonia or nicotine. Electrophoretic analysis of the ¹²⁵I-labeled polypeptides released after insemination indicates that the cell surface protein fraction contains a minimum of 10 polypeptides ranging from 22,000 to 200,000 daltons. In five experiments, the cell surface proteins released during the ammonia incubation had no significant effect on the status of protein synthesis in partially activated eggs.     

Polypeptide Composition of Envelopes of Spinach Chloroplasts: Two Major Proteins Occupy 90% of Outer Envelope Membranes

Outer and inner envelope membranes of spinach chloroplasts wereisolated using floatation centrifugation followed by sedimentationsucrose density gradient centrifugation after disruption ofintact chloroplasts by freezing and thawing. Two major fractionswith buoyant densities of 1.11 and 1.08 g cm^–3 and a minorfraction with a density of 1.15 g cm^–3 were obtained.They were identified as innei and outer envelope and thylakoidfractions, respectively, by analyzing their polypeptide compositionby high-resolution SDS-PAGE and the N-terminal sequences oftheir protein components. Due to the refinement of the isolation procedure, most of theribulose-l,5-bisphosphate carboxylase/oxygenasc (RuBisCO), whichhad always been observed as a contaminant, was eliminated fromthe outer envelope fraction. Application of high-resolutionSDS-PAGE revealed that this fraction was rich in the low-molecular-massouter envelope protein, E6.7 [Salomon et at. (1990) Proc. Natl.Acad. Sci. USA 87: 5778] and a protein with a molecular massof 15 kDa which is homologous to the 16 kDa outer envelope proteinof pea [Pohlmeyer et al. (1997) Proc. Natl. Acad. Sci. USA 94:9504]. The two proteins account for 90% of the total proteinspresent in outer envelope membranes. Proteins which are suggestedto function in translocation of nuclear-encoded polypeptideswere not identified in the envelopes from spinach in the presentstudy. Differences in the protein composition of outer envelopemembranes arc discussed based on the developemental stages ofchloroplasts. ¹Present address: Biological Function Section, Kansai AdvancedResearch Center, Communications Research Laboratory, Ministryof Posts and Telecommunications, Kobe, Hyogo, 651-24 Japan.     

Isolation and partial characterization of a rat hepatoma heparan sulfate proteoglycan

A proteoglycan was isolated from a Morris rat hepatoma by sequential precipitations with ammonium sulfate and cetyl pyridinium chloride followed by chromatography on Sepharose CL-4B and DEAE-cellulose. The proteoglycan has a molecular weight of about 1.5 × 10⁵ with 40,000 molecular weight glycosaminoglycan side chains, identified as heparan sulfate based on resistance to chondroitinase and susceptibility to nitrous acid treatment. Immunological studies showed that the protein core of this proteoglycan is immunologically distinct from a rat yolk sac tumor chondroitin sulfate proteoglycan (Å. Oldberg, E. G. Hayman, and E. Ruoslahti, 1981,J. Biol. Chem.256, 10847–10852), but resembles a heparan sulfate proteoglycan isolated from a basement membrane-producing mouse tumor (J. R. Hassell, P.M. Robey, H.-J. Barrach, J. Wilczek, S. R. Rennard, and G. R. Martin, 1980, Proc. Nat. Acad. Sci. USA77, 4494–4498).     

The cellular anatomy of embryos of the nematode Caenorhabditis elegans. Analysis and reconstruction of serial section electron micrographs

As described from light microscopy, embryogenesis of the free-living soil nematode Caenorhabditis elegans follows a strictly determinate cleavage pattern, producing a newly hatched juvenile with about 550 cells arranged quite predictably. In this communication we present results on the electron microscopy of C. elegans embryos and introduce methods for fixing, embedding, and serially sectioning embryos encased in the egg shell. Fixation at elevated temperature either with osmium tetroxide alone or with glutaraldehyde followed by osmium tetroxide gives reproducible results with embryos in all developmental stages so far tested, from the fertilized egg to hatching. Eighteen wild-type eggs at various stages have been sectioned to date. We have achieved using newly developed procedures for analyzing electron micrographs of serial sections detailed reconstructions of the cellular anatomy of complete embryos of a metazoan organism. Three-dimensional serial section reconstructions were made with a computer system. We characterize and map the 24 cells of an early-stage embryo in this report. Additionally, we can specify the lineage history of all cells of this embyro by matching the reconstructed three-dimensional arrangement of this series to a living embryo at this stage, where cell lineage has been observed with Nomarski optics and analyzed using videotape (U. Deppe, E. Schierenberg, T. Cole, C. Krieg, D. Schmitt, B. Yoder, and G. von Ehrenstein, 1978, Proc. Nat. Acad. Sci. USA75, 376–380). In addition, cytoplasmic and nuclear morphological features such as incomplete membranes between sister cells, rounding-off of the cytoplasm, and chromatin condensation patterns have been correlated with cell division. Mapping of such structures presents a new method by which supplementary lineage information can be obtained directly from an electron micrographic series.