Involvement of p21ras distinguishes positive and negative selection in thymocytes.

Small molecular weight GTP binding proteins of the ras family have been implicated in signal transduction from the T cell antigen receptor (TCR). To test the importance of p21ras in the control of thymocyte development, we generated mice expressing a dominant-negative p21ras protein (H-rasN17) in T lineage cells under the control of the lck proximal promoter. Proliferation of thymocytes from lck-H-rasN17 mice in response to TCR stimulation was nearly completely blocked, confirming the importance of p21ras in mediating TCR-derived signals in mature CD4+8- or CD8+4- thymocytes. In contrast, some TCR-derived signals proceeded unimpaired in the CD4+8+ thymocytes of mice expressing dominant-negative p21ras. Analysis of thymocyte development in mice made doubly transgenic for the H-Y-specific TCR and lck-H-rasN17 demonstrated that antigen-specific negative selection occurs normally in the presence of p21H-rasN17. Superantigen-induced negative selection in vivo also proceeded unhindered in H-rasN17 thymocytes. In contrast, positive selection of thymocytes in the H-Y mice was severely compromised by the presence of p21H-rasN17. These observations demonstrate that positive and negative selection, two conceptually antithetical consequences of TCR stimulation, are biochemically distinguishable.     

Action Spectra for Conversions of Phycochrome c from Nostoc muscorum

The reversibly photochromic pigment, phycochrome c, was extracted from the blue-green alga Nostoc muscorum strain A. Action spectra were determined for in vitro conversions of the pigment from the short wavelength to the long wavelength form and vice versa. The action peak for the absorbance decrease at 650 nm is at 630 nm. During this decrease there is only a slight increase of the absorbance in the green region. Green and yellow light (maximum efficiency at 580 nm) completely restores absorbance at 650 nm. The observations are explained by the existence of three spectrally different forms of phycochrome c: P^c₆₃₀ and P^c₆₅₀ which equilibrate in darkness and P^c₅₈₀ which is reversibly photoconvertible to P^c₆₃₀. We have also measured the absorbance changes brought about by saturating irradiations with light of various wavelengths (“photostationary state spectrum”). Extreme photostationary states were obtained with about 650 nm and 500 nm light.     

Effects of Frost Hardening and Dehardening on Photosynthetic Electron Transport and Fluorescence Properties in Isolated Chloroplasts of Pinus silvestris

Photosynthetic electron transport and low-temperature fluorescence emission properties have been analyzed in isolated chloroplasts during the course of frost hardening and dehardening of Pinus silvestris L. Both the partial electron-transport reactions (H₂O DPIP and Asc./DPIP NADP) and the overall electron transport (H₂O — NAPD) showed decreasing capacities during the course of hardening. Upon exposing the plants to ?5°C and high irradiance a block in the electron-transport chain between the two photosystems developed, whereas the partial reactions still showed activities. The decrease in activity of PSl was accompanied by a decrease in P700 content, as determined by light oxidation of P700, which indicates a correlation between the two changes. Hardening also induced changes in the in vivo chlorophyll organization. During the course of hardening the fluorescence emission bands F692 and F726 decreased relative to F680. These changes were more pronounced if the plants were treated in high than in low irradiance. This suggests a greater destruction of the chlorophyll antennae in close association with the two photoreactions than in the so-called light-harvesting chlorophyll a/b antenna. During dehardening basically the reverse of the changes observed during hardening occurred. The recovery of secondary needles was complete, whereas primary needles only partly recovered.     

Sedimentation studies of giant DNA molecules present in unsheared whole-cell lysates of D. melanogaster cells.

We have used band sedimentation in shallow density gradients of CsCl in the preparative centrifuge to analyze the distribution of sedimentation coefficients present in tritium labelled DNA from D. melanogaster cells. The cells were lysed according to the method of Kavenoff and Zimm to preserve very high molecular weight DNA. Sedimentation measurements have been carried out as a function of speed of centrifugation. The resulting distribution functions have been interpreted with the aid of the Zimm-Schumaker equation for the speed dependence of the sedimentation coefficient of very high molecular weight DNA. Low-speed centrifugation (3000 rpm) indicates that DNA molecules from the lysate are evenly distributed over values of S_20,w from 0 to 514S. This distribution is very sensitive to changes in speed of centrifugation and is transformed into a bimodal distribution at 12,080 rpm. Analysis of this transformation allows us to postulate that perhaps 55% of the DNA in the lysate may have molecular weights in excess of 40 × 10⁹ g/mol. Some of these molecules may also possess a variety of configurations including partially replicated branched structures.     

An inhibition enzyme immunoassay for myelin basic protein

An improved Enzyme Immunoassay for Myelin Basic Protein is described. Myelin Basic Protein covalently attached to glass balls, and Myelin Basic Protein in samples compete with each other for binding of a peroxidase conjugated anti Myelin Basic Protein antibody. The peroxidase activity on the balls is then inversely proportional to the amount of Myelin Basic Protein in the sample. A detection limit of 0.6 ng/ml is demonstrated for diluent or spinal fluid. For plasma a dilution step increases this to 1.8 ng/ml. Both the coated balls and the peroxidase conjugate are stable for long periods. The assay requires no expensive equipment. Although the assay appears to be valid for subcellular fractions spinal fluid and plasma, successful detection of Myelin Basic Protection peptides in clinical samples may require careful selection of suitable antisera. The assay would be very suitable for eventual use with an appropriate monoclonal antibody.     

Deposition and transport of radionuclides within an upland drainage basin in mid-Wales

The deposition of radiocaesium from nuclear weapons testing and the Chernobyl accident upon the Llyn Llygad Rheidol catchment in mid-Wales is described. Inventories of soil cores from the catchment support estimates of total atmospheric fallout. The mean inventory of weapons testing ¹³⁷Cs in lake sediment cores is broadly similar to that in soil cores. The inventory of Chernobyl fallout in sediment cores is significantly lower and raises questions concerning the residence time of ¹³⁷Cs in the catchment soils and lake waters. ¹³⁷Cs and ²⁴¹Am activities in a sediment core record the 1963 peak of fallout from nuclear weapons testing. The association of the peak activities of ¹³⁷Cs and ²⁴¹Am in the sediments with the falloot maximum is confirmed by ²¹⁰Pb dating. The ²¹⁰Pb dates also reveal a significant increase in sediment accumulation rates over the past 20 years.     

Control of cell volume in the J774 macrophage by microtubule disassembly and cyclic AMP

We have explored the possibilities that cell volume is regulated by the status of microtubule assembly and cyclic AMP metabolism and may be coordinated with shape change. Treatment of J774.2 mouse macrophages with colchicine caused rapid microtubule disassembly and was associated with a striking increase (from 15-20 to more than 90 percent) in the proportion of cells with a large protuberance at one pole. This provided a simple experimental system in which shape changes occurred in virtually an entire cell population in suspension. Parallel changes in cell volume could then be quantified by isotope dilution techniques. We found that the shape change caused by colchicine was accompanied by a decrease in cell volume of approximately 20 percent. Nocodozole, but not lumicolchicine, caused identical changes in both cell shape and cell volume. The volume loss was not due to cell lysis nor to inhibition of pinocytosis. The mechanism of volume loss was also examined. Colchicine induced a small but reproducible increase in activity of the ouabain-sensitive Na(+), K(+)-dependent ATPase. However, inhibition of this enzyme/transport system by ouabain did not change cell volume nor did it block the colchicines-induced decrease in volume. One the other hand, SITS (4’acetamido, 4-isothiocyano 2,2’ disulfonic acid stilbene), an inhibitor of anion transport, inhibited the effects of colchicines, thus suggesting a role for an anion transport system in cell volume regulation. Because colchicine is known to activate adenylate cyclase in several systems and because cell shape changes are often induced by hormones that elevate cyclic AMP, we also examined the effects of cyclic AMP on cell volume. Agents that act to increase syclic AMP (cholera toxin, which activates adenylate cyclase; IBMX, and inhibitor of phosphodiesterase; and dibutyryl cyclic AMP) all caused a volume decrease comparable to that of colchicine. To define the effective metabolic pathway, we studied two mutants of J774.2, one deficient in adenylate cyclase and the other exhibiting markedly reduced activity of cyclic AMP-dependent protein kinase. Cholera toxin did not produce a volume change in either mutant. Cyclic AMP produced a decrease in the cyclase-deficient line comparable to that in wild type, but did not cause a volume change in the kinase- deficient line. This analysis established separate roles for cyclic AMP and colchicine. The volume decrease induced by cyclic AMP requires the action of a cyclic AMP-dependent protein kinase. Colchicine, on the other hand, induced a comparable volume change in both mutants and wild type, and thus does not require the kinase.     

Leghaemoglobin within bacteroid-enclosing membrane envelopes from soybean root nodules

Methods are reported for the preparation from soybean (Glycine max (L.) Merr.) root nodules, of well-washed, intact membrane envelopes containing bacteroids. The intact envelopes are of much lower density than the bacteroids within and therefore only low speed centrifugation (approx. 150 g) may be used. The optimum osmotic strength is 600 mOsm/kg H₂O. The envelope contents were recovered following mild osmotic shock and-or hard centrifugal packing at >10,000 g. Extracts prepared in this way contained leghaemoglobin (identified spectrophotometrically), low-molecular-weight fluorescent materials and other components which are yet to be identified. Envelope leghaemoglobin did not react with specific antibody until the envelopes were ruptured. ¹³¹I-Labelled leghaemoglobin or bovine serum albumin, added during initial breakage of nodule cells, was not released when envelopes were ruptured to release leghaemoglobin. It is therefore concluded that this leghaemoglobin is located within the envelope space and did not arise from adhering or occluded cytosol leghaemoglobin. Based on the number and dimensions of microscopically intact envelopes in these preparations, the concentration within that space was in the range 178–523 M. Based on these estimates, leghaemoglobin within envelopes represented about one third of the total amount present in the nodule cells. Flat-bed isoelectric focusing of partially-purified envelope leghaemoglobin demonstrated that the latter contained all of the leghaemoglobin components previously reported for soybean nodules and an additional minor component focusing between leghaemoglobins a and b.     

Leghemoglobin. An electron paramagnetic resonance and optical spectral study of the free protein and its complexes with nicotinate and acetate.

Electron paramagnetic resonance (EPR) and optical spectra are used as probes of the heme and its ligands in ferric and ferrous leghemoglobin. The proximal ligand to the heme iron atom of ferric soybean leghemoglobin is identified as imidazole by comparison of the EPR of leghemoglobin hydroxide, azide, and cyanide with the corresponding derivatives of human hemoglobin. Optical spectra show that ferric soybean leghemoglobin near room temperature is almost entirely in the high spin state. At 77 K the optical spectrum is that of a low spin compound, while at 1.6 K the EPR is that of a low spin form resembling bis-imidazole heme. Acetate binds to ferric leghemoglobin to form a high spin complex as judged from the optical spectrum. The EPR of this complex is that of high spin ferric heme in a nearly axial environment. The complexes of ferrous leghemoglobin with substituted pyridines exhibit optical absorption maxima near 685 nm, whose absorption maxima and extinctions are strongly dependent on the nature of the substitutents of the pyridine ring; electron withdrawing groups on the pyridine ring shift the absorption maxima to lower energy. A crystal field analysis of the EPR of nicotinate derivatives of ferric leghemoblobin demonstrates that the pyridine nitrogen is also bound to the heme iron in the ferric state. These findings lead us to picture leghemoglobin as a somewhat flexible molecule in which the transition region between the E and F helices may act as a hinge, opening a small amount at higher temperature to a stable configuration in which the protein is high spin and can accommodate exogenous ligand molecules and closing at low temperature to a second stable configuration in which the protein is low spin and in which close approach of the E helix permits the distal histidine to become the principal sixth ligand.