Activation of antifreeze proteins from larvae of the beetle Dendroides canadensis

Summary Purified antifreeze proteins (AFPs) from the larvae of the beetle Dendroides canadensis do not produce the high levels of antifreeze activity seen in the hemolymph of overwintering larvae, even when the purified AFPs are assayed at very high concentrations. However, addition of certain proteins or agar (at concentrations sufficiently low that the gel state does not result) to the Dendroides AFP resulted in a 2–3-fold increase in activity. A 70-kDa protein with AFP-activating capabilities was purified from Dendroides larvae. Addition of this endogenous activator protein to a 4 mg·ml^-1 solution of AFP increased the activity of the AFPs to values comparable to those of the hemolymph of overwintering larvae. Data derived from a modified immunoblot technique demonstrate that the activators bind to the AFP, or vice versa. Formation of this association must allow the AFP to block ice crystal growth by binding to the surface of potential seed crystals in the normal fashion. However, because the AFP-activator complex is much larger than the AFP alone, the complex probably blocks a greater surface area of the crystal and is thus a more efficient antifreeze.Abbreviations AFP antifreeze protein - BSA bovine serum albumine - DEAE diethylaminoethyl - Ig immunoglubolin - LPIN lipoprotein ice nucleator - PIN protein ice nucleator - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - TH thermal hysteresis     

Experimental effects of elevated salinity on three benthic invertebrates in Pyramid Lake,Nevada

Salinity of Pyramid Lake increased from 3.7 to 5.5 between 1933 and 1980. Concern over future reductions in overall species richness prompted experiments to assess responses of dominant lake organisms to elevated salinity. Salinity tolerances of three important benthic invertebrates, Hyalella aztecta, Chironomus utahensis, and Heterocypris sp., were tested in controlled laboratory bioassays and also in a semi-natural environment consisting of large (47 m³) mesocosms.Densities of H. azteca in mesocosms were significantly lower at salinities of 8.0 and 11.0 compared with 5.6 controls in year one, but not in 8.5 salinity mesocosms in year two. The 96-h LC₅₀ for H. azteca was high at 19.5. Short-term mortalities of C. utahensis were 100% at salinities of 13.3 and greater. Fifty-seven percent fewer larvae matured from third to fourth instar at 8.9 than at 5.5 salinity in 17 day subacute bioassays. Furthermore, larval chironomid densities and emergence of adults from mesocosms were significantly reduced at salinities of 8.0 and higher compared with controls. Mortality of Heterocypris sp. was 50% at a salinity of 18.6 in laboratory bioassays and populations in mesocosms ranged between 40 and 100% lower at salinities of 8.0 and 11.0 than in controls.Multiple generation mesocosm experiments indicated all three invertebrates were more sensitive to elevated salinity than results of short-term bioassays. Our studies suggest populations of these invertebrates may be reduced from present levels if Pyramid Lake's salinity were to double, although none are expected to be extirpated. Food habit shifts and reduced production of lake fishes are likely consequences of salinity-induced disruption in the benthic invertebrate forage base.     

Effect of Meloidogyne incognita and Importance of the Inoculum on the Yield of Eggplant

The relationship between population densities of race 1 of Meloidogyne incognita and yield of eggplant was studied. Microplots were infested with finely chopped nematode-infected pepper roots to give population densities of 0, 0.062, 0.125, 0.25, 0.50, 1, 2, 4, 8, 16, 32, 64, and 128 eggs and juveniles/cm³ soil. Both plant growth and yield were suppressed by the nematode. A tolerance limit of 0.054 eggs and juveniles/cm³ soil and a minimum relative yield of 0.05 at four or more eggs and juveniles/cm³ soil were derived by fitting the data with the equation y = m + (1 - m)z^P⁻T. Maximum nematode reproduction rate was 12,300. Hatch of eggs from egg masses in water or from sodium hypochlorite dissolved egg masses was similar (41% and 39%), but egg viability was significantly greater from egg masses in water (58%) than from sodium hypochlorite dissolved egg masses (12%) after 4 weeks. Greater numbers of nematodes were collected from roots of tomatoes from soil infested with entire egg masses than from tomato roots from soil infested with egg masses dissolved by sodium hypochlorite.     

Effects of NaCl stress on proline and cation accumulation in salt sensitive and tolerant turfgrasses

Summary Concentrations of proline, sodium and potassium in shoot tissues of five turfgrass species were measured following exposure to 170 mM NaCl salinity stress. Salt tolerant ‘Fults’ alkaligrass and ‘Dawson’ red fescue restricted the accumulation of Na-ions to significatnly low levels compared to the salt sensitive Kentucky bluegrasses (‘Adelphi’ and ‘Ram I’) and ‘Jamestown’ red fescue. Accumulation of proline began in all species within 24 h of initiation of salt stress but at a more rapid rate and higher overall concentration for ‘Fults’ alkaligrass. Proline levels were variable and too low in relation to sodium accumulations to have any significant osmoregulatory role in salt tolerance among all cultivars tested with the possible exception of alkaligrass.     

Calcium-Activated Neutral Proteinase of Human Brain: Subunit Structure and Enzymatic Properties of Multiple Molecular Forms

Abstract Calcium-activated neutral proteinase (CANP) was purified 2,625-fold from postmortem human cerebral cortex by a procedure involving chromatography on diethylaminoethyl (DEAE)-cellulose, phenyl-Sepharose, Ultrogel AcA-44, and DEAE-Biogel A. The major active form of CANP exhibited a molecular weight of 94–100 kilodaltons (Kd) by gel filtration on Sephacryl 300 and consisted of 78-Kd and 27-Kd subunits. Two-dimensional gel electrophoresis resolved the small subunit into two molecular species with different isoelectric points. CANP degraded most human cytoskeletal proteins but was particularly active toward fodrin and the neurofilament protein subunits (145 Kd > 200 Kd > 70 Kd). The enzyme required 175 μMCa²⁺ for half-maximal activation and 2 mM Ca²⁺ for optimal activity toward [methl-¹⁴C]azocasein. Other divalent metal ions were poor activators of the enzyme, and some, including copper, lead, and zinc, strongly inhibited the enzyme. Aluminum, a neurotoxic ion that induces neurofilament accumulations in mammalian brain, inhibited the enzyme 47% at 1 mM and 100% at 5 mM A second CANP form lacking the 27-Kd subunit was partially resolved from the 100-Kd heterodimer during DEAE-Biogel A chromatography. The 78-Kd monomer exhibited the same specific activity, calcium ion requirement, pH optimum, and specificity for cytoskeletal proteins as the 100-Kd heterodimer, suggesting that the 27-Kd subunit is not essential for the major catalytic properties of the enzyme. The rapid autolysis of the 27-Kd subunit to a 18-Kd intermediate when CANP is exposed to calcium may explain differences between our results and previous reports, which describe brain mCANP in other species as a 76-80-Kd monomer or a heterodimer containing 76-80-Kd and 17-20-Kd subunits. The similarity of the 100-Kd human brain CANP to CANPs in nonneural tissues indicates that the heterodimeric form is relatively conserved among various tissues and species.     

Differential response of Trifolium species to 4-(2,4-dichlorophenoxy)butyric acid treatments

The differential response of white clover ( Trifolium repens L. cv. Regal Ladino) and berseem clover ( Trifolium alexandrinum L. cv. Mississippi ecotype) was investigated by treating greenhouse cultured plants with 4-(2,4-dichlorophenoxy)butyric acid (2,4-DB). Berseem clover plants were significantly injured by a treatment concentration of 0.6 kg ha^-1 of 2,4-DB, whereas white clover plants were not injured by treatment levels below 2.4 kg ha^-1. The metabolism of 2,4-DB in cell suspension cultures of white clover and berseem clover was investigated using [ring-¹⁴C]-2,4-DB and non-labeled 2,4-DB. White clover cell cultures metabolized ca 4-fold more 2,4-DB than berseem cultures over a 44-h treatment period. The decrease in berseem cell population was 4-fold greater than the decrease in white clover cell population in response to the 8 μ M 2,4-DB treatment. The herbicide and its [ring-¹⁴C]-labeled metabolites were isolated from treated cells and medium after 44 h by partition and thin-layer chromatography. White clover cells metabolized 90% of the [¹⁴C]-2,4-DB and berseem clover cells metabolized 22% of the herbicide. The major portion of the radiolabel was in the glycoside fractions from extracts of both species. The differential response of Trifolium species to 2,4-DB is implied to be due to the differential rate of 2,4-DB metabolism to a glycoside by the clover plants.