Peptide purification by gel filtration in aqueous pyridine--ammonia.

A method is described for the purification of peptides by gel filtration on Sephadex. The success of the method is due mainly to the use of 70% ($\text{v}\text{v}$) pyridine-30% ($\text{v}\text{v}$) 1 m aqueous ammonia, an excellent volatile solvent for peptides which does not degrade Sephadex. The method has been used to purify all of the major peptides of cowpea chlorotic mottle virus coat protein, after an initial fractionation by ion-exchange chromatography, and selected separations are used to illustrate the degree of fractionation which can be achieved.     

Extraction of pure ribosomal protein and removal of Coomassie blue from acrylamide gels

A method has been developed to recover pure ribosomal proteins from two-dimensional polyacrylamide electrophoresis slabs. Proteins and Coomassie blue were extracted from the gels and separated by passing them through a Sephadex G-25 column. All the steps were performed in the presence of 66% ($\text{v}\text{v}$) acetic acid. The technique has been applied to Escherichia coll ribosomal proteins labeled with ¹²⁵I.     

Solubility of the intramitochondrially synthesized protein and other membrane proteins of rat liver mitochondria in acidic or neutral chloroform-methanol

Almost all protein species of submitochondrial particles from rat liver identified by SDS-polyacrylamide gel electrophoresis were extracted into acidic /2 mM/HCl/ chloroform:methanol /2:1, $\text{v}\text{v}$/, whereas a single protein /or lipoprotein/ with molecular weight of 9.000 was extracted into neutral chloroform-methanol mixture. Evidence for intramitochondrial synthesis of this hydrophobic protein in rat liver $\text{in vivo}$ is presented.     

High performance liquid chromatography,chemical laboratory practice: By Heinz Engelhardt. Springer-Verlag,New York/Berlin, 1979. 248 pp. $29.80

A gel filtration method has been developed for the complete removal of sodium dodecyl sulfate (SDS) from proteins and peptides. The protein or peptide (20 μg–10 mg) containing SDS (up to 30–60 mg) is dissolved in a mixture of propionic acid, formic acid, and water (2:1:2, $\text{v}\text{v}$). Under these conditions, protein-SDS (or peptide-SDS) complexes, as well as SDS micelles, are dissociated. Subsequently, protein and SDS can be separated on a small Sephadex G-25 superfine column. The recovery of protein is typically 90% or more.     

Selenium binding proteins in rat tissues

The 100,000 × g extracts of rat intestine and colon were incubated $\text{in}\text{vitro}$ with Na₂[⁷⁵Se]O₃. Chromatography of this material on a Sephadex G-100 column produced three radioactive peaks corresponding to molecular weights of 17,000, 68,000 and > 90,000. The 17,000 peak corresponded to a protein which sedimented in the 2S region of a 5–20% ($\text{w}\text{v}$) linear sucrose density gradient. Selenium binding to this protein was specific, stable and sensitive to thiol inhibitors such as p-chloromercuriphenylsulfonic acid (1 mM) and iodoacetamide (2 mM). Chromatography of rat serum - [⁷⁵Se] complex on Sephadex G-100 yielded only two radioactive peaks that corresponded to molecular weights of 68,000 and > 90,000. The 2S selenium binding protein of intestine and colon may mediate the biological functions of selenium in those tissues.     

Cryopreservation of Plasmodium chabaudi I: Protection by glycerol and dimethyl sulfoxide during cooling and by glucose following thawing

Two additives, glycerol and dimethyl sulfoxide (Me₂SO), were investigated for toxic and protective effects for the intraerythrocytic stages of Plasmodium chabaudi. After incubation for 15 min, at 0 °C in Me₂SO and at 37 °C in glycerol, with various concentrations of these additives, half the blood from each treatment was cryopreserved in glass capillary tubes cooled at approximately 3600 °C min^?1 by plunging into liquid nitrogen. Warming was rapid, approximately 12000 °C min^?1, produced by agitation in a water bath at 40 °C for 1 min. The effect of dilution in phosphate-buffered saline (PBS) supplemented with various concentrations (5 to 25% $\text{v}\text{v}$) of glucose was also investigated in conjunction with the two cryoprotectants. Survival of both the frozen and the unfrozen control parasites was assayed by the mean time taken for the parasitemia in groups of five mice to reach a level of 2% following intraperitoneal injection of 10⁶ parasitized erythrocytes into each mouse. Glycerol was toxic at concentrations above 10% $\text{v}\text{v}$ and Me₂SO above approximately 15%. The use of glucose in the recovery medium resulted in a substantial improvement in the survival of frozen and unfrozen parasites previously incubated in either cryoprotectant. The amount of glucose required varied with the concentration of additive used, and optimum survival of cryopreserved parasites was obtaind with 10% $\text{v}\text{v}$ glycerol or 15% $\text{v}\text{v}$ Me₂SO and with 15% $\text{w}\text{v}$ glucose in the diluent medium.     

Isolation of ACTH1-39,ACTH1-38 and CLIP from the calf anterior pituitary

Calf anterior pituitaries were defatted and homogenized and peptides were adsorbed from the homogenate supernatant onto octadecylsilyl-silica. After elution, the resulting extract was subjected to gradient elution reversed-phase high pressure liquid chromatography (RP-HPLC) using aqueous acetonitrile containing 0.1% ($\text{v}\text{v}$) trifluoroacetic acid (TFA). Radioimmunoassay of column fractions for corticotropin (ACTH) revealed three major areas of immunoreactivity. Each was purified to homogeneity by gradient elution RP-HPLC employing aqueous acetonitrile containing either 0.13% heptafluorobutyric acid ($\text{v}\text{v}$) or 0.1% TFA ($\text{v}\text{v}$). Amino acid analysis and exopeptidase and trypsin digestions revealed the three forms of corticotropin to be ACTH_1–38, corticotropin-like intermediary lobe peptide, (CLIP, ACTH_18–39) and ACTH_1–39. ³H-labeled ACTH_1–39 did not give rise to either ³H-ACTH_1–38 or ³H-CLIP during isolation.     

Effect of Aspergillus flavus mycotoxin on Culex mosquito larvae

An isolate of Aspergillus flavus var. columnaris recovered from moribund larvae of the mosquito Culex peus produced metabolites which caused mortality in larvae of C. peus and C. tarsalis. Production of these toxins in vitro on solid substrate rice was based on culture techniques used in making Ang kak. The effect of incubation time on mycotoxin production was determined for cultures fermented from 3 to 13 days. Mycotoxins produced by isolate FU-051 were extracted by refluxing the spent medium with chloroform. Fractionation on a silicic acid column with a methanol-chloroform (3:97 $\text{v}\text{v}$) solvent mixture yielded two toxic fractions. Toxic activity was determined using a biological assay with mosquito larvae.     

Extraction of lipids from mammalian liver using nontoxic solvents

A simple method for extracting and purifying lipids from rat liver in a single step using nontoxic solvents is described. The method consists of homogenizing the puliverized tissue with a mixture of tricholotrifluoroethane (Cl₂CF-CClF₂) and isopropyl alcohol ($\text{1 : 1,}\text{v}\text{v}$). Just enough water is added to the lipid extract to produce a biphasic system. Pure lipid extract is obtained by isolating the lower layer from the aqueous upper phase which contains the non-lipid materials. The described method compares favourably with that of Folch et al., both quantitatively and qualitatively. The solvent system used also has the advantage of being less toxic than the widely used chloroform/methanol system, which makes it safer for prolonged use. The new method is simple, efficient and reproducible.     

Effects of cytochalasin B on division and calcium carbonate extrusion in a calcareous alga.

Cytochalasin B (CB) (100 μg/ml) reversibly blocked cell division and cuased the formation of abnormal cytoplasmic bodies in the alga Cricosphaera carterae. Concentrations of 20 μg/ml and 40 μg/ml CB were without effects. In the presence of CB, calcified bodies (coccoliths) which form in Golgi vesicles and are normally extruded through the plasma membrane were not extruded and accumulated within the cell. CB appeared to alter the membranes of Golgi vesicles containing coccoliths. DMSO (10% $\text{v}\text{v}$), the solvent for CB, was without effect on cell division and coccolith extrusion. A concentration of 20% $\text{v}\text{v}$ DMSO inhibited cell division irreversibly.     

Effect of detergents on antibody-antigen interaction.

The effect of the different detergent mixtures on immunodiffusion and immunoprecipitation was studied. The anionic detergent sodium dodecyl sulfate at concentrations above 0.2% ($\text{w}\text{v}$) inhibits the reaction between antigen and antibody by more than 90%. Nonionic detergents at a concentration of 1% ($\text{w}\text{v}$) have little or no detectable effect. In contrast, when we used mixtures of various concentrations of ionic and nonionic detergents the inhibitory effect of the ionic detergent decreased.     

Survival of electrical activity of deep frozen fetal mouse hearts after microwave thawing

Hearts removed from 17–19 day fetal mice were frozen in liquid nitrogen and tested for electrical activity after rewarming. After exposure to various cryoprotective agents, hearts were cooled at 0.5–0.7 °C/min. to ?100 °C and then stored in liquid nitrogen for periods between 72 and 216 hr. Exposure to controlled microwaves at 2450 MHz or immersion in a water bath at 25 C was used in thawing. Electrical activity was studied for periods as long as 90 days after subcutaneous implantation into the ear of syngeneic adult mice. Overall, 59% of 54 frozen-thawed fetal hearts showed strong electrical activity after 30 days when the cryoprotective solution that had been used contained 10% ($\text{v}\text{v}$) dimethylsulfoxide (DMSO) and 10% ($\text{v}\text{v}$) fetal calf serum in Hepes buffer. This system consists of a multicellular structure that is nourished by diffusion; it is well suited for the evaluation of different cryoprotective agents and for various thawing techniques.     

Liquid scintillation counting of aqueous samples using Triton-containing scintillants

Markedly unstable count rates were observed using a toluene-Triton (2:1, $\text{v}\text{v}$) scintillant during counting of water-soluble radioactive compounds when < 5% ($\text{v}\text{v}$) water was present, because of the separation of phases. Efficiency correction in these instances could not be made by using ³H₂O as internal standard, because under the same conditions count rates with tritiated water were stable. Increasing water to ≥6% stabilized the count rates. With toluene-Triton (2:1, $\text{v}\text{v}$) scintillant, the water level should preferably be maintained between either 6 and 12 or 18 and 24% for ¹⁴C- and ³H-labeled compounds for counting at 6°C or at ambient temperature (but only between 6 and 12% for ³H counting at room temperature). With a “Tritosol” (Anal. Biochem.63, 555 (1975) modified to contain 35 ml of ethylene glycol, 140 ml of ethanol, 250 ml of Triton X-100, 575 ml of xylene, 3 g of PPO, and ±200 mg of POPOP, water levels of up to 23% were acceptable for ¹⁴C and ³H for counting at room temperature or at 6°C. Within these limitations, with the toluene-Triton or with the modified Tritosol as scintillant, both polar and apolar radioactive compounds exhibited similar efficiencies and gave quench-correction curves, based on the external standard ratio, that were linear for both ¹⁴C and ³H-labeled compounds.     

Microdetermination of norepinephrine, 3,4-dihydroxyphenylethylamine, and 5-hydroxyyptamine from single extracts of specific rat brain areas

The conditions reported by Toru and Aprison (4) for extracting ACh in specific brain areas were tested to determine whether 5-HT, NE, and dopamine were also extracted quantitatively. It was found that the extraction solution used in brain ACh determinations, 15% 1N formic acid plus 85% acetone ($\text{v}\text{v}$), was also excellent for extraction of NE, 5-HT, and dopamine from different brain areas. Experimental conditions are given for the microdetermination of all three biogenic amines in such a single extract of a specific rat brain area. The methods are based on previously published fluorometric methods; these have been scaled down or modified slightly to permit analyses of small aliquots. The concentration of 5-HT, NE, and dopamine in the telencephalon, diencephalon plus mesencephalon, pons plus medulla oblongata, and cerebellum of the rat are also reported using the described micromethods after extraction with 15% 1 N formic acid plus 85% acetone ($\text{v}\text{v}$).     

Membrane leakage of solutes after thermal shock or freezing

Washed human erythrocytes were cooled at different rates from +37 °C to 0 °C in hypertonic solutions of either NaCl (1.2 m) or of a mixture of sucrose (40% $\text{w}\text{v}$) with NaCl (2.53% $\text{w}\text{v}$). Thermal shock hemolysis was measured and the surviving cells were examined for their mass and cell water content and also for net movements of sodium, potassium, and ¹⁴C-sucrose. The results were compared with those obtained from cells in sucrose (40% $\text{w}\text{v}$) initially, cooled at different rates to ?196 °C and rapidly thawed.The cells cooled to 0 °C in NaCl (1.2 m) showed maximal hemolysis at the fastest cooling rate studied (39 °C/min). In addition in the surviving cells this cooling rate induced the greatest uptake of ¹⁴C-sucrose and increase in cell water and cell mass and also entry of sodium and loss of cell potassium. A different dependence on cooling rate was seen with the cells cooled from +37 °C to 0 °C in sucrose (40% $\text{w}\text{v}$) with NaCl (2.53% $\text{w}\text{v}$). In this solution, survival decreased both at slow and fast cooling rates correlating with the greatest uptake of cell sucrose and increase in cell water. There was extensive loss of cell potassium and uptake of sodium at all cooling rates, the cation concentrations across the cell membrane approaching unity.The cells frozen to ?196 °C at different cooling rates in sucrose (40% $\text{w}\text{v}$) initially, also showed sucrose and water entry on thawing together with a loss of cell potassium and an uptake of cell sodium. More sucrose entered the cells cooled slowly (1.8 ° C/min) than those cooled rapidly (318 ° C/min).These results show that cooling to 0 °C in hypertonic solutions (thermal shock) and freezing to ?196 °C both induce membrane leaks to sucrose as well as to sodium and potassium. These leaks are not induced by the hypertonic solutions themselves but are due to the effects of the added stress of the temperature reduction on the membranes modified by the hypertonic solutions. The effects of cooling rate are explicable in terms of the different times of exposure to the hypertonic solutions. These results indicate that the damage observed after thermal shock or slow freezing is of a similar nature.     

Effect of protected protein supplements on some testicular traits in Brahman cross bulls

Differences in a number of testicular traits were examined in 12 Brahman cross (F₂ generation $\text{1}\text{2}$ and $\text{3}\text{4}$ BX) bulls fed either poor-quality native pasture (NP) hay or NP hay with a protected protein supplement. Supplementation for 60 days significantly (P < 0.05) increased roughage dry matter intake (7.7 $\text{v}$ 5.6 kg/head/day), enabling maintenance of liveweight, whereas control animals lost 40 kg. There were significant (P < 0.05) decreases in scrotal circumference (1.5cm) and testicular consistency (0.8 score) in the control group, in which testes weights at slaughter were significantly (P < 0.05) less (373 g $\text{v}$ 459 g), with corresponding lower epididymal weights (37.4 g $\text{v}$ 43.5 g). Estimates of daily sperm production per gram (DSPG) were similar for both groups, and testis daily sperm production (DSP) was somewhat but not significantly (P>0.05) lower in the control group (4.3 × 10⁹$\text{v}$ 6.0 × 10⁹) as a result of lower testis weights. Total epididymal sperm storage capacity was also lower in control bulls (17.2 × 10⁹$\text{v}$ 27.0 × 10⁹), but only significantly (P < 0.05) in relation to cauda sperm reserves (8.5 × 10⁹$\text{v}$ 13.6 × 10⁹). Luteinizing hormone (LH) and testosterone responses to gonadotrophin releasing hormone (GnRH) treatment were similar for both groups, although LH responses to GnRH were greater in $\text{1}\text{2}$ BX than in $\text{3}\text{4}$ BX bulls.     

Determination of prednisolone,prednisone, and cortisol in human plasma by high-performance liquid chromatography

A method is described for the measurement of prednisone and prednisolone in human plasma by high-performance liquid chromatography (hplc). An ether/dichloromethane extract (60:40, $\text{v}\text{v}$) of plasma is evaporated to dryness and chromatographed on a 250 × 4.5-mm Whatman Partisil column with uv detection at 239 nm. Prednisone, prednisolone cortisol, and the internal standard dexamethasone are satisfactorily resolved with the elution system of water-saturated dichloromethane/ethanol (95:4, $\text{v}\text{v}$). The hplc method can be used for plasma prednisolone concentrations as low as 25 ng/ml. The values correlate well with those obtained by a radioimmunoassay procedure for prednisolone.     

Determination of inorganic phosphate in the presence of nonionic detergents: A modified isobutanol-benzene extraction method

Inorganic phosphate can be determined either radiometrically or spectrophotometrically after extraction of its complex with molybdate into an organic phase. Triton X-100 interferes with this extraction. Determination of the inorganic phosphate can be carried out in the presence of up to 0.8% ($\text{w}\text{v}$) Triton X-100 by modification of the method: After addition of the silicotungstate, the sample is centrifuged, the yellow oily phase removed, and a sufficient amount of silicotungstate added again. Lubrol WX interfered even more drastically than Triton X-100, but the modified method was effective in the presence of 0.04% ($\text{w}\text{v}$) Lubrol WX. The method was useful in biochemical assays such as those of ATPase and cyclic nucleotide-dependent phosphodiesterase.     

Schistosoma mansoni: Cryopreservation of schistosomula by two-step addition of ethanediol and rapid cooling

A simple, inexpensive, and highly effective technique for the Cryopreservation of schistosomula of Schistosoma mansoni is outlined by experiments designed to clarify the role of each of the steps involved. The technique consists of incubating schistosomula in 10% ($\text{v}\text{v}$) ethanediol for 10 min at 37 C followed by 5 min at 0 C and for a further 10 min in 35% ($\text{v}\text{v}$) ethanediol at 0 C. The schistosomular suspension is then aliquotted in 20-μl drops onto 40 × 5.5-mm glass slivers prepared from standard microscope coverslips, each drop being spread out to cover an area of approximately 15 × 4 mm. These glass slivers are then dropped directly into liquid nitrogen giving a cooling rate of approximately 5000 C min^?1. Survival is further improved if the schistosomula are at least 90 min old before Cryopreservation and if the frozen organisms are thawed in culture medium prewarmed to +42 C. Levels of survival obtained with this technique are consistently high: 44 to 60% as assessed by motility. From 400 ± 11 cryopreserved schistosomula injected intramuscularly into eight mice, a mean of 54.5 ± 16.3 adult worms were recovered representing an infection level of 13.7%, and which in turn represents 47.4% of the unfrozen control level.     

The effects of different concentrations of glycerol and dimethylsulfoxide on the metabolic activities of kidney slices

Kidney slices either were exposed to the cryoprotectants for 1 hr at room temperature and subsequently washed and incubated in fresh KR buffer containing only the radioactive metabolic tracers, or were immediately incubated for 2 hr at 37 °C in KR buffer containing the cryoprotectant and the tracers. Exposure to glycerol by incubation of kidney slices in Krebs-Ringer bicarbonate buffer containing varying concentrations of glycerol from 0 to 70% ($\text{v}\text{v}$) resulted in a pronounced inhibitory effect on the protein synthesizing activity, while thymidine incorporation into DNA and the α-aminoisobutyric acid uptake through the cell membranes were less affected. Exposure of the tissue to buffer containing dimethylsulfoxide (Me₂SO) in concentrations of 10 to 20% ($\text{v}\text{v}$) resulted in a stimulatory effect on metabolism. At higher concentrations, Me₂SO was toxic resulting in damaging effects on protein and DNA synthesis as well as on membrane integrity. The stimulatory effects of exposure to low concentrations of Me₂SO on protein and DNA synthesis in kidney slices were concluded to be the result of an increased transport of precursors through the cell membranes.