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Studies in sheep on the absorption of magnesium from a low molecular weight fraction of the reticulo-rumen contents.

1. Six sheep, three animals per diet, were prepared with rumen fistulas and fed on frozen grass or grass-maize pellets to give magnesium intakes of 1.79 and 2.23 g/d respectively. The mean apparent availabilities of Mg in sheep fed on frozen grass and grass-maize pellets were 0.31 and 0.36 respectively. 2. The rumen contents were fractionated by straining the digesta through linen cloth and then differentially centrifuged to give 20,000 g and 100,000 g supernatant fractions. 3. In all sheep, regardless of diet, at 4 and 16 h after a meal, 50 and 60% respectively of the total Mg in the rumen contents was found in strained rumen fluid while 30 and 38% respectively of the total Mg was found in the 100,000 g supernatant fraction. 4. The net absorption of Mg from the temporarily isolated and washed reticulo-rumen was studied using either 100,000 g supernatant fractions of rumen contents from sheep fed on one or other of the two diets, or inorganic buffers containing the same concentration of Mg and other macroelements. 5. The Mg was readily absorbed from the 100,000 g supernatant fraction placed in the rumen with the rate of absorption being 7.3 mumol/l per min (505 mg/d) from the supernatant fraction obtained from sheep fed on frozen grass and 11.3 mumol/l per min (781 mg/d) from the supernatant fraction from sheep fed on grass-maize pellets. In the same sheep, the previously described rates of Mg absorption from the 100,000 g supernatant fraction were similar to those obtained from the comparable inorganic buffers. 6. The effects of varying concentrations of potassium and sodium on the net absorption rate of Mg (as 24Mg) and on the one-way efflux of Mg (as 28Mg) from supernatant fractions or rumen fluid and inorganic buffers were investigated using the temporarily-isolated and washed rumen in three sheep. Although the net absorption rate of 24Mg from supernatant fractions or buffers containing similar K concentrations varied significantly between sheep, a similar percentage decrease in the absorption rates of both 24Mg and 28Mg was found for each sheep as the K concentration was increased. 7. One pair of sheep was fed on the frozen grass and the other pair was fed on the grass-maize pellets. Their daily intakes of K were then increased to 50 g/d for 14 d by intrarumen infusion of potassium chloride. In three of the four sheep the plasma Mg concentration fell within 12 h of the start of the KCl administration.(ABSTRACT TRUNCATED AT 400 WORDS)

Absorption↗

The shortening fraction of myocardial fibers and its layered distribution, as derived from cine-MR imaged left ventriculograms. An approach for evaluating globar left ventricular function.

PURPOSE: To evaluate the mean shortening fraction and its SD through the wall calculated from multiple cine-MR views, as an estimate of left ventricular globar function. MATERIAL AND METHODS: The average myocardial fiber shortening fraction was calculated by means of a simple truncated ellipsoid nested shell model. Left ventricular parameters, acquired by cine-MR imaging, from 20 healthy volunteers served as input. Fiber angles, ventricular torsion and a gradient increase in wall thickening from epicard to endocard were part of the model. RESULTS: The average fiber shortening fraction was 0.203 (0.158-0.246) +/- 0.021 diastolic lengths. It varied only moderately with variations in fiber angle values and not at all when the torsion angles were varied within physiological limits. The average shortening fraction correlates well with the systolic increase in chamber oblonguity (k = 0.837), with the ejection fraction (k = 0.877), and even better with the calculated wall thickening (k = 0.973). The average epicardial shortening fraction 0.169 (0.142-0.202) +/- 0.016 increased gradually through the wall to the endocardial value 0.250 (0.212-0.290) +/- 0.024. The increase in chamber length-width ratio from diastole to systole reduced the SD of the shortening fraction through the wall layers to a minimum. CONCLUSION: The fiber shortening fraction expresses the layered contraction of the myocardial wall, the wall thickening, and also the endocardial wall motion. The ejection fraction expresses only the latter. The shortening fraction and its SD through the wall may prove a valuable additional tool for estimating ventricular globar function.

Adult↗

Apoptosis, energy metabolism, and fraction of radiobiologically hypoxic cells: a study of human melanoma multicellular spheroids.

The magnitude of the fraction of radiobiologically hypoxic cells in tumours is generally believed to reflect the efficiency of the vascular network. Theoretical studies have suggested that the hypoxic fraction might also be influenced by biological properties of the tumour cells. Quantitative experimental results of cell energy metabolism, hypoxia- induced apoptosis, and radiobiological hypoxia are reported here. Human melanoma multicellular spheroids (BEX-c and WIX-c) were used as tumour models to avoid confounding effects of the vascular network. Radiobiological studies showed that the fractions of hypoxic cells in 1000-microM spheroids were 32 +/- 12% (BEX-c) and 2.5 +/- 1.1% (WIX-c). The spheroid hypoxic volume fractions (28 +/- 6% (BEX-c) and 1.4 +/- 7% (WIX-c)), calculated from the rate of oxygen consumption per cell, the cell packing density, and the thickness of the viable rim, were similar to the fractions of radiobiologically hypoxic cells. Large differences between tumours in fraction of hypoxic cells are therefore not necessarily a result of differences in the efficiency of the vascular network. Studies of monolayer cell cultures, performed to identify the biological properties of the BEX-c and WIX-c cells leading to this large difference in fraction of hypoxic cells, gave the following results: (1) WIX-c showed lower cell surviving fractions after exposure to hypoxia than BEX-c, (2) WIX-c showed higher glucose uptake and lactate release rates than BEX-c both under aerobic and hypoxic conditions, and (3) hypoxia induced apoptosis in WIX-c but not in BEX-c. These observations suggested that the difference between BEX-c and WIX-c spheroids in fraction of hypoxic cells resulted partly from differences in cell energy metabolism and partly from a difference in capacity to retain viability under hypoxic stress. The induction of apoptosis by hypoxia was identified as a phenomenon which has an important influence on the magnitude of the fraction of radiobiologically hypoxic cells in multicellular spheroids.

Apoptosis↗

Response of the central nervous system to fractionated boron neutron capture irradiation: studies with borocaptate sodium.

The response of the central nervous system (CNS) to fractionated doses of boron neutron capture (BNC) irradiation was assessed using a rat spinal cord model. The thermal neutron beam at the Brookhaven Medical Research Reactor (BMRR) was used for the spinal cord irradiations, with borocaptate sodium (BSH) as the neutron capture agent. Irradiations were given as a single dose or as two or four equal fractions. The ED50 for radiation-induced myeloparesis, as indicated by limb paralysis within 7 months, after a single exposure to thermal neutrons in the presence of BSH (blood boron-10 content approximately 70 micrograms/g) was 27.2 +/- 0.9 Gy. This was expressed as the total physical dose to the blood. Dividing the radiation dose into two consecutive daily fractions or four fractions given over 1 week, resulted in ED50 = 32.0 +/- 1.4 and 31.5 +/- 0.4 Gy respectively. Although there was no significant dose sparing in moving from two to four fractions, there was a dose increment of approximately 17% as compared with single-dose irradiation. The variation in the relative biological effectiveness of the thermal neutron beam, with dose per fraction, was established using data from a previous study with single and fractionated doses of thermal neutrons in the absence of a neutron capture agent. This varied from 1.40 to 3.74 for thermal neutron dose per fraction in the range 13.6-1.5 Gy. Previously published CBE factors for both BSH and BPA have been recalculated in the present report to take into account the change in the RBE of the thermal neutron beam with dose. In all cases the recalculated CBE factors were lower than those obtained previously. Values for this parameter increased with fraction number. In the case of BSH, the CBE factor increased from 0.36 +/- 0.03 after a single-dose to 0.51 +/- 0.06 after four fractions.

Animals↗

Pathways and kinetics of redistribution of cobalt among solid-phase fractions in arid-zone soils under saturated regime.

An adequate supply of Co in pasture is important to the health of grazing animals. Bio-availability of Co in soils is largely depended upon its distribution among solid-phase fractions. Distribution of cobalt in six arid-zone soils and its redistribution among the solid-phase fractions during long-term saturated paste incubation were studied. Cobalt was fractionated by a selective sequential dissolution procedure into six empirically defined fractions. Concentrations of total Co and Mn or Fe, and Co and Mn fractionation pattern were strongly correlated in the soils. During saturated incubation, Co in soils was redistributed mainly from the Mn oxide bound, and to some extent, Fe oxide bound and organic matter bound fractions into the carbonate bound fraction. During saturated incubation, significant correlations were found between concentrations of Co and Mn in the Mn oxide bound, Fe oxide bound and carbonate bound fractions. Also, significant correlations between concentrations of Co and Fe in the Fe oxide bound fraction were present. However, a negative correlation between concentrations of Co and Fe in the Mn oxide bound fraction was observed. The rates of redistribution of Co between these solid-phase components were initially high: major changes occurred in the first 3 days in the sandy soil and the first 18 days in the loessial soil. Afterwards, the rates of change slowed but changes in redistribution continued during the rest of the study period of one year.

Adsorption↗

Correlations between plasma protein fractions, antibody titers, and the passive transfer of delayed and immediate cutaneous reactivity to tuberculin PPD and tuberculopolysaccharides.

GUINEA PIGS SENSITIZED WITH TUBERCLE BACILLI DEMONSTRATE A DUAL ALLERGIC RESPONSE MEDIATED BY TWO CHEMICALLY DISTINCT PLASMA FRACTIONS: 1. Antibody to tuberculopolysaccharide is located exclusively in fraction II (gamma globulin). This fraction will passively transfer systemic anaphylaxis and urticarial type skin reactivity to tuberculopolysaccharide, and contains the Middlebrook-Dubos antibody. 2. Antibody to tuberculoprotein is contained exclusively in a new plasma fraction called fraction IV-10. By Cohn's Method X, fraction IV-10 is a part of fraction IV (alpha globulin) and to a lesser extent V (albumin). This fraction will passively transfer to normal guinea pigs a delayed type skin sensitivity to tuberculin PPD which is maximal between 18 and 30 hours, and it contains the Boyden antibody. When fractions II and IV-10 are combined, the antibody to tuberculopolysaccharide inhibits the passive transfer of delayed type reactivity. Combination of these two fractions does not alter their separate in vitro hemagglutinating properties. Adsorption of IV-10 with Boyden sensitized cells removes its ability to transfer delayed type tuberculin sensitivity. Adsorption of II with Middlebrook-Dubos-sensitized cells removes its capacity to effect passive transfer of immediate type reactivity to tuberculopolysaccharides.

Animals↗

Shortening fraction: its dependence on the Starling mechanism.

The influence of increasing muscle length (ML) from L0 to 15 to 20% of L0 and calcium concentration (Ca2+) from 1.34 to 10 mmol . litre-1 on shortening fraction has been analysed in cat papillary muscles. Shortening fraction was calculated by dividing the amount of shortening by the muscle length at which that shortening occurred. When the muscle shortened at contrast total load, increasing muscle length from approximately or equal to L0 to approximately or equal to 15% above L0, increased the shortening fraction from 0.2 +/- 0.1% to 7.1 +/- 0.7% (P less than 0.01) and from 1.0 +/- 0.5% to 12.2 +/- 0.5% (P less than 0.01) at low and high (Ca2+) respectively. The highest shortening fraction values obtained (7 and 12%) correspond to calculated ejection fraction values of 20 and 32% respectively. At a given muscle length, increasing (Ca2+) significantly increased the shortening fraction (P less than 0.01). At constant afterload the shortening fraction increased from 3.5 +/- 1% to 9.1 +/- 1.9% when the muscle length changed from approximately or equal to 5% to approximately or equal to 20% above L0 and from 3,3 +/- 1.6% to 14.3 +/- 0.7% when the muscle was stretched from L0 to approximately or equal to 20% above L0 at low and high calcium respectively. Shortening fraction values of 9 and 14% correspond to calculated ejection fraction values of 25 and 37% respectively. The results indicate that the shortening fraction is altered not only by changes in cardiac contractility but also by the Starling mechanism.

Animals↗

Segregation of rapidly acetylated histones into a chromatin fraction released from intact nuclei by the action of micrococcal nuclease.

It has been previously shown that micrococcal nuclease digestion and subsequent fractionation of hen oviduct nuclei generates fractions enriched (first supernatant fraction - 1SF) and depleted (second supernatant fraction - 2SF) in ovalbumin genes, while a third fraction, the pellet fraction, contains about the same level of this gene as whole chromatin (Bloom and Anderson (1978) Cell 15, 141-150). We have utilized this fractionation method in an attempt to assess the extent and kinetics of histone acetylation associated with chromatin from the 1SF, 2SF, and pellet fraction. Hepatoma Tissue Culture (HTC) cells were labelled for 30 minutes in vivo with 3H-acetate, nuclei isolated and the chromatin fractionated. The specific activity of the histones in the 1SF was slightly greater than that of the 2SF (1.2 to 1.6 fold difference) independent of the length of nuclease digestion. If the labelling period is followed by short (10 to 60 minute) treatment of the cells with sodium butyrate, the more rapidly as well as more extensively acetylated histones are also preferentially found in the 1SF. This is in part the result of segregation of chromatin particles into the 1SF as the histones associated with these particles become hyperacetylated. That is, the extent of histone acetylation regulates the distribution of chromatin in the 1SF, 2SF and pellet fraction.

Acetates↗

Additional predictive value of both left and right ventricular ejection fractions on long-term survival in idiopathic dilated cardiomyopathy.

BACKGROUND: In idiopathic dilated cardiomyopathy, long-term outcome is poor and left ventricular ejection fraction is a major powerful predictor of survival. However, right ventricular function might also play an important role in the long-term prognosis of this disease. AIM: The aim of this study was to determine the role of right ventricular parameters, mainly right ventricular ejection fraction, on survival in idiopathic cardiomyopathy. METHODS: We prospectively assessed long-term follow-up and predictors of survival in 62 consecutive patients referred from 1990 to 1992 for evaluation of idiopathic dilated cardiomyopathy, including haemodynamic evaluation, thermodilution right ventricular ejection fraction and volume measurements. RESULTS: At the time of catheterization, dyspnoea class III or IV was present in 60% of the patients, atrial fibrillation in 19% and complete left bundle branch block in 35%. Left ventricular ejection fraction was 30 +/- 10% and right ventricular ejection fraction was 30 +/- 16%. During follow-up (2.2 +/- 1.3 years), 15 patients (24%) had heart transplantation and nine (14%) died before cardiac transplantation. Cumulative survival rate without heart transplantation was 74% and 56% at 1 and 4 years, respectively. In univariate analysis, survival was related to: dyspnoea class I or II (P < 0.04), absence of complete left bundle branch block (P < 0.05), administration of lower doses of furosemide (P < 0.01), high left ventricular ejection fraction (P < 0.001), low pulmonary artery pressure (P < 0.002), high cardiac index (P < 0.006), and low right ventricular volumes (P < 0.001). Multivariate analysis showed only two independent predictors of survival: left ventricular ejection fraction (P < 0.001) and right ventricular ejection fraction (P < 0.004). CONCLUSION: In addition to left ventricular ejection fraction, right ventricular ejection fraction appears to be a complementary predictor of survival in idiopathic dilated cardiomyopathy, suggesting the importance of assessing right ventricular function in this disease.

Adult↗

Soluble proteins from fowl feather keratin. I. Fractionation and properties.

A method is described for the fractionation of reduced and alkylated proteins of fowl feather. Fowl feather extracts were chromatographed on a Sephadex G-75 column in 4 M urea containing 1 M NaCl and separated into four fractions, GF-1, 2, 3, and 4. The elution patterns were used to compare the components of different feather parts, barbs, rachis + medulla, and calamus. In all cases, GF-3 was the main fraction and the percentages with respect to the total peak area found for barbs, rachis + medulla, and calamus were about 65%, 74%, and 93%, respectively. Each of the fractions was examined by polyacrylamide disc gel electrophoresis and all were heterogeneous. The slowly moving bands mainly corresponded to fraction GF-1, intermediate bands to GF-2 and 3, and faster bands to GF-4. Many other polypeptide chains, which have not been found previously, were newly separated from three minor fractions of fowl body feather. The molecular weights of fractions GF-2 and 3 were estimated by calibrated gel filtration to be 33,000 and 10,500, respectively. Marked differences were found in the amino acid compositions of various fractions from fowl feather. The GF-1 fraction and insoluble residue had very similar compositions; in both cases the contents of serine, glycine, and proline were lower and those of helix-favoring amino acids, namely, lysine, tyrosine, and methionine, were higher than those found in other fractions.

Alkylation↗

Guar meal germ and hull fractions differently affect growth performance and intestinal viscosity of broiler chickens.

High concentrations of guar meal in poultry diets deleteriously affect growth, feed intake, and digesta viscosity. These effects are attributed to residual gum in the meal. A 2 x 5 factorial experiment investigated the impacts of two guar meal fractions (germ and hull) at five inclusion levels (0, 2.5, 5.0, 7.5, and 10.0%) on intestinal viscosity, measures of growth, and feed conversion in broiler chickens fed to 20 d of age. Growth and feed conversion ratio were not affected by inclusion of as much as 7.5% of the germ fraction into poultry diets, while inclusion of the hull fraction reduced growth at all concentrations. The hull fraction increased intestinal viscosity at all inclusion levels fed, although feed conversion was not affected until the inclusion rate exceeded 5.0%. The germ fraction significantly increased intestinal viscosity at 7.5 and 10% inclusion rates. When germ fraction was fed, relative organ weights remained constant through all concentrations except for the ventriculus and duodenum at 7.5 and 10% inclusion levels. Relative pancreas weight was significantly increased at the 10% level of the hull fraction. Increases in intestinal viscosity corresponded with growth depression. These results suggest that residual gum was responsible for some deleterious effects seen when guar meal was fed. The germ fraction was a superior ingredient when compared with the hull fraction. The guar meal germ fraction constituting as much as 7.5% of the diet supported growth and feed conversion measures similar to those observed with a typical corn-soybean poultry ration.

Animal Nutritional Physiological Phenomena↗

Intra- and interindividual variability in the free fraction of cyclosporine in plasma in recipients of renal transplants.

The protein binding of cyclosporine (CyA) in plasma was studied in serial samples for 6 months after transplantation in 66 renal transplant recipients. Analysis was performed with a recently developed method of equilibrium dialysis in steel chambers. Among the 1,848 samples, the free fraction ranged from 0.5 to 4.2%, with a median of 1.30%. The free fraction of CyA was highest immediately after transplantation (1.66 +/- 0.49%). Diabetics had a higher free fraction of CyA than nondiabetics during the first 2 weeks after transplantation. A weak, but significant, covariation was observed between the serum concentration of albumin or bilirubin and the free fraction of CyA (r = -0.47 and r = 0.39, respectively; p less than 0.01). There was also a significant correlation between serum HDL cholesterol (r = -0.39 to -0.48; p less than 0.01), as well as apolipoprotein A1 (r = -0.35 to -0.49; p less than 0.01), and the free fraction of CyA. A significant drop in the free fraction of CyA was observed immediately prior to acute rejection episodes as compared with 1 week earlier (p less than 0.01), but there was no difference in the free fraction of CyA between patients who lost their graft and those who did not. There was an overall eightfold variation in the free fraction of CyA in plasma, with an up to fivefold intraindividual variation and a 2.3-fold variation in the mean free fraction between individuals. A significant covariation was observed between the free fraction of CyA and the concentration of binding proteins in plasma, as well as factors such as time after transplantation, diagnosis, and clinical events.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

Death, mode of death, morbidity and requirement for rehospitalization during 2 years after coronary artery bypass grafting in relation to preoperative ejection fraction.

OBJECTIVE: To describe the impact of ejection fraction on the prognosis during 2 years after coronary artery bypass grafting (CABG). PATIENTS: All patients in western Sweden who underwent CABG without concomitant valve surgery between June 1988 and June 1991. RESULTS: In all, 2121 patients were operated upon and information on ejection fraction was available for 1961 patients (92%). Of these patients, 178 (9%) had an ejection fraction < 40%, 517 (26%) an ejection fraction of 40-59% and 1266 (65%) an ejection fraction > or = 60%. In these groups the mortalities during the first 30 days after CABG were 5.1, 4.3 and 2.2%, respectively (P < 0.01). The corresponding values for mortalities between 30 days and 2 years were 7.7, 4.3 and 3.3%, respectively (P < 0.01). Patients with a lower ejection fraction were more frequently men and more frequently had a history of cardiovascular disease. In multivariate analysis the preoperative ejection fraction was an independent predictor for total 2-year mortality. Patients with a low ejection fraction died more frequently in association with ventricular fibrillation. Morbidity was, with the exception of that for rehospitalization due to heart failure and infection, not associated significantly with the preoperative ejection fraction. CONCLUSION: During the 2 years after CABG a low preoperative ejection fraction was associated with a higher mortality, but the association with morbidity was more complex.

Adult↗

Immunogenic properties of Brucella melitensis cell-wall fractions in BALB/c mice.

The immunogenicity of several Brucella melitensis cell-wall (CW) fractions was tested in BALB/c mice. These CW fractions were smooth lipopolysaccharide (S-LPS) fraction from smooth (S) B. melitensis strain 16M, sodium dodecyl sulphate-insoluble (SDS-I) CW fraction from B. melitensis strain 16M (S) undigested or digested with pepsin, and SDS-I CW fraction from rough (R) B. melitensis strain H38. The B. melitensis SDS-I CW fraction contained two major outer-membrane proteins (OMPs) of 25-27 kDa and 31-34 kDa, peptidoglycan (PG) and a small quantity (1.5%) of LPS. One month after immunisation, mice were challenged with virulent B. melitensis strain H38 (S) and Brucella spleen counts were recorded on days 28 and 49 after challenge. Before challenge, as measured by ELISA, the highest antibody responses to S-LPS were observed in mice immunised with SDS-I CW fraction from B. melitensis strain 16M (S), whether digested with pepsin or undigested. All immunised mice, except those immunised with the SDS-I CW fraction from the R strain, showed higher IgG1 than IgG2a antibody responses to S-LPS (IgG1:IgG2a ratio 3.64-7.71). Antibody responses to the 25-27-kDa OMP were very low, with the highest responses in the mice immunised with the SDS-I CW fraction from the R strain. These results indicated that, in BALB/c mice, these CW fractions probably induced Th2-dependent more than Th1-dependent antibody responses.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Biological activities of lipopolysaccharide fractionated by preparative acrylamide gel electrophoresis.

Lipopolysaccharide from a smooth strain of Salmonella minnesota was fractionated into two major fractions and one intermediate fraction by using sodium dodecylsulfate-polyacrylamide gel electrophoresis. On the basis of the study by Hitchcock and Brown, it was deduced that the top fraction was mainly long O-side chain LPS and the bottom fraction was O-side chain-less LPS. The middle fraction was a mixture of both short O-side chain LPS and O-side chain-less LPS. The antigenic properties and biological activities were not altered in this fractionation procedure. Comparison of the biological activities of the top fraction with those of the bottom fraction revealed that the bottom fraction had higher activity in polyclonal B-cell activation and spleen-swelling effect and that there was no significant difference in adjuvant activity, ability to render macrophages cytotoxic, induction of colony-stimulating factor and the ability to induce the Schwartzmann reaction. It was suggested that O-side chain makes no contribution to the latter biological activities including adjuvant activity of S. minnesota LPS.

Adjuvants, Immunologic↗

Poly(A)-associated RNA from the mitochondrial fraction of the fungus Trichoderma.

Total RNA was extracted from purified mitochondrial and cytoplasmic fractions of germinating conidia of Trichoderma viride and bound to oligo(dT)-cellulose at 22 and 4 degrees C. Under chromatographic conditions which retained very short poly(A) segments (i.e., 4 degrees C), up to 10% of short-term 32PO4-lebeled RNA from the mitochondrial fraction were selectively bound. The poly(A)-associated RNAs from the mitochondrial and cytoplasmic fractions showed the following characteristics. (a) On polyacrylamide gels mitochondrial fraction RNA had a distinctive pattern with a major peak at about 22 S and a smaller one at about 29 S; in contrast, cytoplasmic fraction RNA was heterogenously distributed along the gel. (b) The poly(A) segment released by RNAase digestion of mitochondrial fraction poly(A)-associated RNA migrated on polyacrylamide gels as molecules 20-25-nucleotides long, while that of the cytoplasmic fraction showed an apparent size of 50-60 nucleotides. (c) Mitochondrial fraction RNA bound to oligo(dT)-cellulose in the cold had a guanine + cytosine content of 21% versus 34% for bulk mitochondrial RNA and 48% for cytoplasmic poly(A)-associated RNA; the oligo(dT)-bound RNAs were further identified by their high percentages of adenine residues (46% for the mitochondria and 30% for the cytoplasm). (d) The poly(A)-associated RNA fraction was translated, in vitro, in a cell-free protein-synthesizing system from wheat germ. The products induced by cytoplasmic RNA showed a complex pattern on polyacrylamide gels of many polypeptides ranging in molecular weights from 10000 to 40000. The pattern induced by mitochondrial fraction RNA however, was much simpler, revealing two discrete, main products: a major one at Mr approximately 13000 and a minor one at Mr approximately 20000.

Chromatography, Affinity↗

Lipid A fractions analyzed by a technique involving thin-layer chromatography and enzyme-linked immunosorbent assay.

A modified enzyme-linked immunosorbent assay (ELISA), with alkaline phosphatase as enzyme, was used for the study of antigenicity of lipid A fractions directly on thin-layer chromatographic (TLC) plates. For visualization a gel slab containing the enzyme substrate was placed on the plate containing enzyme-conjugated antibodies. The plate was read by a thin-layer chromatogram spectrophotometer. The immunoassay was both highly specific and quite sensitive. Sensitivity was superior to levels obtained by staining the plate with molybdenum blue (for phosphate) or orcinol (for carbohydrate). Fractions of lipid A from Escherichia coli 0111, Shigella flexneri or Salmonella minnesota R595, after being separated by thin-layer chromatography, were analyzed using rabbit anti-(lipid A) serum. Patterns obtained by scanning the same plates for phosphate staining and for the TLC-ELISA corresponded well. For comparison with TLC-ELISA, an inhibition assay was run using a tube ELISA. The tube ELISA, run in aqueous medium, showed that fractions 6-8 (those having the highest RF values) had the least activities. In contrast, TLC-ELISA did not detect large differences between fractions 2-7. This discrepancy probably reflected limited aqueous solubility of fractions 6 and 7. We conclude that TLC-ELISA might reveal antigenic activities of lipids that could be missed by other methods. The data suggested that all fractions, except for fraction 8, were similar in their antigenicity by TLC-ELISA. Differences in antigenicity between the fractions occurred when the fractions were tested in free form in an aqueous environment and these differences possibly could have been due to different solubilities of individual fractions.

Chromatography, Thin Layer↗

Two hydrophobic low-molecular-mass protein fractions of pulmonary surfactant. Characterization and biophysical activity.

Hydrophobic low-molecular-mass proteins were isolated from minced pig lungs and separated into two fractions. Electrophoresis of protein fraction 1 showed two major bands. Calculations of molecular masses from the electrophoretic mobilities are unreliable because of the extreme hydrophobicity of the peptides. However, the two bands were at positions corresponding to apparent molecular masses of about 3 kDa and 14 kDa, while sequence degradation disclosed only one major structure. Electrophoretic separation of protein fraction 2 revealed one band, at an apparent molecular mass of about 6 kDa. Microheterogeneities at the N terminus of both fractions were observed. However, the two fractions had different N-terminal structures and amino acid compositions. Consequently they are concluded to represent different polypeptides without common segments. Bronchoalveolar lavage from humans also contains surfactant polypeptides and at least the fraction 2 peptide is highly similar in human and porcine surfactants. Artificial surfactant preparations, obtained by recombination of protein fraction 1 or 2 with a mixture of synthetic phospholipids, were evaluated with the pulsating bubble method and in experiments on artificially ventilated premature newborn rabbits. The addition of protein fraction 1 to the phospholipid mixture improved surface adsorption from more than 300 s to about 2 s and reduced minimum surface tension from more than 20 mN/m to nearly 0 as measured with a pulsating bubble. When this surfactant preparation was instilled into the airways of newborn rabbits, the tidal volumes at insufflation pressure 25 cm H2O was increased about twentyfold compared to the volumes obtained in non-treated controls. Preparations based on protein fraction 1 had better in vitro and in vivo properties than those based on protein fraction 2. Both these protein-based preparations were decidedly more effective than phospholipids alone.

Amino Acid Sequence↗