Probing the electronic structure and conformational flexibility of individual light-harvesting 3 complexes by optical single-molecule spectroscopy.

We present fluorescence-excitation spectra of individual light-harvesting 3 (LH3 or B800-820) complexes of Rhodopseudomonas acidophila at 1.2 K. The optical single-molecule studies were employed to investigate the electronic structure as well as the conformational flexibility of the individual pigment-protein complexes. The optical spectra resemble those of individual light-harvesting 2 (LH2) complexes, in agreement with the structural similarity of both types of complexes. Although variations among the LH3 spectra are large, there is a distinct difference in the spectral features of the 800 and 820 nm region that appears in all the complexes studied. In the B800 region 4-6 narrow bands are present whereas in the B820 region a limited number of relatively broad bands are observed. These observations can generally be interpreted in terms of localized excitations in the 800 nm region and delocalized excitations in the 820 nm region. The observed heterogeneous spectral behavior, especially in the B820 band, indicates that the B820 pigments of LH3 are sensitive to light-induced local conformational changes. It is suggested that a rotation of the C(3)-acetyl chain of a BChl a pigment bound to the beta-subunit of the light-harvesting complex is the origin of the conformational flexibility and affects the optical properties of the whole pigment-protein complex.

Features of amino acid metabolism in Moniliformis moniliformis (Acanthocephala) in vitro.

Experiments to investigate the metabolism of glycine, L-glutamic acid and L-aspartic acid by Moniliformis moniliformis were carried out by incubating adult worms aerobically for 3 h at 37 degrees C in Tyrode's solution containing either [U-14C]glycine, L-[U-14C]glutamic acid, L-[U-14C]aspartic acid or L-[4-14C]aspartic acid. Much of the glycine and glutamic acid was absorbed by the worms, but little of either was metabolized. Aspartic acid was readily taken up and metabolized. After incubating with L[U-14C]aspartic acid, most radioactivity was found in ethanol and a volatile compound, presumed to be carbon dioxide, with smaller amounts in lactate, alanine, acetate, malate, glucose and succinate. After incubating with L-[4-14C]aspartic acid, most radioactivity was found in lactate and the presumed CO2 with small amounts in alanine, malate and succinate. No radioactivity was found associated with ethanol or acetate. Possible metabolic pathways and suggestions for a relationship between the metabolism of aspartate with that of alanine and serine in this parasite are discussed.

Linked metabolism of L-serine and L-alanine by Moniliformis moniliformis (Acanthocephala) in vitro.

Experiments to investigate the linked metabolism of L-serine and L-alanine by Moniliformis moniliformis in vitro were carried out by incubating adult worms aerobically for 3 h at 37 degrees C in Tyrode's solution containing L-[U-14C]alanine and other amino acids. When present in the medium alone, alanine was totally removed by the worms and metabolized almost entirely to ethanol and a compound identified as carbon dioxide. When present in the medium with serine and no other amino acids, alanine was largely metabolized as before but additional alanine, believed to originate from serine, was excreted. The same results were obtained with serine and 16 other amino acids in the incubation medium.

Genetic interaction between non-MHC T- and B-cell alloantigens in response to Rous sarcomas in chickens.

Chickens of Regional Poultry Research Laboratory (RPRL) inbred line 6(3) regress sarcomas induced by Bryan high-titer Rous sarcoma virus to a greater extent than chickens of line RPRL 100, although these lines are identical for the major histocompatibility B complex. They differ, however, at three independent autosomal loci: Ly-4 and Th-1 determine the surface alloantigens of partly overlapping subsets of T lymphocytes, and Bu-1 determines a surface alloantigen of B lymphocytes. The association of genotypes at these loci with quantitative variation in their ability to regress Rous sarcomas was tested in segregating F4 generation progeny derived from crosses of lines 100 and 6(3). The Ly-4 and Bu-1 genotypes showed association with Rous sarcoma regression, but the Th-1 genotype did not. Chickens of the Ly-4a/Ly-4a, Bu-1b/Bu-1b and Ly-4b/Ly-4b, Bu-1a/Bu-1a genotypes had a significantly higher regressor ability than the other two double homozygous genotypes. These results indicate that higher regression is associated with (1) interaction between the Ly-4 and Bu-1 loci, and (2) complementation between either the line 6 Ly-4a allele and the line 100 Bu-1b allele, or the line 100 Ly-4b allele and the line 6 Bu-1a allele.

The design and construction of an oxygen micro-electrode for monitoring 20-100 microliter volumes.

Full instructions are given for the construction of a micro-respirometer. It is based on a membrane-covered platinum electrode of 10 micron diameter that consumes little oxygen. The reaction chamber, made entirely of glass, can be varied in volume between 20 and 100 microliter by adjusting the sealer assembly. Although specifically designed to study the properties of small samples of mitochondria from tiny organisms, it can be used equally well either for oxygen dissolved in aqueous solutions or dry gas mixtures containing oxygen. Measurements can be made with about one-hundredth the amount of material required for commercially available macro-respirometers, and when connected via an impedance-matching device to a pen recorder it gives a stable output of 100-200 mV in air.

Selective metabolism of L-serine by Moniliformis (Acanthocephala) in vitro.

In a series of in vitro experiments, adult male and female Moniliformis dubius were incubated at pH 6.88 and 37 degrees C for 3 h in a 2.5 mM solution of 18 amino acids. Fifteen of these were absorbed slightly from the medium, but L-serine was almost completely absorbed while the concentrations of glycine and alanine in the medium increased during the course of the incubation. By using L-[U-14C]serine, it was found that labelled ethanol and CO2 were the main end-products of metabolism excreted into the medium, with smaller amounts of labelled alanine, lactate and acetate. Small amounts of cystathionine with high specific radioactivity were found in extracts of the worms at the end of incubation, together with other radioactive metabolites including glucose, ethanol, lactate, succinate, malate, serine, glycine and alanine. Ammonia was found to be an excretory product of the amino acid metabolism of M. dubius. Possible metabolic pathways and suggestions for the significance of serine metabolism in this parasite are discussed.

The use of faecal egg counts for estimating worm burdens in sheep infected with Haemonchus contortus.

Clun forest sheep, worm-free from birth, were given a single dose of 20 000 infective larvae of Haemonchus contortus. The total number of eggs/day in the faeces was recorded for 21 infections and data on population size, sex ratio and individual worm development were collected from 76 sheep. The female to male ratio was 1.28 +/- 0.07 (S.E.). The relation between increase in worm size and uterine egg content was linear. The number of eggs present in the uteri was found to be an accurate measure of eggs passed. It was shown that the daily faecal egg output is related to total parasite weight and is not a measure of the number of individuals present.

The estimation of low levels of 14C in biological samples by alkaline extraction and liquid scintillation counting.

A liquid extraction method capable of accurately estimating low levels of 14C in blood and faecal samples is described. The lower limit of detection is about 4 pCi of 14C per g wet wt of ovine faeces and the lowest level of quantitative determination is 33 pCi per g. Extraction of soluble material is efficient and 14C present in the extract as n-[1-14C]hexadecane, D-[U-14C]glucose, [1-14C]stearic acid, [1-14C]methionine, or Na2 14CO3 is estimated without loss. The aqueous extraction medium used contains 2.5% (v/v) Synperonic NP9 (a non-ionic emulsifier) and 3.5% NaOH and samples are decolorised using hydrogen peroxide. The extract is counted without neutralisation in an inexpensive scintillation mixture based on an emulsifiable oil rather than on an aromatic solvent. It consists of White Spirit 100 (53.8% v/v), Synperonic NP9 (30.8% v/v), octan-2-ol (10.8% v/v), benzyl alcohol (4.6% v/v) and 2,5-diphenyl oxazole (3 g/l). Ethanediol added with the aqueous alkaline extract acts as a third cosurfactant. The advantage of this mixture over one based on an aromatic solvent is that it has a much larger capacity for the concentrated blood and faecal extracts.

The development, composition and maintenance of experimental populations of Haemonchus contortus in sheep.

Clun Forest sheep, aged between 3 and 18 months and worm-free from birth, were given a single dose of 25 000 infective larvae of the nematode Haemonchus contortus. The host animals were killed between 4 and 100 days after infection and the nematode populations were examined to determine size and composition. The relation between worm body length, dry weight and age was studied and growth curves were constructed. Variations in the sex ratio for infections of different ages were noted. No evidence was found for a relation between rate of growth and population density. The rate of expulsion was determined and its variability discussed.

A new technique for the extraction of Nematodirus battus eggs from sheep faeces.

Details are given of a method for the separation of the eggs of the nematode Nematodirus battus from sheep faeces. Faecal pellets obtained from worm-free lambs experimentally infected with N. battus were homogenized and passed through a graded series of metal sieves. The material retained in a 53 mum aperture sieve was transferred to a cylindrical column and the eggs were cleaned and separated from other suspended material by controlled differential flotation using tap water. A model experiment indicated that, within limits, the principle held for any size of tower. The density distribution for eggs at the morula stage of development was determined. Suggested flow rates for a specified tower are given.

The glucose metabolism of adult Ostertagia circumcincta, in vitro.

Adult Ostertagia circumcincta from freshly killed sheep were incubated at 39 degrees C in a medium containing inorganic salts, antibiotics and D-[U-14C] glucose. The worms appeared healthy even after incubation for as long as 72 h. All the radioactivity was recovered either within the worms or in the incubation vessel in the form of metabolic products or unmetabolized glucose. Incubations were carried out at low oxygen tension except for those in which CO2 was measured. These were either aerobic or anaerobic. In terms of both quantity and radioactivity the main metabolic products of glucose were CO2, propan-1-ol, acetate and propionate. Smaller amounts of ethanol, lactate and succinate were formed. The results are compared with those found for the similar nematode Haemonchus contortus.

The role of carbon dioxide in the metabolism of adult Haemonchus contortus, in vitro.

Adult Haemonchus contortus worms simultaneously excrete and fix CO2. Their initial content of CO2 was measured as 4.55 mumoles/100 mg wet weight and their excretion rate in air as 1 mumol/100 mg wet weight/h for at least 4 h. When the worms were incubated either aerobically or anaerobically with 14CO2 most of the metabolized radioactivity was associated with propan-1-ol and propionate but small amounts were found in succinate and lactate. No radioactivity was associated with ethanol or acetate, two major catabolites of glucose. Stepwise degradation of the metabolized radioactive propanol and propionate showed that all the radioactivity in both compounds was associated with carbon atom no. 1. These results show that H. contortus has much in common with the anaerobic energy metabolism of Ascaris lumbricoides but they are not inconsistent with the utilization of the tricarboxylic acid cycle by the worm. H. contortus worms were found to metabolize their excretory products. When they were incubated with either [2,3-14C]succinate or [2-14C]acetate, 14CO2 was excreted under aerobic but not under anaerobic conditions. These results are consistent with a pathway similar to that used by Ascaris operating aloneunder anaerobic conditions and together with the tricarboxylic acid cycle under aerobic conditions.

The energy metabolism of adult Haemonchus contortus, in vitro.

A comparison was made of the major excretory products when adult Haemonchus contortus worms were incubated with D-[U-14C]glucose under aerobic and anaerobic conditions. Catabolites measured were propan-1-ol, acetate, n-propionate and CO2 and the only major difference was that nearly twice as much CO2 both in terms of quantity and radioactivity was excreted under aerobic than anaerobic conditions. The worms were also much more physically active under aerobic conditions. When worms were incubated under aerobic conditions with increasing amounts of fluoroacetate their CO2 production was progressively reduced to the anaerobic level. Their movement and their ability to clump together was also progressively reduced. After aerobic incubation with fluoroacetate and D-[U-14C]glucose the quantity and radioactivity of citrate within worms increased greatly. When worms were similarly incubated anaerobically no increase in citrate occurred, no radioactivity was associated with the citrate and the worms appeared physically unaffected. When worms were incubated aerobically with fluoro[1(-14)C]acetate they produced radioactive fluorocitrate.

Molecular structure and morphology of glycogen isolated from the cestode, Moniezia expansa.

A particulate polysaccharide was isolated by differential centrifugation and alkali extraction from homogenates of the cestode Moniezia expansa. The polysaccharide had the structure of a glycogen. Its chemical properties, infra-red spectrum and optical rotation showed that it consisted of alpha-1,4- and alpha-1,6-linked glucopyranose units. Examination of the complex with iodine and the precipitate with concanavalin-A showed that the structure was highly branched. Oxidation with periodate and hydrolysis with alpha- and beta-amylase were used to measure mean chain lengths. For the particulate preparation the average chain length was 12-9 glucose units and the exterior and interior chain lengths were 9-0 and 2-9 units respectively. The particulate preparation had a very high sedimentation constant (s20, w = 910) with a smaller component at about s20, w = 600, but the alkali extracted material had an s20, w = 61 similar to that shown by alkali degradation of the particulate preparation. The morphology of the particulate material was similar to that of rat liver glycogen, alpha, beta- and possible gamma-particles being identified by electron microscopy. The alpha-particles were relatively stable under acidic conditions remaining intact down to pH 2-5. At pH 1-7 the alpha-particles dissociated into their constituent beta-particles with a consequent decrease in the opalescence of the solution. The nitrogen content of 0-9% was high for a glycogen.

Observations on the development of Haemonchus contortus in young sheep given a single infection.

Worm free lambs 2-4 months old were given a single infection of 50,000 Haemonchus contortus larvae. The changes in packed red cell volume were compared with those of uninfected sheep and the infected sheep were divided into two groups according to the rate of change of packed cell volume. In group 1, packed cell volume fell sharply from the 10th day of infection and the sheep eventually died. In group 2, the fall was less marked and the packed cell volume returned to normal after about 40 days. The rates of increase of parasite body length and dry weight were compared for the two groups. Parasites in group 1 sheep grew more slowly than their group 2 contemporaries. Accurate planning of metabolic studies on H. contortus was made possible by using this information to predict the course of an infection and the size of the worms. Parasite development was also measured in sheep for which the packed cell volume was not recorded. Computer analysis showed that adult worm length increase did not follow a simple growth pattern starting from the last ecdysis, but consisted of a rapid elongation followed by a simple negative exponential increase. Dry weight increase also followed a negative exponential during the second phase. Measurements of dry weight as a percentage of wet weight indicated that the rapid elongation was possibly linked with cell enlargement by the uptake of water.

The metabolism of fluoroacetate in lettuce.

1. Whole lettuce plants were incubated with (1) [1-(14)C]acetate, (2) fluoroacetate followed by [1-(14)C]acetate, (3) fluoro[1-(14)C]acetate, (4) fluoro[2-(14)C]acetate or (5) S-carboxy[(14)C]methylglutathione. 2. Fluoroacetate did not affect the expiration of (14)CO(2) from [1-(14)C]acetate and only a small amount of (14)CO(2) was produced from either fluoro[1-(14)C]-acetate or fluoro[2-(14)C]acetate in 43h. 3. Fluoroacetate at 50mg/kg wet wt. doubled the plant citrate concentration after 43h incubation, and depending on the age and size of the plant 50-100% of the compound was metabolized. 4. With both fluoro[1-(14)C]acetate and fluoro[2-(14)C]acetate all the radioactivity except that in the CO(2) was found in the water-soluble acid fraction. About 2% was in fluorocitrate and the remainder, apart from unchanged fluoroacetate, was in a number of compounds devoid of fluorine but containing nitrogen and sulphur. These were peptide-like and could be separated by chromatography on an amino acid analyser. 5. Identical compounds were obtained from the spontaneous reaction between iodo[2-(14)C]acetate and glutathione, the major product being S-carboxymethylglutathione. 6. S-Carboxymethylcysteine was also isolated and its mass spectrum compared with a commercial sample. 7. Reaction rates of all the monohaloacetates with glutathione were studied at pH7 at 25 degrees C. No reaction was observed with fluoroacetate. 8. The metabolism of fluoroacetate by lettuce is discussed in relation to that of aliphatic and aromatic halogen compounds, including fluoroacetate, by mammalian liver and to the metabolism of fluoroacetate by different plants reported by other workers.