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Studies on the amino acids metabolism of larval stages of the tapeworm Taenia crassiceps
292
LABORATORY MEETING
The parasites were isolated from citrated mouse blood, suspended in a buffered saline + horse serum medium containing 5 per cent. ferritin, and incubated for 15 min. at 37°C. Controls were incubated without ferritin. T h e trypanosomes were then osmium-fixed, embedded and sectioned for electron microscopy. Localization of the ferritin tracer was observed in our material as follows: i) Extracellularly, ferritin molecules showed a marked affinity for the invaginated part of the cell membrane around the base of the flagellum. Only occasional molecules seemed to be adhering to other parts of the external cell surface. ii) In the cytoplasm, membrane-bound dense bodies and vesicles (provisionally identified as lysosomes) contained numerous ferritin molecules. Such vesicles were confined to the part of the cell between the base of the flagellum and the nucleus. iii) I n a few cells the ferritin occurred free in the cytoplasm, as well as in lysosome-like vesicles; but it did not occur in the nucleus, perinuclear space or endoplasmic reticulum. T h e localization of ferritin is consistent with the notion that T. rhodesiense ingests macromolecules by phagocytosis; and the findings indicate as sole point of entry the depression, or reservoir, at the base of the flagellum. This is also a part of the cell surface from which pellicular fibrils are absent. The following sequence is proposed: i) Local adsorption of protein molecules in the reservoir. ii) Their incorporation, by phagocytosis at this site, into lysosome-like vesicles. iii) Intracellular migration and coalescence of the vesicles, perhaps followed by release of partially digested materials into the cytoplasm.
Dr. F. Hawking, Dr. P. J. Walker and Mr. M. J. Worms: N e w s m a l l a n i m a l s for laboratory experiment, viz: Herpestes sanguineus (African black tailed m o n g o o s e ) , host of filarial w o r m , Monnigofilaria setariosa.
Orizomys goeldi, Proechimys guyanensis, f r o m Brazil. Tharanomys surdaster, Congo tree rat, host of Plasmodium berghei; c o l o n i z e d in laboratory.
Mr. M. J. Worms: Echinostoma malayanum (?) i n a n imported m o n g o o s e . Trematodes recovered from the intestine of a male 'dwarf Indian' mongoose purchased in London are identified as Echinostoma mala3~anum Lieper 1911. The parasites are broad (8.5-9.7~ x 2.6-3.3~0 with deeply lobed testes, a cirrus sac extending beyond the acetabulum posteriorly and a head collar bearing 43 spines. This parasite has been reported in man, pigs, dog, rats and shrews in India and Malaya. This is the first record from a mongoose.
OTHER
EXHIBITORS
Dr. A n g e l a E. R. T a y l o r a n d Mr. W. D. G. H a y n e s ( Q u e e n E l i z a b e t h C o l l e g e , B i o l o g y Dept.): Studies on the a m i n o acids m e t a b o l i s m of larval stages of the t a p e w o r m Taenia
crassiceps. The demonstration showed a two-dimensional chromatogram (butanol-acetic/phenol) of the free amino acids of Taenia crassiceps larvae, after their extraction from whole larvae by 70 per cent. alcohol. The presence of alanine, threonine, glyeine, glutamic acid, taurine, serine, lysine, histidine, aspartic acid, valine, leucine/isoleucine, tyrosine, methionine/phenylalanine/tryptophanwere indicated. Traces of cysteic acid, glutamine, ornithine, citralline, arginine and proline were also present. Further studies were demonstrated graphically. These have been mainly concerned with the uptake, utilization and loss of 14C L-valine by T. crassiceps larvae. The technique involved incubation of the larvae in Hank's saline (pH 7.4) containing 14C L-valine (0.2 ~e/ml) in the presence or absence of other amino acids. T h e activity of the valine inside the larvae was determined (after extraction for three days into 70 per c e n t . alcohol) using a scintillation counter. Graph I showed the rate of absorption of 14C L-valine by T. crassiceps larvae. They were incubated in two concentrations (10-2M and 10-4M) over a period of 128 minutes. I n both cases valine was concentrated against a gradient indicating a process of active transport; this was markedly apparent within 4-8 minutes of incubation. T h e rate of uptake began to fall off after 64 minutes but this was due to the fact that by this time the valine was being incorporated into larval protein.
LABORATORY MEETING
293
Graph II showed the rate of incorporation, over a period of 8 hours, of 14C L-valine into the protein of T. crassiceps larvae both before and after in vitro culture for 5 days. Ap~ reciable activity could be found in the protein after 2 hours incubation. Hydrolysates of this protein yielded activity only in the valine fraction. Although the rate of valine incorporation fell after 24 hours in vitro, by 5 days it had recovered to about that shown before culture. Graph I I I demonstrated the effect of temperature on the uptake of 14C L-valine by these larvae over a temperature range 11-43°C. Q10 values were 3.8 for 11-25°C, 2.6 for 25-37°C and 3.4 for 37-43°C; these were higher than would be expected for a simple chemical process, thus indicating that an enzymic system is probably involved during uptake. Graph I V showed that when larvae, which had been incubated in active valine for 30 minutes, were incubated in non-active media, valine leaked out. The leakage was greater when L-valine, L-methionine or L-lysine were present in the surrounding medium than when the larvae were incubated in Hank's saline alone. Finally a histogram was exhibited showing the effect of the presence of other amino acids on the absorption of 14C L-valine by these larvae. The results indicated that uptake of valine was inhibited by most uncharged amino acids but not by those having charged side chains. Other studies with L-valine and L-methionine indicated that inhibition of valine by methionine is of a competitive nature. These studies are being continued.
Jones and M r . P . J . E . Bendell (Brunel College, Biology Dept.) (Introduced by Dr. J. D. Gillett): Automatic recording of mosquito activity.
M r . M . D . R.
An automatic method of recording mosquito activity using flight sound has been described by Jones (1964, ft. Ins. Physiol., 10, 343). The flight sound is amplified and used to operate a simple pen recorder. Other sounds and electronic interference are reduced by a combination of filter circuits and sound-proofing. The apparatus has been modified to allow simultaneous recording from two chambers, each containing one or more mosquitoes. This makes it possible to record the activity of experimental and control insects at the same time. The method has been applied to the testing of irritability to insecticides in various species and strains of Anopheles. A great many of the disadvantages of direct observation are ddminated and a permanent record is produced from which information may be obtained about the timing, number and length of flights. This method can also be applied to the study of circadian rhythms of activity in mosquitoes.
Dr. G. H. V. Clarke (University College Hospital): Film: Skin conditions found in onchocerciasis. This film shows several cases of onchodermatitis and some views of a village and a river typical of an endemic area. We are shown how to take a skin snip and then in a photo-micrograph very numerous and very active microfilariae emerging from the infected skin. This is a most instructive and interesting sequence and should enable anyone to make the diagnosis of onchodermatitis with confidence. A high power view of a single microfilaria is next shown and the film ends with a child leading a blind man by the h a n d - - a tragic sequel of untreated onchocerciasis. The film is 16 mm., in colour, and has a spoken commentary. It was first shown at the International Congress of Dermatology in Washington in 1962.
Mr. B. E. Brooker and Dr. K. Viekerman (University College, London): Acid phosphatasc in trypanosomes. Acid phosphatase may be demonstrated in the cytoplasm of trypanosomes by cytochemical techniques. This enzyme is used as a marker for the class of organelles termed lysosomes. Lysosomes are cytoplasmic particles containing acid hydrolases. They are bounded by a membrane which normally renders the enzymes inaccessible to surrounding substrates, but become active in intracellular digestion when they coalesce with a food vacuole formed by pinocytosis or phagocytosis. Under certain conditions the membrane is broken down or made permeable to the enzymes resulting in autolysis of the cell. In the present investigation bloodstream forms of Trypanosoma brucei were fixed in buffered 2.5 per cent. glutaraldehyde and tested for acid phosphatase activity by the Gomori technique (Holt, 1959) or Burstone's naphthol AS phosphate method (Burstone, 1962). Controls were incubated without the phosphate-containing substrate or with sodium fluoride as enzyme inhibitor. Deposits
LABORATORY MEETING
The parasites were isolated from citrated mouse blood, suspended in a buffered saline + horse serum medium containing 5 per cent. ferritin, and incubated for 15 min. at 37°C. Controls were incubated without ferritin. T h e trypanosomes were then osmium-fixed, embedded and sectioned for electron microscopy. Localization of the ferritin tracer was observed in our material as follows: i) Extracellularly, ferritin molecules showed a marked affinity for the invaginated part of the cell membrane around the base of the flagellum. Only occasional molecules seemed to be adhering to other parts of the external cell surface. ii) In the cytoplasm, membrane-bound dense bodies and vesicles (provisionally identified as lysosomes) contained numerous ferritin molecules. Such vesicles were confined to the part of the cell between the base of the flagellum and the nucleus. iii) I n a few cells the ferritin occurred free in the cytoplasm, as well as in lysosome-like vesicles; but it did not occur in the nucleus, perinuclear space or endoplasmic reticulum. T h e localization of ferritin is consistent with the notion that T. rhodesiense ingests macromolecules by phagocytosis; and the findings indicate as sole point of entry the depression, or reservoir, at the base of the flagellum. This is also a part of the cell surface from which pellicular fibrils are absent. The following sequence is proposed: i) Local adsorption of protein molecules in the reservoir. ii) Their incorporation, by phagocytosis at this site, into lysosome-like vesicles. iii) Intracellular migration and coalescence of the vesicles, perhaps followed by release of partially digested materials into the cytoplasm.
Dr. F. Hawking, Dr. P. J. Walker and Mr. M. J. Worms: N e w s m a l l a n i m a l s for laboratory experiment, viz: Herpestes sanguineus (African black tailed m o n g o o s e ) , host of filarial w o r m , Monnigofilaria setariosa.
Orizomys goeldi, Proechimys guyanensis, f r o m Brazil. Tharanomys surdaster, Congo tree rat, host of Plasmodium berghei; c o l o n i z e d in laboratory.
Mr. M. J. Worms: Echinostoma malayanum (?) i n a n imported m o n g o o s e . Trematodes recovered from the intestine of a male 'dwarf Indian' mongoose purchased in London are identified as Echinostoma mala3~anum Lieper 1911. The parasites are broad (8.5-9.7~ x 2.6-3.3~0 with deeply lobed testes, a cirrus sac extending beyond the acetabulum posteriorly and a head collar bearing 43 spines. This parasite has been reported in man, pigs, dog, rats and shrews in India and Malaya. This is the first record from a mongoose.
OTHER
EXHIBITORS
Dr. A n g e l a E. R. T a y l o r a n d Mr. W. D. G. H a y n e s ( Q u e e n E l i z a b e t h C o l l e g e , B i o l o g y Dept.): Studies on the a m i n o acids m e t a b o l i s m of larval stages of the t a p e w o r m Taenia
crassiceps. The demonstration showed a two-dimensional chromatogram (butanol-acetic/phenol) of the free amino acids of Taenia crassiceps larvae, after their extraction from whole larvae by 70 per cent. alcohol. The presence of alanine, threonine, glyeine, glutamic acid, taurine, serine, lysine, histidine, aspartic acid, valine, leucine/isoleucine, tyrosine, methionine/phenylalanine/tryptophanwere indicated. Traces of cysteic acid, glutamine, ornithine, citralline, arginine and proline were also present. Further studies were demonstrated graphically. These have been mainly concerned with the uptake, utilization and loss of 14C L-valine by T. crassiceps larvae. The technique involved incubation of the larvae in Hank's saline (pH 7.4) containing 14C L-valine (0.2 ~e/ml) in the presence or absence of other amino acids. T h e activity of the valine inside the larvae was determined (after extraction for three days into 70 per c e n t . alcohol) using a scintillation counter. Graph I showed the rate of absorption of 14C L-valine by T. crassiceps larvae. They were incubated in two concentrations (10-2M and 10-4M) over a period of 128 minutes. I n both cases valine was concentrated against a gradient indicating a process of active transport; this was markedly apparent within 4-8 minutes of incubation. T h e rate of uptake began to fall off after 64 minutes but this was due to the fact that by this time the valine was being incorporated into larval protein.
LABORATORY MEETING
293
Graph II showed the rate of incorporation, over a period of 8 hours, of 14C L-valine into the protein of T. crassiceps larvae both before and after in vitro culture for 5 days. Ap~ reciable activity could be found in the protein after 2 hours incubation. Hydrolysates of this protein yielded activity only in the valine fraction. Although the rate of valine incorporation fell after 24 hours in vitro, by 5 days it had recovered to about that shown before culture. Graph I I I demonstrated the effect of temperature on the uptake of 14C L-valine by these larvae over a temperature range 11-43°C. Q10 values were 3.8 for 11-25°C, 2.6 for 25-37°C and 3.4 for 37-43°C; these were higher than would be expected for a simple chemical process, thus indicating that an enzymic system is probably involved during uptake. Graph I V showed that when larvae, which had been incubated in active valine for 30 minutes, were incubated in non-active media, valine leaked out. The leakage was greater when L-valine, L-methionine or L-lysine were present in the surrounding medium than when the larvae were incubated in Hank's saline alone. Finally a histogram was exhibited showing the effect of the presence of other amino acids on the absorption of 14C L-valine by these larvae. The results indicated that uptake of valine was inhibited by most uncharged amino acids but not by those having charged side chains. Other studies with L-valine and L-methionine indicated that inhibition of valine by methionine is of a competitive nature. These studies are being continued.
Jones and M r . P . J . E . Bendell (Brunel College, Biology Dept.) (Introduced by Dr. J. D. Gillett): Automatic recording of mosquito activity.
M r . M . D . R.
An automatic method of recording mosquito activity using flight sound has been described by Jones (1964, ft. Ins. Physiol., 10, 343). The flight sound is amplified and used to operate a simple pen recorder. Other sounds and electronic interference are reduced by a combination of filter circuits and sound-proofing. The apparatus has been modified to allow simultaneous recording from two chambers, each containing one or more mosquitoes. This makes it possible to record the activity of experimental and control insects at the same time. The method has been applied to the testing of irritability to insecticides in various species and strains of Anopheles. A great many of the disadvantages of direct observation are ddminated and a permanent record is produced from which information may be obtained about the timing, number and length of flights. This method can also be applied to the study of circadian rhythms of activity in mosquitoes.
Dr. G. H. V. Clarke (University College Hospital): Film: Skin conditions found in onchocerciasis. This film shows several cases of onchodermatitis and some views of a village and a river typical of an endemic area. We are shown how to take a skin snip and then in a photo-micrograph very numerous and very active microfilariae emerging from the infected skin. This is a most instructive and interesting sequence and should enable anyone to make the diagnosis of onchodermatitis with confidence. A high power view of a single microfilaria is next shown and the film ends with a child leading a blind man by the h a n d - - a tragic sequel of untreated onchocerciasis. The film is 16 mm., in colour, and has a spoken commentary. It was first shown at the International Congress of Dermatology in Washington in 1962.
Mr. B. E. Brooker and Dr. K. Viekerman (University College, London): Acid phosphatasc in trypanosomes. Acid phosphatase may be demonstrated in the cytoplasm of trypanosomes by cytochemical techniques. This enzyme is used as a marker for the class of organelles termed lysosomes. Lysosomes are cytoplasmic particles containing acid hydrolases. They are bounded by a membrane which normally renders the enzymes inaccessible to surrounding substrates, but become active in intracellular digestion when they coalesce with a food vacuole formed by pinocytosis or phagocytosis. Under certain conditions the membrane is broken down or made permeable to the enzymes resulting in autolysis of the cell. In the present investigation bloodstream forms of Trypanosoma brucei were fixed in buffered 2.5 per cent. glutaraldehyde and tested for acid phosphatase activity by the Gomori technique (Holt, 1959) or Burstone's naphthol AS phosphate method (Burstone, 1962). Controls were incubated without the phosphate-containing substrate or with sodium fluoride as enzyme inhibitor. Deposits