Stuart M. Krassner

A simple monophasic medium for axenic culture of hemoflagellates.

An inexpensive, easily prepared monophasic medium which supports continuous culture of Leishmania donovanì (approximately 4 X 10(7) cells/ml/day 5) and L. tarentolae (approximately 3.5 X 10(7) cells/ml/day 5) promastigotes has been developed. This medium, designated HO-MEM, is a modified Eagles minimal essential medium with Spinners salts and 10% fetal calf serum. Epimastigotes of Trypanosoma cruzi, Costa Rica strain, also grew well in HO-MEM (approximately 2 X 10(7) cells/ml/day 5) but cells of the Corpus Christi strain grew poorly in the medium. The initial inoculum for all of the above culture systems was 3 X 10(5) cells which means that there was approximately 100-fold increase in cell number within 5 days of culture. Average doubling time for L. donovani in HO-MEM during log growth phase was 10 to 12 hr; this shortened to 9.25 hr at mid-log growth (day 2). The optimal pH range for L. donovani was 7.2 to 7.4 and optimal culture temperatures were 25 to 26 C. A change was seen in the average size distribution of L. donovani promastigotes during the growth cycle with smaller cells predominating each succeding day of culture. HO-MEM is a good medium for transformation of L. donovani amastigotes to promastigotes (60 to 80% in 48 hr).

Bacterial clearance in the California sea hare, Aplysia californica☆☆☆

Abstract Aplysia californica normally contains sterile hemolymph. It is capable of completely clearing in vivo at least 4 marine bacteria from the hemolymph, but was unable to completely clear the terrestrial bacterium Serratia marcescens during the period tested. Clearance occurred most rapidly at high temperatures (≈18–20°C) and was accelerated by previous exposure to the bacterium. The latter indicates some type of primitive anamnestic response. Concomitant with early rapid bacterial clearance was a depression of serum agglutinin titers and a decrease in circulating hemocytes; levels of these two variables later return to normal values. Agglutinin titer levels return to normal values even after 3 bacterial challenges. Hemocyte numbers and agglutinin titers return to preinjection levels more rapidly at higher temperatures. Although A. californica serum exhibited no lytic activity, opsonic factors which enhanced phagocytosis of chicken red blood cells were found. A mechanism of bacterial clearance by the sea hare involving agglutinin and opsonin activity is hypothesized.

Characterization of a natural agglutinin present in the hemolymph of the California sea hare, Aplysia californica

Abstract A substance in the serum of the marine gastropod, Aplysia californica , capable of agglutinating marine bacteria and vertebrate red blood cells was subjected to physicochemical analysis in order to ascertain its possible nature. Our studies indicate that agglutinating activity is due to a heterogeneous group of high molecular weight molecules with two activity peaks exhibiting sedimentation coefficients centering ∼18.5 S and ∼31.0 S. This material has a protein component associated with the active site since it is sensitive to heat, p H extremes, and extraction with 2-mercaptoethanol, phenol, chloroform, and trichloracetic acid. Its physicochemical characteristics are different from other known invertebrate agglutinating substances and from classical vertebrate antibody.

Proline metabolism in Trypanosoma cruzi epimastigotes.

Abstract 1. 1. Oxygen uptake by epimastigotes of Trypanosoma cruzi , Costa Rica strain, was stimulated by l -proline, l -glutamate and to a lesser degree by l -aspartate. 2. 2. l -proline reversed partially malonate-induced inhibition of respiration but did not reverse KCN-induced inhibition. 3. 3. Labeled proline, glutamate, aspartate, cystine and lysine were detected by thin-layer chromatography in the free amino acid pool from cells incubated with l -proline- 14 C. 4. 4. Labeled tricarboxylic acid intermediates were also found by this method in extracts from organisms incubated with l -proline- 14 C which contained also pyruvate. 5. 5. Labeled alanine was not found in epimastigotes incubated with l -proline- 14 C, l -glutamate- 14 C or aspartate- 14 C indicating a difference between T. cruzi amino acid metabolism and that found in other hemoflagellates. 6. 6. Labeled l -proline was not found in epimastigotes incubated with d -glucose- 14 C, glutamate- 14 C or aspartate- 14 C indicating that the flow of carbon from proline to glutamate and aspartate may not be reversible. 7. 7. All results suggest the presence of a proline-glutamate inter-conversion pathway in T. cruzi epimastigotes.

Proline metabolism in Leishmania tarentolae

Abstract Leptomonads of Leishmania tarentolae were able to grow in Tragers defined medium C deficient in glucose or l -proline, but not without both. In addition, inositol and glycine could be deleted without any adverse effect as long as either glucose or l -proline was present. Cells could grow in methionine-free media only if l -proline was present. Respiration studies indicated that l -proline, glutamate, and aspartate were the only amino acids in medium C capable of stimulating O2 uptake by L. tarentolae. Methionine and serine appeared to inhibit respiration. d -proline and hydroxyproline could not substitute for the l form. Thin layer chromatography separation of free amino acids from cells incubated in 14C l -proline contained radioactive proline, glutamate, and alanine. Separations of tricarboxylic acid cycle acids contained a number of radioactive intermediates including α-ketoglutarate and malate as well as pyruvate. Although Δ1-pyrroline-5-carboxylic acid could not be found in chromatograms, an increased positive O-aminobenzaldehyde reaction was found in cell extracts incubated in l -proline. The above results suggest the presence of a proline-glutamate interconversion oxidation pathway similar to the proline oxidase metabolic system found in a variety of organisms. Alanihe-14C was found in cells incubated with 14C l -proline. Alanine is thought to be derived from pyruvate because of: (a) The presence of 14C Krebs cycle acids, (including malate) and pyruvate; (b) demonstration of an NADP dependent malic enzyme, and (c) formation of only 14C alanine by cells incubated in 14C sodium pyruvate.

A preliminary physicochemical characterization of an agglutinin found in the hemolymph of the crayfish Procambarus clarkii

Abstract An agglutinin found in the serum of the crayfish Procambarus clarkii , capable of agglutinating marine bacteria and vertebrate chicken and rabbit red blood cells (RBC), was studied to determine some of its physical and chemical properties. Tests to analyze its nature were (1) p H extreme and heat stability, (2) freezing and thawing, (3) dialysis against 0.01 m Tris buffer, 0.15 M NaCl, (4) chloroform, toluene, xylene, trichloroacetic acid (TCA), ether, and phenol extraction, (5) urea, trypsin, and pronase incubation. Agglutinin activity was normal between p H 6.4 and 10.4; it was inactivated at 70°C in 30 min and by extraction with phenol and 10% TCA. Tests with RBC, three marine bacteria, and the terrestrial pathogenic bacterium Serratia marcescens showed little cross absorption activity (exception between chicken RBC and the marine bacterium 628). Agglutinin molecular weight greater than 150,000 was estimated, using Sephadex G-200 column chromatography. These results indicate that a large molecular weight material, probably containing protein, was responsible for agglutination. Crayfish serum and hemolymph were tested against two marine bacteria (628 and Fr) and one terrestrial bacterium ( S. marcescens ) for the presence of bactericidins. None of the bacteria were killed by either the serum or the hemolymph.

Experimental wound repair in the black abalone, Haliotis cracherodii☆

Histological sections of incisions made in the mantle and foot of the black abalone, Haliotis cracherodii, revealed a number of differences in wound healing of the two tissues. Initial muscular closure of the wound was more pronounced in the mantle as was invagination of the cut edges. Within 8 hr post-incision, leukocytes lined the wound surface, initiating granulation tissue formation. A layer of squamous epithelium covered this reparative tissue in the wound cavity within 16 days. Leukocytes and collagen deposition filled the foot wound cavity, pushing the new epithelium upward until it was even with the normal epithelial surface. Mucous cell secretion indicated that the new epithelium was functional 2 months after incision. Granulation tissue did not fill up the mantle wound cavity, so that the epithelial surface was still depressed 75 days post-incision. Granulation tissue formation in the foot was far more elaborate than that of the mantle during all phases of wound healing. In both structures epithelial differentation and vascularization of the granulation tissue occurred within 2 months after incision. Muscle development in the healing wound appeared to result from new muscle formation within the granulation tissue rather than by gradual infiltration of muscle fibers from the surrounding tissue. The healed tissue in both structures appeared to be functional.

Cellular defense reactions to particulate materials in the California sea hare, Aplysia californica

Abstract Cellular mechanisms of defense in a marine gastropod, the California sea hare Aplysia californica , were studied histologically following injections of either carmine or India ink. Sea hares were capable of mobilizing an efficient cellular defense against either of these foreign bodies injected intramuscularly into the foot. Carmine was gathered into large nodules and walled off, while ink was sequestered in situ within individual phagocytes. However, injections of India ink made directly into the body cavity resulted in death to these mollusks. In addition to the circulating phagocytes observed in most invertebrates, A. californica has primitive lymphoid tissue at the base of the gill.

Antibacterial factors in the sipunculid worms Golfingia gouldii and Dendrostomum pyroides.

Abstract The coelomic fluid of Golfingia gouldii (an east coast sipunculid worm) exhibited in vitro antibacterial activity against several marine bacteria. This activity is heat labile and is lost after trypsinization. It is resistant to treatment with pepsin, lipase, or toluene extraction and appears to be unaffected by freezing and thawing. Dialysis against seawater also causes the loss of antibacterial activity. Dendrostomum pyroides (a west coast sipunculid) exhibits a similar type of in vitro antibacterial activity in its coelomic fluid, suggesting that nonspecific antimicrobial substances may be common in this phylum.

Hemin deprivation in culture stages of the hemoflagellate, Leishmania tarentolae

Abstract 1. Intact iron porphyrins appear to be necessary for culture of promastigote stages of Leishmania tarentolae grown in Brain Heart Infusion (BHI). The iron porphyrins catalase and peroxidase could substitute for hemin in BHI whereas cytochrome c could not. 2. Both cytochrome c and protoporphyrin IX inhibited the growth of L. tarentolae in BHI containing hemin. 3. Cells grown in hemin-free BHI grew poorly, exhibited increased length, showed higher respiratory rates and lost a major electrophoretic protein band. 4. It was not possible to grow the organism for more than one subculture in hemin deficient BHI. δ-Amino-levulinic acid (ALA), a key intermediate in porphyrin biosynthesis, could not substitute for hemin. This was not due to a permeability barrier since 3 H-ALA was taken up by L. tarentolae . 5. Label from incorporated 3 H-ALA was found in hemin and porphyrin fractions of cell extracts although the counts were very low. 6. Free porphyrins were found in cell extracts of L. tarentolae separated by thin layer chromatography; one of these showed an R f value similar to that of uroporphyrin.