Augmentation of albumin but not fibrinogen synthesis by corticosteroids in patients with hepatocellular disease.

Simultaneous studies of albumin and fibrinogen metabolism have been conducted using the carbonate-(14)C method before and after a 13 day course of prednisolone in eight patients with hepatocellular disease. Initially six patients were hypoalbuminemic. The mean plasma albumin and fibrinogen concentrations and albumin and fibrinogen synthetic rates were all lower than the corresponding values in a group of control subjects. Prednisolone therapy was associated with significant increases in the plasma concentration and synthetic rate of albumin but changes in the intravascular albumin pools were not significant. It is inferred that a low synthetic rate of albumin in a patient with liver disease does not necessarily represent the maximum capacity of the diseased liver to synthesize this protein. Changes in the plasma concentration, intravascular pool, and synthetic rate of fibrinogen were small and inconsistent. The data are compatible with a selective action of corticosteroids on hepatic protein metabolism and with the existence of different mechanisms for the control of albumin and fibrinogen synthesis.

Myeloma-like lesions of the kidney. Occurrence in a case of acinic cell adenocarcinoma of the pancreas.

A 54-year-old man with acinic cell adenocarcinoma of the pancreas died of oliguric renal failure associated with myeloma-like renal lesions. Electron microscopical study of the tumor cells disclosed rich rough-surfaced endoplasmic reticulum and membrane-bound secretory granules, which indicated active protein synthesis and suggested that abnormal proteins produced by the tumor cells were the underlying cause of the renal lesions. Rapid deterioration of renal function ensued after intravenous pyelography, as is usual in the syndrome of myeloma-like lesions of the kidneys. This case presents further evidence for the occurrence of "myeloma kidney" in association with tumors other than plasma cell myeloma.

Glycosaminoglycans of tegumental fractions of Hymenolepis diminuta.

The teguments of 6 and 10 day-old Hymenolepis diminuta were removed with Triton X-100 and separated into brush border and vesicular fractions by differential centrifugation. Glycosaminoglycans (GAG) isolated from these tissues and from the denuded carcass were treated with specific GAG-degrading enzymes and other chemical agents and analyzed by sodium dodecyl sulfate-polyacrylamide, agarose gel and cellulose acetate electrophoresis. Both 6 and 10 day-old worm carcasses contained chondroitin sulfate, heparin/heparan sulfate and hyaluronic acid. The 10 day-old worm brush border and vesicle fractions contained chondroitin sulfate but no heparin-like material. Colorimetric analysis showed that the carcasses of both 6 and 10 day-old worms contained uronic acid. About 98% of the detectable uronic acid of 10 day-old worms was found in the carcass, and only 2% in the brush border fraction. No uronic acid was detected in the other tegumental fractions.

Characterization of polysaccharides of the eggs and adults of Hymenolepis diminuta.

Polysaccharides and other complex carbohydrates were released by proteolysis of the chloroform-methanol insoluble residue of 10 day-old worms and eggs of Hymenolepis diminuta. Gas-liquid chromatographic analysis of alditol acetate derivatives of monosaccharides released from the polysaccharides by hydrolysis revealed that in the 10 day-old worm, glucose was the most abundant sugar, followed by galactose, glucosamine, galactosamine, fucose and possibly rhamnose. Mannose was least abundant and xylose was absent. In the egg, glucose and galactose were equally abundant, followed by the same sugars found in 10 day-old worms, and xylose was present. Uronic acid was detected in both fractions by specific chemical tests. None of the saccharide material from eggs and worms was susceptible to degradation by Streptomyces hyaluronidase, chondroitinase AC, and slightly susceptible to chondroitinase ABC, as shown by electrophoretic analysis on composite 2.2% acrylamide-agarose slab gels and 4.5/12.5% polyacrylamide gels before and after enzymatic treatment. One of the gel-separable bands, however, was degradable by both nitrous acid and Flavobacterium heparinase. Both bands from eggs were degradable by nitrous acid. These results suggest that eggs contain heparin and/or heparan sulfate and perhaps dermatan sulfate and that 10 day-old worms also have these polyglycans but possibly not chondroitin sulfate or hyaluronic acid.

The gene family encoding eggshell proteins of Schistosoma japonicum.

The four closely related genes encoding eggshell proteins in the human parasite Schistosoma japonicum are described. A cDNA and a genomic DNA library were constructed and members of the eggshell protein gene family isolated. The four genes in this family do not contain introns, and differ in organization and nucleotide sequence from the related set of genes in Schistosoma mansoni and Schistosoma haematobium. The coding sequences of two of the S. japonicum genes and their flanking regions were determined. Transcription start sites for these genes were shown by primer extension analysis to occur 47 and 50 nucleotides in front of the start codon. A female-specific component in nuclear extracts binds to a DNA fragment containing conserved sequences upstream of the transcription start sites. The deduced protein sequences of 207 and 212 amino acids are composed of 50% glycine with continuous glycine regions as long as 11 residues. In vitro translations of male and female RNAs revealed female-specific translation products, the sizes of which were consistent with the eggshell proteins.

A simple method of obtaining an enriched fraction of tegumental brush border from Hymenolepis diminuta.

A method for isolating an enriched preparation of tegumental brush border from the tapeworm, Hymenolepis diminuta, is described. Combining incubation of whole tapeworms in Krebs-Ringer/tris-maleate solution containing a hemolytic saponin, low shear-force agitation, and differential centrifugation, a pellet is obtained at 2,500 g which contains a significant concentration of surface brush border. The content of brush border in this fraction is identified by the presence of numerous microvilli, increased specific radioactivity after surface tagging with 3H-Concanavalin A, and relatively little mitochondrial contamination (succinic dehydrogenase). Based on morphological criteria, fractions sedimenting with greater force contain dense vesicles and mitochondria from the outer portion of the tegument.

Lipids from subcellular fractions of the tegument of Hymenolepis diminuta.

Lipids comprised 37% and 22.1%, respectively, of the day weights of brush border- and vesicle-rich fractions separated by differential centrifugation of isolated H. diminuta tegument. Neutral lipids of both fractions were rich in cholesterol, but also contained small amounts of glycerides, sterol esters, and (in brush borders) free fatty acids. Phosphatidyl ethanolamine was the most prevalent polar lipid in both fractions, and was particularly abundant (63.4% of total polar lipids) in vesicles; sphingomyelin, not previously reported from H. diminuta, was also present. Polar lipids of both tegumental fractions resembled each other but differed from whole worm polar lipids in fatty acid composition. Tegumental polar lipids contained lower levels of long-chain, polyunsaturated fatty acids than reported for corresponding lipids of whole worms.

Responses of Megalodiscus temperatus miracidia to amino and sialic acids found in snail-conditioned water.

Analysis of snail-conditioned water (SCW) from Helisoma trivolvis revealed 17 free amino acids. Those in great concentration were glycine, serine, and alanine. The concentration of sialic acid was found to be twice that of the most abundant amino acid. The behavior of miracidia of Megalodiscus temperatus, measured by the contact with return method, to agar cylinders containing single amino acids and sialic acid indicated greater responses to polar molecules charged either positively or negatively at neutral pH. The molecules elicting the greatest response were aspartic, glutamic, and sialic acid. No correlation was found between concentration of amino acids in H. trivolvis SCW and response of M. temperatus miracidia.

Molecular characterization of Echinostoma caproni and E. paraensei by random amplification of polymorphic DNA (RAPD) analysis.

Genomic DNA from Echinostoma caproni and Echinostoma paraensei was amplified by the polymerase chain reaction using primers (5'-TCGTAGCCAA and 5'-TCACGATGCA), originally found to differentiate species and strain of Schistosoma. The 2 putative species of Echinostoma produced distinct banding patterns clearly distinguishable from one another, thereby suggesting that RAPD (random amplification of polymorphic DNA) analysis may be useful for the identification of echinostome strains and species previously misunderstood or undescribed, and that primers developed for species within a given genus, e.g., Schistosoma, may have broader application in identifying other trematodes.

Comparison of calcium, freeze-thaw, and Triton X-100 tegumental disruption/recovery techniques applied to Schistosoma mansoni.

Three techniques for the disruption/recovery of tegumental free-surface plasmalemma were compared by (i) morphological examination of carcasses and centrifugally-derived isolates, (ii) specific enrichment of bound surface tags (lectin) and of "marker" enzymes for membrane, and (iii) assessment of total protein and lectin recovered by each procedure. Procedures compared included the use of Triton X-100, freezing and thawing, and high ionic strength calcium. Triton X-100 consistently provided the greatest amounts of recovered surface membrane on a per worm basis, whereas calcium retained the highest amounts of alkaline p-nitrophenyl phosphatase, adenosine triphosphatase, and adenosine monophosphatase activity. Ultrastructural examination of membrane isolates and worm carcasses prepared by freezing and thawing indicated that significant amounts of parenchymal material contaminated the membrane fractions. Thus results based on the freeze-thaw technique can be difficult to interpret.

Enzyme polymorphism in Ascaris suum (Nematoda).

The potential of multilocus electrophoretic studies for providing insight into the population biology of parasitic organisms was studied using the swine parasite Ascaris suum suum. Thirty-eight loci encoding enzymatic or nonenzymatic proteins have been resolved in extracts of adult worms by starch-gel electrophoresis. A preliminary study of variation in Ascaris from eastern Iowa revealed an average heterozygosity of 6.6%. Allele frequencies at six polymorphic loci were similar in males and females and genotypic frequencies were in accord with those expected in a single, randomly mating population; however, the significant linkage disequilibrium between Pep-2 and Es-3 suggested that there may be some genetic substructuring within Ascaris from Iowa. Genetic comparisons of Ascaris from Iowa with Ascaris from New Jersey and Maryland indicated slight differences between eastern and midwestern populations, as well as between the east coast localities. Larger samples from more locations are needed before any statistical significance can be attached to these differences; however, qualitative comparisons suggest that the differentiation is a biological reality. Knowledge of the population biology of this and other parasites may contribute to planning effective control programs.

Disruption and removal of the tegument from Schistosoma mansoni with triton X-100.

Tegumental membranes of Schistosoma mansoni were disrupted by 0.2% Triton X-100 in Tris-maleate buffered/Kreb-Ringer's solution. Subsequent differential centrifugation of the disruption solution at 2,500 g and 30,000 g produced two pellets which contained membrane components. Examination of the carcass by scanning electron microscopy revealed that most of the exposed tegument of both male and female worms was removed, while surface membrane protected by close apposition of another surface (i.e., in the gynecophoral canal) remained intact. The parenchymal tissue (e.g., subtegumental muscle and tegumental perikarya), excretory and gut epithelia, and the tegument's basement membrane also remained intact. The selectivity of the disruption suggests that membrane in both pellets originated almost exclusively from the tegument. Although larger morphological features (i.e., surface crypts) present in the intact tegument did not maintain their form in the 2,500 g pellet, the high specific activity of 3H-concanavalin A retained by this fraction, and the presence of numerous spines and large pieces of membrane, suggest that the 2,500 g pellet contained most of the worm's disrupted surface membrane. Transmission electron microscopy demonstrated the presence of dense spinelike material and vesicles of various sizes and densities, as well as some mitochondria in the 30,000 g pellet. Low specific activity of 3H-concanavalin A in the post-30,000 g supernatant suggests that relatively few externally oriented, saccharide-containing molecules were solubilized from tegumental membranes by Triton X-100.

A comparison of Texan and Hawaiian strains of the avian eyefluke, philophthalmus gralli, with a cautionary note on the importation of exotic animals.

Philophthalmus gralli (Mathis and Leger, 1910) was introduced into the San Antonio, Texas area within the last 25-30 years from an unknown foreign source. Strains of P. gralli originally from Texas and Hawaii were compared for differences in morphology, growth patterns, mating compatibility, and isozyme mobilities. Metacercarial cysts of the 2 strains were compared for viability after storage at room temperature. Adult stages of the 2 strains were indistinguishable based on sucker ratios, vitellaria, and egg sizes. In single- and multiple-worm infections, both strains exhibited similar growth patterns when reared in chickens. Adults of the 2 strains, when transplanted in concurrent infections, readily cross-inseminated. Isozyme mobilities of 5 enzymes were identical in all adult worms examined from both strains. After 3 days, metacercarial cysts from both strains rapidly lost their ability to excyst. The cysts of the Texan strain remained viable for 4 days longer than the Hawaiian strain, although excystment rates were low during that period. The evolutionary implications of this imported species are discussed.

[Comparative studies on the repetitive DNA sequences of diploid and triploid forms of Paragonimus westermani by restriction endonuclease and Southern blotting].

Restriction enzyme digestion of total genomic DNA of two chromosomes forms of Paragonimus westermani showed the presence of homologous highly repeated DNA in both diploid and triploid forms. Southern blotting analysis provided further evidence that the distribution of restriction enzyme sites (with 3 enzymes) on repetitive sequence of DNA of both forms were similar. However, with Pstl, Ddel, HaeIII and HpaII, their polymorphism revealed differences which were also found in each form tested separately with the hybridization technique. The present study, at the molecular level, supports the previously reported biological and biochemical results that they might be considered as different isolates or forms. It is suggested that PstI, DdeI, HaeIII and HpaII digestion pattern might be useful to distinguish the diploid from the triploid forms of Paragonimus westermani.

Metam sodium reduces viability and infectivity of Eimeria oocysts.

Metam sodium (MS, sodium N-methyldithiocarbamate) is a widely used soil pesticide. Fumigation or chemical sterilization of poultry litter containing infectious oocysts could be an effective strategy to block the transmission of avian coccidia. In the current study, the effect of MS on the viability and infectivity of ocysts was investigated. The development of isolated, unsporulated oocysts of both Eimeria tenella and Eimeria maxima was inhibited, in a dose-related manner (IC(50) 8 to 14 microg/ml), by exposure to aqueous MS. Most treated oocysts failed to develop beyond early stages of sporulation. To determine the effect of MS on infectivity, isolated oocysts of E. tenella , Eimeria acervulina , and E. maxima were exposed for 24 hr to aqueous concentrations of MS ranging from 0 to 1,000 microg/ml. Treated oocysts were inoculated into chickens, and parameters of coccidiosis infection were compared to chickens inoculated with equal numbers of untreated oocysts. In a dose-related manner, MS significantly reduced the infectivity of oocysts with maximum effect observed at a dose of 300 microg/ml. When a mixture of oocysts containing 3 coccidian species was exposed to 300 microg/ml MS, from 0 to 24 hr, infectivity of oocysts was significantly reduced after a minimum of 12 hr of exposure. Treatment of aqueous slurries of litter samples obtained from commercial poultry houses, with 300 microg/ml MS for 24 hr, prevented the sporulation of eimerian oocysts in the litter samples relative to untreated control samples. The results indicate that MS could be used to reduce coccidial contamination of poultry litter.