Expression of immunological markers on leukemic cells before and after cryopreservation and thawing

We have studied the effects of cryopreservation on the viability and on the expression of surface antigens of acute leukemia cells. Marrow samples were obtained at initial diagnosis from 89 patients with acute myeloid leukemia (AML), acute undifferentiated leukemia (AUL), and acute lymphoid leukemia (ALL). In AML, the mean viability was greater than 90% in the types M1, M4, and M5 of the French-American-British classification, 79% in M2, and 3% in M3 types. The viability was 74% in AUL. In ALL, the viability was 95% for pre-B leukemias, but only 2% in T-cell leukemias. The expression of myeloid antigens was studied before and after freezing and thawing using three monoclonal antibodies (NHL30.5, against poorly differentiated granulocytic leukemias, VIMC6 against differentiated granulocytic leukemias and granulocytes; and UCHM1 or CRIS-6, against monocytic leukemias and monocytes). The percentage of cells stained by NHL30.5 and UCHM1 or CRIS-6 was very similar before and after cryopreservation. For VIMC6, the mean staining after cryopreservation was 60% of the initial one. In pre-B ALL, the stainings by anti common ALL antigen before and after cryopreservation were also very similar. We conclude that leukemic cryopreserved cells are suitable for immunologic studies. The recovery is, however, very low in promyelocytic AML and T-cell ALL.     

Immunochemical study of galanin in the cat digestive tract and autonomic ganglia

The presence of galanin was examined in the cat gut and related autonomic nervous structures using radioimmunoassay (RIA) and high performance liquid chromatography (HPLC). In the gut wall, the concentration of galanin-like immunoreactivity (GAL-LI) was assayed separately in the muscular layers with the nervous plexuses and in the mucosa and ranged from 0.35 to 0.55 pmol/g wet tissue. In the autonomic nervous structures, GAL-LI concentrations ranged from 0.22 (thoracic spinal ganglia) to 0.81 (inferior mesenteric ganglion) pmol/g wet tissue. The presence of galanin was checked by HPLC in the antrum, intestine, and colon. HPLC of extractable material revealed a major peak coeluting with the synthetic porcine peptide and minor earlier peaks representing likely different molecular forms of galanin. Our study strengthens the notion that galanin acts in nervous control of the cat gut functions.     

Changes in morphology and in polyphosphoinositide turnover of human erythrocytes after cholesterol depletion

Human erythrocytes were cholesterol-depleted (5-25%) by incubation with phosphatidylcholine vesicles in media containing Ca2+ at different concentrations (0, 28 nM, 5 microM or 1 mM). After removal of the vesicles, the cells were reincubated with [32P]phosphate in the same media. Control (incubated in buffer alone) and cholesterol-maintained erythrocytes (incubated with cholesterol/phosphatidylcholine vesicles) were treated similarly. Cholesterol depletion induced the conversion of the cells into stomatocytes III and spherostomatocytes and decreased the turnover rate of phosphatidylinositol phosphate and of phosphatidylinositol bisphosphate. None of these effects were observed in cholesterol-maintained cells. In cholesterol-depleted cells, they occurred without changes in the ATP specific activity or in the polyphosphoinositide concentrations. Moreover, these modifications of shape and of lipid metabolism were proportional to the extent of the cholesterol depletion and were independent of the external Ca2+ concentration. In contrast, other effects of cholesterol depletion, a decrease in the turnover rate of phosphatidic acid, a decrease in diacylglycerol and in phosphatidic acid concentrations were dependent on the external Ca2+ concentration. Thus it appears that the shape change was not correlated with a change in the concentrations of these phospholipids or of diacylglycerol and therefore cannot be explained by a bilayer couple mechanism involving these phospholipids. However, the spherostomatocytic transformation was correlated with the decrease in the turnover rate of the polyphosphoinositides, but not with the turnover rate of phosphatidic acid, suggesting a role for the turnover of the polyphosphoinositides in the maintenance of the erythrocyte shape.     

Freeze-fracture studies of frog atrial fibres.

The freeze-fracturing technique was used to characterize the junctional devices involved in the electrical coupling of frog atrial fibres. These fibres are connected by a type of junction which can be interpreted as a morphological variant of the "gap junction" or "nexus". The most characteristic features are rows of 9-nm junctional particles forming single or anastomosed circular profiles on the inner membrane face, and corresponding pits on the outer membrane face. Very seldom aggregates consisting of few geometrically disposed 9-nm particles are found. The significance of the junctional structures in the atrial fibres is discussed, with respect to present knowledge about junctional features of gap junctions in various tissues, including embryonic ones.     

Revisiting the Central Metabolism of the Bloodstream Forms of Trypanosoma brucei: Production of Acetate in the Mitochondrion Is Essential for Parasite Viability

Background

The bloodstream forms of Trypanosoma brucei, the causative agent of sleeping sickness, rely solely on glycolysis for ATP production. It is generally accepted that pyruvate is the major end-product excreted from glucose metabolism by the proliferative long-slender bloodstream forms of the parasite, with virtually no production of succinate and acetate, the main end-products excreted from glycolysis by all the other trypanosomatid adaptative forms, including the procyclic insect form of T. brucei.

Methodology/Principal Findings

A comparative NMR analysis showed that the bloodstream long-slender and procyclic trypanosomes excreted equivalent amounts of acetate and succinate from glucose metabolism. Key enzymes of acetate production from glucose-derived pyruvate and threonine are expressed in the mitochondrion of the long-slender forms, which produces 1.4-times more acetate from glucose than from threonine in the presence of an equal amount of both carbon sources. By using a combination of reverse genetics and NMR analyses, we showed that mitochondrial production of acetate is essential for the long-slender forms, since blocking of acetate biosynthesis from both carbon sources induces cell death. This was confirmed in the absence of threonine by the lethal phenotype of RNAi-mediated depletion of the pyruvate dehydrogenase, which is involved in glucose-derived acetate production. In addition, we showed that de novo fatty acid biosynthesis from acetate is essential for this parasite, as demonstrated by a lethal phenotype and metabolic analyses of RNAi-mediated depletion of acetyl-CoA synthetase, catalyzing the first cytosolic step of this pathway.

Conclusions/Significance

Acetate produced in the mitochondrion from glucose and threonine is synthetically essential for the long-slender mammalian forms of T. brucei to feed the essential fatty acid biosynthesis through the “acetate shuttle” that was recently described in the procyclic insect form of the parasite. Consequently, key enzymatic steps of this pathway, particularly acetyl-CoA synthetase, constitute new attractive drug targets against trypanosomiasis.     

Research Commentary: Association of Zoonotic Pathogens with Fresh, Estuarine, and Marine Macroaggregates

Aquatic macroaggregates (flocs ≥0.5 mm) provide an important mechanism for vertical flux of nutrients and organic matter in aquatic ecosystems, yet their role in the transport and fate of zoonotic pathogens is largely unknown. Terrestrial pathogens that enter coastal waters through contaminated freshwater runoff may be especially prone to flocculation due to fluid dynamics and electrochemical changes that occur where fresh and marine waters mix. In this study, laboratory experiments were conducted to evaluate whether zoonotic pathogens (Cryptosporidium, Giardia, Salmonella) and a virus surrogate (PP7) are associated with aquatic macroaggregates and whether pathogen aggregation is enhanced in saline waters. Targeted microorganisms showed increased association with macroaggregates in estuarine and marine waters, as compared with an ultrapure water control and natural freshwater. Enrichment factor estimations demonstrated that pathogens are 2–4 orders of magnitude more concentrated in aggregates than in the estuarine and marine water surrounding the aggregates. Pathogen incorporation into aquatic macroaggregates may influence their transmission to susceptible hosts through settling and subsequent accumulation in zones where aggregation is greatest, as well as via enhanced uptake by invertebrates that serve as prey for marine animals or as seafood for humans.     

Habitat Management to Reduce Human Exposure to <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis> and Western Conenose Bugs (<Emphasis Type="Italic">Triatoma protracta</Emphasis>)

Chagas disease, which manifests as cardiomyopathy and severe gastrointestinal dysfunction, is caused by Trypanosoma cruzi, a vector-borne parasite. In California, the vector Triatoma protracta frequently colonizes woodrat (Neotoma spp.) lodges, but may also invade nearby residences, feeding upon humans and creating the dual risk of bite-induced anaphylaxis and T. cruzi transmission. Our research aimed to assess T. cruzi presence in woodrats in a previously unstudied northern California area, statistically evaluate woodrat microhabitat use with respect to vegetation parameters, and provide guidance for habitat modifications to mitigate public health risks associated with Tr. protracta exposure. Blood samples from big-eared woodrats (N. macrotis) trapped on rural private properties yielded a T. cruzi prevalence of 14.3%. Microhabitat analyses suggest that modifying vegetation to reduce understory density within a 40 meter radius of human residences might minimize woodrat lodge construction within this buffer area, potentially decreasing human exposure to Tr. protracta.     

Southern Sea Otter as a Sentinel of Marine Ecosystem Health

The southern sea otter (Enhydra lutris nereis) is listed as threatened under the Endangered Species Act (ESA) and is a keystone species, strongly influencing the abundance and diversity of the other species within its kelp forest ecosystem. This is accomplished primarily by preying upon urchins that eat the kelp stipe and holdfast, which can reduce a kelp forest to an urchin barren. Sea otters are very susceptible to marine pollutants such as petroleum, which may be directly toxic and/or alter their furs insulating properties. Sea otters are an excellent sentinel species. They eat approximately 25% of their body weight per day in shellfish and other invertebrates, and can concentrate and integrate chemical contaminants. In addition, they appear to be susceptible to a number of diseases and parasites that may have anthropogenic origins, and shellfish may serve as an intermediary for some of these infections. Many of the shellfish the otters eat are also harvested for human food. In their role as sentinels, sea otter health has implications for human health, economic sustainability of shellfisheries, as well as overall marine ecosystem health. The recent southern sea otter decline has been viewed with some alarm by conservationists and, indeed, recovery seems a long way off. High mortality rather than depressed recruitment appears to underlie the decline. A good deal of debate has centered on the role of infectious diseases and parasites, exposure to contaminants, nutrition and prey availability, net and pot fishery interactions, and other sources of mortality. Current research is being done related to major classes of mortality, various types of pollutants and some specific organisms causing southern sea otter mortality, and their implications for marine ecosystem health and sustainability.     

Diagnosis and seroprevalence of leptospirosis in California sea lions from coastal California

The sensitivity and specificity of the microscopic agglutination test (MAT) as a method for detection of exposure to Leptospira spp. in California sea lions (Zalophus californianus) were determined. Sera came from individuals that demonstrated clinical signs of renal disease, had lesions suggestive of leptospirosis at necropsy, and had visible leptospires in silver stained kidney sections as positive controls. Sera from unexposed captive individuals were used as negative controls. The test was 100% sensitive at 1:3,200 for confirming renal infection and 100% specific at negative < 1:100 for detection of Leptospira interrogans scrovar pomona antibodies by MAT in California sea lions. Leptospira interrogans serovar pomona was used as a screening serovar because it has been isolated previously from the kidneys and placentas of California sea lions, and there appears to be cross-reactivity between serovar pomona and other serovars. Sera from 225 free-ranging California sea lions presented to one of three participating California (USA) coastal marine mammal rehabilitation centers in 1996 were then evaluated for antibodies to serovar pomona using the MAT. The overall seroprevalence was 38.2% (86/225), although the prevalence varied among locations from 100% (38/38) in animals at the Marine Mammal Care Center (Fort MacArthur, California, USA) to 0% (0/14) at SeaWorld California (San Diego, California). At The Marine Mammal Center (Sausalito, California) [prevalence 27.8% (48/173)], the majority of seropositive animals were subadults and adults, and males were 4.7 times more likely to be seropositive to serovar pomona than females. When combining results from all three centers, subadult and adult animals were more likely to be seropositive than pups and juvenile sea lions, and the highest proportion of seropositive animals presented during the autumn months. Serum elevations of blood urea nitrogen, creatinine, phosphorus, and/or calcium were associated with seropositivity to serovar pomona. We found no association between potassium or sodium levels and seropositivity.