The susceptibility of Salvelinus fontinalis (Mitchill) to Gyrodactylus salaris Malmberg (Platyhelminthes; Monogenea) under experimental conditions

The host specificity of Gyrodaclylus Solaris is examined experimentally with respect to its ability to infect the brook trout, Salvelinus fontinalis . The parasite readily attached to and reproduced on parr of this host and infections grew for c. 20 days from first monitoring (c. 30 days from first infection) before declining. Parasites could persist on this host for up to 70 days before finally disappearing. The pattern of infection resembled that seen in many other gyrodactylid species on their normal hosts, and suggested the action of a host response, In this respect infections of G. salaris on parr of S. fontinalis , anadromous Salvelinus alpinus, Oncorhynchus mykiss, Thymallus thymallus and Baltic Salmo salar follow a normal pattern, while infections of Norwegian S. salar are unusual in a continued unchecked growth, until the host dies, under pooled laboratory conditions.     

Time Course of Adaptations in Dopamine Biosynthesis, Metabolism, and Release Following Nigrostriatal Lesions: Implications for Behavioral Recovery from Brain Injury

Alterations in neostriatal dopamine metabolism, release, and biosynthesis were determined 3, 5, or 18 days following partial, unilateral destruction of the rat nigrostriatal dopamine projection. Concentrations of dopamine and each of its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 3-methoxytyramine (3-MT) were markedly decreased in the lesioned striata at 3, 5, or 18 days postoperation. The decline in striatal high-affinity [3H]dopamine uptake closely matched the depletion of dopamine at 3 and 18 days postoperation. However, neither DOPAC, HVA, nor 3-MT concentrations were decreased to as great an extent as dopamine at any time following lesions that depleted the dopamine innervation of the striatum by greater than 80%. In these more severely lesioned animals, dopamine metabolism, estimated from the ratio of DOPAC or HVA to dopamine, was increased two- to four-fold in the injured hemisphere compared with the intact hemisphere. Dopamine release, estimated by the ratio of 3-MT to dopamine, was more increased, by five- to sixfold. Importantly, the HVA/dopamine, DOPAC/dopamine, and 3-MT/dopamine ratios did not differ between 3 and 18 days postlesioning. The rate of in vivo dopamine biosynthesis, as estimated by striatal DOPA accumulation following 3,4-dihydroxyphenylalanine (DOPA) decarboxylase inhibition with NSD 1015, was increased by 2.6- to 2.7-fold in the surviving dopamine terminals but again equally at 3 and 18 days postoperation. Thus, maximal increases in dopamine metabolism, release, and biosynthesis occur rapidly within neostriatal terminals that survive a lesion. This mobilization of dopaminergic function could contribute to the recovery from the behavioral deficits of partial denervation by increasing the availability of dopamine to neostriatal dopamine receptors. However, these presynaptic compensations are not sufficient to account for the protracted (at least 3-week) time course of sensorimotor recovery that has been observed following partial nigrostriatal lesion.     

Phosphatidylinositol Phosphodiesterase (Phospholipase C) Activity in the Pineal Gland: Characterization and Photoneural Regulation

Phosphatidylinositol phosphodiesterase (PL-C) appears to be a key element in the adrenergic regulation of pineal cyclic AMP levels. In the present study, the rat pineal enzyme was characterized using exogenous [3H]phosphatidylinositol (0.5 mM) as substrate. Half the enzyme activity was found in the cytosolic fraction, but the highest specific concentration was associated with the membrane fraction. Two pH optima (5.5 and 7.5) of enzyme activity were observed for the membrane fraction but only one in the cytosol fraction (pH 5.5). Enzyme activity in both fractions was Ca2+ dependent. In the case of the membrane protein in pH 7.5, the enzyme activity was sensitive to changes in Ca2+ in the 10-100 nM range. Addition of an equimolar concentration of phosphatidylinositol 4-phosphate nearly completely inhibited the hydrolysis of [3H]phosphatidylinositol; other phospholipids (1.0 mM) were less potent. This may reflect our present finding that [3H]phosphatidylinositol 4-phosphate is a better substrate than [3H]phosphatidylinositol for the enzyme. Stimulus deprivation (2 weeks of constant light or superior cervical ganglionectomy) reduced the cytosolic activity by 30% and had no effect on the membrane-associated enzyme.     

Purification, properties, and cellular localization of Euglena ferredoxin-NADP reductase

Ferredoxin-NADP reductase from Euglena gracilis Klebs var. Bacillaris Cori purified to apparent homogeneity, yields a typical 36 kDa and an unusual 15 kDa polypeptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, exhibits a typical flavoprotein spectrum, contains FAD, and catalyzes NADPH-dependent iodonitrotetrazolium-violet diaphorase, NADPH-specific ferredoxin-dependent cytochrome-c-550 reductase and NADPH-NAD transhydrogenase activities. Rabbit antibody to the purified FNR blocks these activities specifically and also blocks the iodonitrotetrazolium-violet diaphorase activity of Euglena chloroplasts completely. The low iodonitrotetrazolium-violet diaphorase activity in the plastidless mutant, W₁₀BSmL, is mitochondrial and is not specifically blocked by the ferredoxin-NADP reductase antibody. Dark-grown non-dividing (resting) wild-type Euglena cells show a 4-fold increase in ferredoxin-NADP reductase activity during greening at 970 lx. Half of the low ferredoxin-NADP reductase activity in dark-grown cells is initially soluble, but by the end of chloroplast development nearly all of the enzyme is membrane-bound. The binding of ferredoxin-NADP reductase on exposure to light correlates with the extent of thylakoid membrane formation. Immunoblots of wild-type extracts during greening indicate that the 15 kDa polypeptide increases in the same manner as the extent of reductase binding to thylakoid membranes.     

Inhibition of phosphoglucose isomerase allozymes from the wing polymorphic waterstrider,Limnoporus canaliculatus,by pentose shunt metabolites

Inhibition of phosphoglucose isomerase (PGI) allozymes from the wing-polymorphic waterstrider, Limnoporus canaliculatus, by three pentose-shunt metabolites was studied at several different temperatures. This was done to determine if the allozymes exhibited a differential ability to participate in lipid biosynthesis via differential partitioning of carbon flux through the pentose shunt versus glycolysis. 6-Phosphogluconate and erythrose-4-phosphate proved to be strong competitive inhibitors of PGI, while sedoheptulose-7-phosphate was a very weak inhibitor. The PGI allozymes from L. canalicualtus were differentially inhibited by 6-phosphogluconate at two of the three temperatures studied. However, this property does not appear to be an adaptive difference between the allozymes but, rather, a correlated effect resulting from variation in substrate binding. Estimates of reaction rates for the allozymes indicate that the differences in inhibition result in no detectable differences in reaction velocities. Thus, no evidence in support of the hypothesis that PGI allozymes from Limnoporus canaliculatus were adapted to function in different metabolic capacities via differential inhibition was obtained in this study. However, the importance of this characteristic in allozymic adaptation in natural populations remains an open question.Supported by NSF Grant DEB 7908802 and UPHS Grant GM 21133 to R. K. Koehn and an NSF dissertation improvement grant to A. J. Zera.     

Natural killer (NK) cell immunodeficiency in patients with chronic myelogenous leukemia

Summary Defective natural killer (NK) cell populations from patients with chronic myelogenous leukemia (CML), that reacted with both HNK-1⁺ and B73.1⁺ antibodies, were obtained by a flluorescence-activated cell sorter (FACS). These fractions, along with NK fractions from normal donors which reacted with both antibodies, were expanded as bulk cultures or clones by limiting dilution, for 4 weeks in the presence of 10% interleukin 2 (IL 2), human type AB plasma, and irradiated human allogeneic mononuclear cells. Successfully established clones from patients with CML, with lytic activity against autologous and more differentiated neoplastic granulocytes, were generated more efficiently from B73.1⁺ than from HNK-1⁺ subsets. However, there were no significant differences among the generations of B73.1⁺ and HNK-1⁺ clones for both patients and normal donors with lytic activity against NK susceptible K-562 targets. Fresh myeloblast preparations from a blast crisis were found to be more susceptible to lysis by IL 2-proliferative B73.1⁺ and HNK-1⁺ clones than were fresh myelocyte preparations from a chronic phase CML patient, which were lytically susceptible to only B73.1⁺ clones. B73.1⁺ and HNK-1⁺ subsets from CML patients demonstrated major histocompatibility complex nonrestricted killing, and showed the following predominant phenotypes: B73.1⁺T3⁺T8⁺ or B73.1⁺T3⁺T8^– from B73.1⁺ subsets; and HNK-1^–T3⁺T8⁺ (initially HNK-1⁺) from HNK-1⁺ subsets. In contrast, B73.1⁺ and HNK-1⁺ clones from normal donors showed the following predominant phenotypes: B73.1⁺T3^–T8^–; and HNK-1^–T3^–T8^– or HNK-1^–T3^–T8⁺ (initially all HNK-1⁺). Short-term in vitro IL 2 or interferon treatment of fresh NK defective subsets from CML patients resulted in minimal cytotoxic augmentation. In contrast, defective NK cells from CML patients, whether HNK-1⁺ or B73.1⁺ subsets, proliferated with complete regeneration of cytolytic activity after a 3–4 week exposure to IL 2, but differed in phenotypic profiles as compared to those of normal donors. These observations imply that not only fresh defective NK cells but also the cytotoxically restored clones from CML patients are derived from different NK subsets and may represent undifferentiated forms of NK cells that may be arrested at an early stage of development by yet unknown mechanism(s). In vitro substantiation of autologous leukemia cell killing by IL 2-proliferative NK cell clones is encouraging and may allow for new in vivo immunotherapeutic modalities in CML patients.     

Localization and cloning of Xp21 deletion breakpoints involved in muscular dystrophy

Summary Twenty-nine deletion breakpoints were mapped in 220 kb of the DXS164 locus relative to potential exons of the Duchenne and Becker muscular dystrophy gene. Four deletion junction fragments were isolated to acquire outlying Xp21 loci on both the terminal and centromere side of the DXS164 locus. The junction loci were used for chromosome walking, searches for DNA polymorphisms, and mapping against deletion and translocation breakpoints. Forty-four unrelated deletions were analyzed using the junction loci as hybridization probes to map the endpoints between cloned Xp21 loci. DNA polymorphisms from the DXS164 and junction loci were used to follow the segregation of a mutation in a family that represents a recombinant. Both the physical and genetic data point to a very large size for this X-linked muscular dystrophy locus.