Sub-typing of animal and human Campylobacter spp. using RAPD

R.H. MADDEN, L. MORAN AND P. SCATES. 1996. Based on a 10-mer primer (5'- CCTGTTAGCC-3'), a random amplified polymorphic DNA (RAPD) method for typing Campylobacter coli isolated from pigs was developed. The method proved effective with a high discrimination and good reproducibility. In contrast with serotyping no untypable strains were found out of a total of 269 isolates (veterinary, food and clinical) examined. The method was also successfully applied to typing Campylobacter jejuni from a similar range of sources.     

A genetic linkage map for Pinus radiata based on RFLP, RAPD, and microsatellite markers

A genetic linkage map for radiata pine (Pinus radiata D. Don) has been constructed using segregation data from a three-generation outbred pedigree. A total of 208 loci were analyzed including 165 restriction fragment length polymorphism (RFLP), 41 random amplified polymorphic DNA (RAPD) and 2 microsatellite markers. The markers were assembled into 22 linkage groups of 2 or more loci and covered a total distance of 1382 cM. Thirteen loci were unlinked to any other marker. Of the RFLP loci that were mapped, 93 were detected by loblolly pine (P. taeda L.) cDNA probes that had been previously mapped or evaluated in that species. The remaining 72 RFLP loci were detected by radiata pine probes from a PstI genomic DNA library. Two hundred and eighty RAPD primers were evaluated, and 41 loci which were segregating in a 11 ratio were mapped. Two microsatellite markers were also placed on the map. This map and the markers derived from it will have wide applicability to genetic studies in P. radiata and other pine species.     

Impact of a Genetically Engineered Bacterium with Enhanced Alkaline Phosphatase Activity on Marine Phytoplankton Communities

An indigenous marine Achromobacter sp. was isolated from coastal Georgia seawater and modified in the laboratory by introduction of a plasmid with a phoA hybrid gene that directed constitutive overproduction of alkaline phosphatase. The effects of this "indigenous" genetically engineered microorganism (GEM) on phosphorus cycling were determined in seawater microcosms following the addition of a model dissolved organic phosphorus compound, glycerol 3-phosphate, at a concentration of 1 or 10 (mu)M. Within 48 h, a 2- to 10-fold increase in the concentration of inorganic phosphate occurred in microcosms containing the GEM (added at an initial density equivalent to 8% of the total bacterial population) relative to controls containing only natural microbial populations, natural populations with the unmodified Achromobacter sp., or natural populations with the Achromobacter sp. containing the plasmid but not the phoA gene. Secondary effects of the GEM on the phytoplankton community were observed after several days, evident as sustained increases in phytoplankton biomass (up to 14-fold) over that in controls. Even in the absence of added glycerol 3-phosphate, a numerically stable GEM population (averaging 3 to 5% of culturable bacteria) was established within 2 to 3 weeks of introduction into seawater. Moreover, alkaline phosphatase activity in microcosms with the GEM was substantially higher than that in controls for up to 25 days, and microcosms containing the GEM maintained the potential for net phosphate accumulation above control levels for longer than 1 month.     

Influence of simulated microgravity on the exercise performance of Fischer 344 rats

Measurements from mission specialists after space flights or from subjects subjected to head down tilt experiments have demonstrated a decrease in exercise performance. Similar decreases have been reported for rats that have participated in simulated microgravity studies using the head down-tail suspended method of Morey-Holton (HDS). Because it is unclear whether older animal populations would exhibit similar responses, we undertook a HDS study with Fischer 344 male rats.     

External morphology of the male and female of Sphyrion lumpi (Krøyer,1845) (Copepoda; Siphonostomatoida; Sphyriidae)

Sphyrion lumpi is a sexually dimorphic, parasitic copepod which causes significant losses in fisheries in the North Atlantic. Studies involving both light and scanning electron microscopy revealed important details of the external morphology of both sexes that are discussed in relation to other members of the family Sphyriidae. Conclusions are drawn regarding the life cycle of S. lumpi. The present finding of this parasite on Urophycis tenuis constitutes a new host record.     

Modulation of small conductance calcium-activated potassium channels in C6 glioma cells

Using the patch clamp technique, we have characterized a small conductance, calcium-activated potassium (SK) channel in the C6 glioma cell line. Elevation of cytosolic Ca²⁺ concentration ([Ca²⁺] _i ) by applications of serotonin or ionomycin induced bursts of channel openings recorded in the cell-attached configuration. These channels underlie the serotonin-induced, [Ca²⁺] _i -activated whole-cell K⁺ conductance described previously. [Ca²⁺] _i directly activated SK channels in inside-out patches with a biphasic concentration dependence. Submicromolar [Ca²⁺] _i induced bursts of channel openings with a unitary conductance of about 25 pS, similar to that of the serotonin-induced channels. Supramicromolar [Ca²⁺] _i caused prolonged openings with a unitary conductance of about 35 pS, resulting in a pronounced increase of the average current in patches exposed to [Ca²⁺] _i above 100 m. The two modes of opening reflect the activity of the same SK channel. The channel conductance depended on external K⁺ concentration with K _Dof 5 m. The channel was slightly permeable to cations other than K⁺, with a permeability ratio for K⁺Ca²⁺Na⁺ of 10.0400.030, respectively. ATP was required to maintain channel activity in outside-out patches but was not essential in inside-out patches. The modulation of SK channels in C6 cells by components in their microenvironment may be related to the role of glial cells in controlling the extracellular milieu in the CNS.The authors are grateful to Dr. M. Segal for continuous support, stimulating discussions and criticism throughout the course of this work, to Dr. I. Steinberg for helpful suggestions and to Dr. H. Jarosch, for helping with the Fortran application. N.M.'s research was supported in part by BARD, the U.S.-Israel Binational Agricultural Research and Development Fund, grant no. IS-1670-89RC.     

Characteristics of Modified Leghemoglobins Isolated from Soybean (Glycine max Merr.) Root Nodules

Hemoprotein derivatives of an abundant soybean (Glycine max Merr.) root nodule leghemoglobin, Lba, were studied for their modified spectral characteristics and physical properties. Three modified hemoprotein derivatives of Lba (Lbam1, Lbam2, and Lbam3) were purified by preparative isoelectric focusing. The ferric forms of these pigments were green and exhibited anomalous spectra in the visible region as compared to the Lba3+ forms. These modified pigments showed a hypochromic shift of 10 nm for the charge transfer absorption maximum; however, differences were not apparent in the Soret region. Upon binding with nicotinate, the [alpha] and [beta] bands were shifted significantly into the red region as compared to the Lba3+ nicotinate complex. The three Lbam fractions were reduced by dithionite or by NADH in the presence of riboflavin. Lbam2+ also bound nicotinate and displayed absorption spectra indistinguishable from those of Lba2+ nicotinate. In contrast to Lba2+, Lbam2+ displayed aberrant spectra when bound with either O2 or CO. These complexes exhibited a prominent charge transfer band at approximately 620 nm and failed to exhibit spectra characteristic of Lba2+O2 and Lba2+CO. The protein moiety of these modified pigments was intact because their tyrosine/tryptophan ratios and their amino acid compositions were identical with those of Lba, nor were differences observed in the peptide profiles resulting from trypsin digests of purified Lba and Lbams. Automated Edman degradation of selected peaks further confirmed the intactness of the protein backbone including the absence of deamination. Pyridine hemochromogen for heme from Lbams could be formed, and the spectra displayed distinct differences compared to those of Lba. A new peak at 580 nm and a loss of a peak at 480 nm were observed for all three Lbams.     

Urinary steroid evaluations to monitor ovarian function in exotic ungulates: X. Pregnancy diagnosis in Perissodactyla

Enzymeimmunoassays (EIAs) for estrone conjugates (EC), pregnanediol-3-glucuronide (PDG), and C-19 and C-21 progesterone metabolites (C-19/C-21) were used to analyze urine samples from four nondomestic equid species, four tapir species, and two rhinoceros species in an attempt to identify if these assays could be used for diagnosing and monitoring pregnancy. The same urine samples were also analyzed for the presence of equine chorionic gonadotropin (eCG) activity, using a field dipstick test and a radioimmunoassay (RIA). The EC EIA was validated for three equid species and the Malayan tapir. Neither the PDG nor the C-19/C-21 EIAs were validated in any species evaluated. In equid species, the EC EIA demonstrated a specificity (the percentage of nonpregnant samples identified correctly) of 100% and a sensitivity (the percentage of pregnant samples identified correctly) of ≥ 88%. With the exception of the Grevy's zebra, the C-19/C-21 EIA showed a similar accuracy in identifying pregnant and nonpregnant equids. The PDG EIA was not sufficiently accurate to merit its use in equids or tapirs for pregnancy diagnosis. From the data collected, it appears analysis of a single urine by both the EC EIA and the C-19/C-21 EIA would be the best method of pregnancy detection during the last 2 trimesters of gestation, in equid species. In tapirs, the C-19/C-21 EIA was slightly more accurate for pregnancy diagnosis than the EC EIA. The C-19/C-21 EIA had a specificity of 93%, but a sensitivity of only 73% in tapir species. None of the EIAs evaluated demonstrated a sufficient specificity or sensitivity to be useful, as presently performed, for pregnancy diagnosis from a single sample in the black rhinoceros. The eCG dipstick used in this study did not prove a sufficiently reliable test for routine pregnancy in nondomestic equids. The eCG RIA results in the Przewalski's horses and the Hartman's mountain zebra were positive early in gestation, and indicate that gonadotropin analysis may be useful for pregnancy detection in these species. Only very low amounts of eCG activity was measured by the eCG RIA in the tapir and rhinoceros urine samples. © 1994 Wiley-Liss, Inc.