Kinetics of Na(+) transport in necturus proximal tubule

The dependence of proximal tubular sodium and fluid readsorption on the Na(+) concentration of the luminal and peritubular fluid was studied in the perfused necturus kidney. Fluid droplets, separated by oil from the tubular contents and identical in composition to the vascular perfusate, were introduced into proximal tubules, reaspirated, and analyzed for Na(+) and [(14)C]mannitol. In addition, fluid transport was measured in short-circuited fluid samples by observing the rate of change in length of the split droplets in the tubular lumen. Both reabsorptive fluid and calculated Na fluxes were simple, storable functions of the perfusate Na(+) concentration (K(m) = 35-39 mM/liter, V(max) = 1.37 control value). Intracellular Na(+), determined by tissue analysis, and open-circuit transepithelial electrical potential differences were also saturable functions of extracellular Na(+). In contrast, net reabsorptive fluid and Na(+) fluxes were linearly dependent on intracellular Na(+) and showed no saturation, even at sharply elevated cellular sodium concentrations. These concentrations were achieved by addition of amphotericin B to the luminal perfusate, a maneuver which increased the rate of Na(+) entry into the tubule cells and caused a proportionate rise in net Na(+) flux. It is concluded that active peritubular sodium transport in proximal tubule cells of necturus is normally unsaturated and remains so even after amphotericin-induced enhancement of luminal Na(+) entry. Transepithelial movement of NaCl may be described by a model with a saturable luminal entry step of Na(+) or NaCl into the cell and a second, unsaturated active transport step of Na(+) across the peritubular cell boundary.     

Pupal development of Ceratitis capitata (Diptera: Tephritidae) and Diachasmimorpha longicaudata (Hymenoptera: Braconidae) at different moisture values in four soil types

This study aimed to evaluate adult emergence and duration of the pupal stage of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann), and emergence of the fruit fly parasitoid, Diachasmimorpha longicaudata (Ashmead), under different moisture conditions in four soil types, using soil water matric potential. Pupal stage duration in C. capitata was influenced differently for males and females. In females, only soil type affected pupal stage duration, which was longer in a clay soil. In males, pupal stage duration was individually influenced by moisture and soil type, with a reduction in pupal stage duration in a heavy clay soil and in a sandy clay, with longer duration in the clay soil. As matric potential decreased, duration of the pupal stage of C. capitata males increased, regardless of soil type. C. capitata emergence was affected by moisture, regardless of soil type, and was higher in drier soils. The emergence of D. longicaudata adults was individually influenced by soil type and moisture factors, and the number of emerged D. longicaudata adults was three times higher in sandy loam and lower in a heavy clay soil. Always, the number of emerged adults was higher at higher moisture conditions. C. capitata and D. longicaudata pupal development was affected by moisture and soil type, which may facilitate pest sampling and allow release areas for the parasitoid to be defined under field conditions.     

Phenotypic characteristics of thermotolerantCampylobacter from human and animal sources

Five reference strains and 314 field strains ofCampylobacter growing at 42°C, but not at 25°C, were characterized by tests of hippurate hydrolysis and sensitivity to nalidixic acid, to metronidazole, and to 2,3,5-triphenyltetrazolium chloride (TTC). All strains but seven were TTC-resistant by the disc test used. Of 168 human isolates, 87% hydrolyzed hippurate; three of these strains were nalidixic acid-resistant. Also hippurate-positive were all 23 strains from ovine abortion, 79% of 43 avian strains, two of six bovine isolates, and one of two strains of equine origin. Seventy-two porcine strains were all hippurate-negative. Metronidazole sensitivity was found to be a variable property in hippurate-positive strains, although present in all but four hippurate-negative strains. A group of eight nalidixic acid-resistant, hippurate-negative isolates of porcine and bovine origin probably represent a new group ofCampylobacter.     

Oligodeoxynucleotide-directed mutagenesis using the yeast transformation system

In this report we describe a highly efficient method for site-specific mutagenesis using the yeast transformation system. The method is based on the observation that Saccharomyces cerevisiae can be transformed at high frequency with single-stranded circular DNA vectors [Singh et al., Gene 20 (1982) 441-449]. The model system studied was the TRP1 gene of S. cerevisiae cloned into a derivative of the phage M13mp9 vector containing the yeast URA3 gene. ARS1, located adjacent to the TRP1 gene, allows the plasmid to replicate autonomously in yeast. Synthetic 5'P-oligodeoxynucleotides, 19 and 35 nucleotides (nt) in length, designed to produce an A----T transversion mutation within the TRP1 gene, were annealed to ss DNA of the M13 vector at a molar ratio of 30:1 and directly transformed into yeast. The intended single nt mutation was obtained at frequencies of 24 and 43%, respectively. The latter approaches the theoretical limit of 50%. In the absence of the 5'-terminal phosphate, both the transformation frequency and the efficiency of mutagenesis by the synthetic oligodeoxynucleotide (oligo) were decreased by 2-4 fold. This procedure completely obviates the need for any enzymatic manipulations in vitro after forming the heteroduplex with the oligo primer containing the desired mutation. For yeast genes, direct phenotypic selection is possible in the recipient strain.     

Use of PCR primers containing a 3'-terminal ribose residue to prevent cross-contamination of amplified sequences.

Cross-contamination with previously amplified products poses a serious limitation in the use of PCR for clinical testing and in certain research applications as well. In the present study we report the use of novel primers containing a 3'-terminal ribose residue to circumvent this problem. Extension of the primer by Taq DNA polymerase generates a cleavable ribonucleotide linkage within the amplified product. Cleavage of the primer by base or with a ribonuclease interferes with further replication of the product should carry over to another sample occur. Primers terminating in any of the 4 ribose residues function equally well as all DNA primers. Taq DNA polymerase is thus able to both efficiently extend and copy the single ribose residue. In translating from all DNA primers to ones containing a 3'-ribose residue no modification of the PCR protocol is required. The products formed can be used in all applications of the PCR. Since neither the original sample DNA, the primers or the extension products are modified by base or ribonuclease treatment both pre- and post-amplification sterilization can be carried out. Pre-amplification treatment with RNase A can yield as high as 10(4)-fold sterilization. Under these conditions the addition of beta-mercaptoethanol or other sulfhydryl reducing agent is necessary to inactivate the enzyme during thermocycling. Post-amplification treatment with NaOH readily yields at least 10(6)-fold sterilization. This alone is sufficient for most, if not all, applications of PCR. It is especially useful for quantitative RT-PCR, since the original target RNA sequence, which may be present in high copy numbers, is also destroyed.     

Cuticular Membrane Fine Structure of Nicotiana tabacum L. Leaves

The cuticular membrane (CM) ofNicotiana tabacumL., includingthe cellin wall (CW), was examined to gain more informationabout the nature and chemical constitution of its fine structurefor possible inclusion in a model system, as recent literaturequestions its function as a major water permeability barrier.Different preparation techniques were used and the results evaluatedto select a method for future studies on tobacco leaf cuticles.Fixation with OsO₄included in the primary fixative, either asa vapour or in combination with other agents, followed by OsO₄aspost-fixative, gave good contrast of the CM. The lamellar structureof the tobacco cuticle proper (CP) was revealed by contrastingwith uranyl acetate and lead citrate. The fine lamellar structureof the CP was very clearly contrasted when KMnO₄was includedin the primary fixative. This was interpreted as indicatingthe tobacco CP to be polar. The reticulate fibrillar patternof the tobacco cuticular layer (CL) containing polysaccharideswas well contrasted when either OsO₄or paraformaldehyde wereincluded in the primary fixative. Cold fixation with glutaraldehydeand dimethyl sulphoxide and post-fixation with OsO₄revealedelectron-opaque material in the outer cutinized, irregularlyoutlined, region of the CW. These ultrahistochemical reactionsare discussed in relation to the known chemical compositionand possible water permeability of the CM. Cuticular fine structure; cuticular transpiration; Nicotiana tabacumL.     

Distribution of individual members of the mosquito Anopheles maculipennis complex in Germany identified by newly developed real‐time PCR assays

Owing to their role as vectors of malaria parasites, species of the Anopheles maculipennis complex (Diptera: Culicidae) Meigen were intensively studied in the past, but with the disappearance of malaria in Germany in the middle of the last century, the interest in this field of research declined. A comprehensive ecological analysis of the current species distribution for Germany is lacking. Between 2010 and 2013, a total of 1445 mosquitoes of the An. maculipennis complex were collected at 72 different sites in Germany. The samples comprise 722 single individuals as well as 723 individuals in 90 pools of up to 25 mosquitoes. All samples were analysed with newly developed species‐specific qPCR assays for the identification of the four German species using nucleotide differences within the internal transcribed spacer 2 (ITS2) ribosomal DNA. All gathered data were used for species distribution modelling. The overall prevalence of An. messeae s.l. was highest with 98.89% of all pools; An. daciae with 6.93% of all individuals and An. messeae s.s. with 69.53%. The prevalence of the other two species was relatively low: An. maculipennis s.s. with 13.30% of all individuals (6.67% of all pools) and An. atroparvus with 1.80% of all individuals (1.11% of all pools).     

Parasitism on medfly by Diachasmimorpha longicaudata (Ashmead) (Hymenoptera: Braconidae) in different guava cultivars

The parasitism efficiency of the Braconidae wasp, Diachasmimorpha longicaudata (Ashmead), was checked on four guava cultivars (Paluma, Sassaoca, Pedro Sato and Kumagai) infested with larvae of medfly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae). Five blocks of eight fruits, each with two fruits of each cultivar, were put inside C. capitata adult cages, during 2h for oviposition, and a week later, when the larvae inside guavas were developed, the fruits were exposed to parasitoids for 24h. The mean fruit weight, larvae mortality, number of pupae, percentage of medfly and parasitoid emergence were evaluated. There was not statistical difference among cultivars to weight, larvae mortality, number of pupae e emergence of medfly. The percentage of parasitism was higher in Pedro Sato cultivar (19.8%) compared with Kumagai cultivar (2.9%), but it was statistically similar to the other cultivars.