Differentiation of Ca2+ pumps linked to plasma membrane and endoplasmic reticulum in the microsomal fraction from intestinal smooth muscle

ATP promotes ⁴⁵Ca uptake by the microsomal fraction from the longitudinal smooth muscle of guinea-pig ileum and this uptake is stimulated by oxalate. As the microsomal fraction is made up of various subcellular entities, we examined the localization of the Ca²⁺-transport activity by density gradient centrifugation, taking advantage of the selective effect of digitonin (at low concentration) on the density of plasmalemmal elements. When the ⁴⁵Ca-uptake activity was measured in the absence of oxalate, its behavior in subfractionation experiments closely paralleled that of the plasmalemmal marker 5′-nucleotidase. In contrast, the additional Ca²⁺-transport activity elicited by oxalate behaved like NADH-cytochrome $\text{c}$ reductase, a putative endoplasmic reticulum marker. The endoplasmic reticulum vesicles constituted only a small part of the membranes in the microsomal fraction, which explains that their Ca²⁺-storage capacity was not detectable in the absence of Ca²⁺-trapping agent. Low digitonin concentrations selectively increased the Ca²⁺ permeability of the plasmalemmal vesicles. The two Ca²⁺-transport activities were further differentiated by their distinct sensitivities to K⁺, vanadate and calmodulin. In this respect, the oxalte-insensitive and oxalate-stimulated Ca²⁺-transport systems resembled, respectively, the sarcolemmal and sarcoplasmic reticulum Ca²⁺ pumps in cardiac and skeletal muscle, in accordance with the subcellular locations established by density gradient centrifugation.     

Pure red cell hypoplasia associated with long-arm deletion of chromosome 5

Summary A further instance of the new hematologic entity of pure red cell hypoplasia with deletion of the long arm of a chromosome 5(5q-) was seen in a 59-year-old white male. Examination of the bone marrow showed depressed erythroid cells with normal granulocytic and megakaryocytic precursors. Chromosomal analysis showed deletion of a chromosome 5 without any apparent translocation. This karyotype may be associated with acute leukemia.     

Primary photochemical processes in isolated reaction centers of Rhodopseudomonas viridis

Picosecond and nanosecond spectroscopic techniques have been used to study the primary electron transfer processes in reaction centers isolated from the photosynthetic bacterium Rhodopseudomonas viridis. Following flash excitation, the first excited singlet state (P1) of the bacteriochlorophyll complex (P) transfers an electron to an intermediate acceptor (I) in less than 20 ps. The radical pair state (P⁺I^?) subsequently transfers an electron to another acceptor (X) in about 230 ps. There is an additional step of unknown significance exhibiting 35 ps kinetics. P⁺ subsequently extracts an electron from a cytochrome, with a time constant of about 270 ns. At low redox potential (X reduced before the flash), the state P⁺I^? (or P^F) lives approx. 15 ns. It decays, in part, into a longer lived state (P^R), which appears to be a triplet state. State P^R decays with an exponential time of approx. 55 μs. After continuous illumination at low redox potential (I and X both reduced), excitation with an 8-ps flash produces absorption changes reflecting the formation of the first excited singlet state, P1. Most of P1 then decays with a time constant of 20 ps. The spectra of the absorbance changes associated with the conversion of P to P1 or P⁺ support the view that P involves two or more interacting bacteriochlorophylls. The absorbance changes associated with the reduction of I to I^? suggest that I is a bacteriopheophytin interacting strongly with one or more bacteriochlorophylls in the reaction center.     

Separation of Linked Markers in Chinese Hamster Cell Hybrids: Mitotic Recombination Is Not Involved

A search for mitotic recombination was carried out using mutant subclones of cultured Chinese hamster ovary cells. Recombination events were sought between the linked loci specifying the enzymes hypoxanthine phosphoribosyl transferase and glucose-6-phosphate dehydrogenase. It was shown by fluctuation analysis that markers at these two loci co-segregate from doubly heterozygous pseudotetraploid hybrid cells more than 90% of the time. The minority class of segregants, which had lost one marker without losing the other, were genetically analyzed to distinguish between the possibilities of mitotic recombination and deletion of chromosomal material. Nine clones in which a linkage disruption had occured were studied, using further cell hybridization and segregation. In three cases, a recessive lethal loss of genetic information was indicated, suggesting the deletion mechanism. In six cases, it was demonstrated that no new linkage relationships had been established concomitant with linkage disruption. Thus, in all nine clones, the evidence indicated that mitotic recombination was not involved in the events that disrupted linkage between these two loci. If mitotic recombination takes place at all in this system, the rate must be less than about 10^-6 per cell per generation.     

Ectopic pairing and evolution of 5S ribosomal RNA genes in the chromosomes of Drosophila funebris

5S ribosomal RNA from Drosophila melanogaster labeled with ¹²⁵I was used to locate the 5S rRNA genes in chromosomes of D. funebris by means of in situ hybridization. Silver grains were observed at three distinct sites, one of which was a recognized reverse repeat. Only one half of the reverse repeat, however, hybridizes with 5S rRNA and the significance of this phenomenon is discussed. A case of ectopic pairing between two different 5S sites in the genome is reported, and the significance of ectopic pairing is considered.The author was a Predoctoral Fellow supported by Grant GM 1974 from the National Institute of General Medical Sciences, National Institutes of Health.Contribution from Oak Ridge National Laboratory, operated by the Union Carbide Corporation for the U.S. Energy Research and Development Administration.     

Developmental requirements of the univoltine species Chrysopa downesi: Photoperiodic stimuli and sensitive stages

Chrysopa downesi reproduces only in the spring and the resulting adults enter an aestival-autumnal-hibernal diapause which is primarily controlled by photoperiod. In the laboratory, constant photoperiods result in diapause induction and maintenance, whereas a series of short days followed by long days prevents or terminates diapause and promotes reproduction. The stages most sensitive to the diapause-averting stimulus are the free-living third instar, the third instar within the cocoon, and the pupa.C. downesi responds in different ways to three aspects of photoperiod: (a) an all-or-none response (diapause prevention or induction) to a sequence of two critical photoperiods, (b) an all-or-none response (diapause prevention or induction) to the difference between the long and short daylengths (a 4 hr difference is sufficient to avert diapause but a 2 hr difference is not), and (c) a quantitative response to the absolute duration of day (or night) length (after the short day requirement is fulfilled the rate of diapause termination is related to daylength).Differences and similarities in phenological adaptations and in photoperiodic responses of C. downesi, C. carnea, and C. harrisii reflect the degree of phylogenetic relationship between these closely related species.     

Genetic Heterozygosity in Unreplicated Bacteriophage λ Recombinants

Bacteriophage crosses using density-labeled parents have been carried out under conditions restricting DNA synthesis. The parental material and genetic contributions to progeny manifesting recombination within a genetic interval sufficiently short to exhibit high negative interference have been examined. The unreplicated products of recombination isolated as phage particles appear to contain long continuous heteroduplex regions which are heterozygous for the closely linked markers. Recombination between closely linked markers seems to be the consequence of the removal of base-pair mismatches that are present within the heteroduplex regions. This localized reduction of heterozygosity within the heteroduplex regions that join the parental components of recombinant DNA molecules can account for high negative interference.     

Alternative phenotypes of male mating behaviour in the two-spotted spider mite

Severe intraspecific competition for mates selects for aggressive individuals but may also lead to the evolution of alternative phenotypes that do not act aggressively, yet manage to acquire matings. The two-spotted spider mite, Tetranychus urticae, shows male mate-guarding behaviour and male–male combat for available females. This may provide opportunity for weaker males to avoid fighting by adopting alternative mating behaviour such as sneaker or satellite tactics as observed in other animals. We investigated male precopulatory behaviour in the two-spotted spider mite by means of video-techniques and found three types of male mating behaviour: territorial, sneaker and opportunistic. Territorial and sneaker males associate with female teleiochrysales and spend much time guarding them. Territorial males are easily disturbed by rival males and engage themselves in fights with them. However, sneaker males are not at all disturbed by rival males, never engage in fights and, strikingly, never face attack by territorial males. Opportunistic males wander around in search of females that are in the teleiochrysalis stage but very close to or at emergence. To quickly classify any given mate-guarding male as territorial or sneaker we developed a method based on the instantaneous response of males to disturbance by a live male mounted on top of a brush. We tested this method against the response of the same males to natural disturbance by two or three other males. Because this method proved to be successful, we used it to collect territorial and sneaker males, and subjected them to morphological analysis to assess whether the various behavioural phenotypes are associated with different morphological characters. However, we found no statistical differences between territorial and sneaker males, concerning the length of the first legs, the stylets, the pedipalps or the body.     

Transmissible spongiform encephalopathy strain-associated diversity of N-terminal proteinase K cleavage sites of PrP(Sc) from scrapie-infected and bovine spongiform encephalopathy-infected mice.

Assessment of the different conformational states of the abnormal prion protein (PrP(Sc)) in the CNS provides an established basis for distinguishing transmissible spongiform encephalopathy (TSE) strains. PrP(Sc) conformers are variably resistant to N-terminal proteinase K (PK) digestion, and analysis of the consensus products (PrP(res)) by immunoassay enables effective, but relatively low-resolution differentiation. Determination of the precise N-terminal amino acid profile (N-TAAP) of PrP(res) presents a potential high-resolution means of TSE-strain typing, and thus of differential disease diagnosis. This approach was evaluated using individual mice affected by model scrapie (22A, ME7, 87V and 79A) and bovine spongiform encephalopathy (BSE) (301V) strains. Nano liquid chromatography-mass spectrometry (LC-MS) was used to determine PrP(res) N-terminal tryptic digestion products. Four major N-terminal tryptic peptides were generated from all mouse TSE strains investigated, corresponding with predominant N-termination of PrP(res) at G(81), G(85), G(89) and G(91). Both the mass spectrometric abundance of the individual peptides and the ratios of pairs of these peptides were evaluated as markers of conformation in relation to their potential for strain discrimination. The yield of peptides was significantly greater for BSE than scrapie strains and the relative quantities of particular peptide pairs differed between strains. Thus, whereas peptide G(91)-K(105) was a dominant peptide from 301V, this was not the case for other strains and, significantly, the ratio of peptides G(91)-K(105):G(89)-K(105) was substantially higher for BSE-infected compared with scrapie-infected mice. These data support the potential of the N-TAAP approach for high-resolution TSE strain typing and differential diagnosis.