KCl transport across and insect epithelium: I. Tracer fluxes and the effects of ion substitutions

Summary Models for active Cl transport across epithelia are often assumed to be universal although they are based on detailed studies of a relatively small number of epithelia from vertebrate animals. Epithelial Cl transport is also important in many invertebrates, but little is known regarding its cellular mechanisms. We used short-circuit current, tracer fluxes and ion substitutions to investigate the basic properties of Cl absorption by locust hindgut, an epithelium which is ideally suited for transport studies. Serosal addition of 1mm adenosine 35-cyclic monophosphate (cAMP), a known stimulant of Cl transport in this tissue, increased short-circuit current (I _sc) and net reabsorptive³⁶Cl flux (J _net ^Cl ) by 1000%. Cl absorption did not exhibit an exchange diffusion component and was highly selective over all anions tested except Br. Several predictions of Na- and HCO₃-coupled models for Cl transport were tested: Cl-dependentI _sc was not affected by sodium removal (<0.05mm) during the first 75 min. Also, a large stimulation ofJ _net ^Cl was elicited by cAMP when recta were bathed for 6 hr in nominally Na-free saline (<0.001 to 0.2mm) and there was no correlation between Cl transport rate and the presence of micromolar quantities of Na contamination. Increased unidirectional influx of³⁶Cl into rectal tissue during cAMP-stimulation was not accompanied by a comparable uptake of²²Na.J _net ^Cl was independent of exogenous CO₂ and HCO₃, but was strongly dependent on the presence of K. These results suggest that the major fraction of Cl transport across this insect epithelium occurs by an unusual K-dependent mechanism that does not directly require Na or HCO₃.     

Modulation of Ionotropic GABA Receptors by 6-Methoxyflavanone and 6-Methoxyflavone

We evaluated the effects of 6-methoxyflavanone and 6-methoxyflavone on wild-type α1/α2β2γ2L GABA_A and ρ1 GABA_C receptors and on mutant ρ1I307S, ρ1W328 M, ρ1I307S/W328 M GABA_C receptors expressed in Xenopus oocytes using two-electrode voltage clamp and radioligand binding. 6-Methoxyflavanone and 6-methoxyflavone act as a flumazenil-insensitive positive allosteric modulator of GABA responses at human recombinant α1β2γ2L and α2β2γ2L GABA_A receptors. However, unlike 6-methoxyflavone, 6-methoxyflavanone was relatively inactive at α1β2 GABA_A receptors. 6-Methoxyflavanone inhibited [³H]-flunitrazepam binding to rat brain membranes. Both flavonoids were found to be inactive as modulators at ρ1, ρ1I307S and ρ1W328 M GABA receptors but acted as positive allosteric modulators of GABA at the benzodiazepine sensitive ρ1I307S/W328 M GABA receptors. This double mutant retains ρ1 properties of being insensitive to bicuculline and antagonised by TPMPA and THIP. Additionally, 6-methoxyflavanone was also a partial agonist at ρ1W328 M GABA receptors. The relative inactivity of 6-methoxyflavanone at α1β2 GABA_A receptors and it’s partial agonist action at ρ1W328 M GABA receptors suggest that it exhibits a unique profile not matched by other flavonoids.     

A whole-genome assembly of the domestic cow, Bos taurus

Background

The genome of the domestic cow, Bos taurus, was sequenced using a mixture of hierarchical and whole-genome shotgun sequencing methods.

Results

We have assembled the 35 million sequence reads and applied a variety of assembly improvement techniques, creating an assembly of 2.86 billion base pairs that has multiple improvements over previous assemblies: it is more complete, covering more of the genome; thousands of gaps have been closed; many erroneous inversions, deletions, and translocations have been corrected; and thousands of single-nucleotide errors have been corrected. Our evaluation using independent metrics demonstrates that the resulting assembly is substantially more accurate and complete than alternative versions.

Conclusions

By using independent mapping data and conserved synteny between the cow and human genomes, we were able to construct an assembly with excellent large-scale contiguity in which a large majority (approximately 91%) of the genome has been placed onto the 30 B. taurus chromosomes. We constructed a new cow-human synteny map that expands upon previous maps. We also identified for the first time a portion of the B. taurus Y chromosome.     

Basolateral K channels in an insect epithelium. Channel density, conductance, and block by barium

K channels in the basolateral membrane of insect hindgut were studied using current fluctuation analysis and microelectrodes. Locust recta were mounted in Ussing-type chambers containing Cl-free saline and cyclic AMP (cAMP). A transepithelial K current was induced by raising serosal [K] under short-circuit conditions. Adding Ba to the mucosal (luminal) side under these conditions had no effect; however, serosal Ba reversibly inhibited the short-circuit current (Isc), increased transepithelial resistance (Rt), and added a Lorentzian component to power density spectra of the Isc. A nonlinear relationship between corner frequency and serosal [Ba] was observed, which suggests that the rate constant for Ba association with basolateral channels increased as [Ba] was elevated. Microelectrode experiments revealed that the basolateral membrane hyperpolarized when Ba was added: this change in membrane potential could explain the nonlinearity of the 2 pi fc vs. [Ba] relationship if external Ba sensed about three-quarters of the basolateral membrane field. Conventional microelectrodes were used to determine the correspondence between transepithelially measured current noise and basolateral membrane conductance fluctuations, and ion-sensitive microelectrodes were used to measure intracellular K activity (acK). From the relationship between the net electrochemical potential for K across the basolateral membrane and the single channel current calculated from noise analysis, we estimate that the conductance of basolateral K channels is approximately 60 pS, and that there are approximately 180 million channels per square centimeter of tissue area.     

The APC/C subunit Cdc16/Cut9 is a contiguous tetratricopeptide repeat superhelix with a homo-dimer interface similar to Cdc27

The anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase responsible for controlling cell cycle transitions, is a multisubunit complex assembled from 13 different proteins. Numerous APC/C subunits incorporate multiple copies of the tetratricopeptide repeat (TPR). Here, we report the crystal structure of Schizosaccharomyces pombe Cut9 (Cdc16/Apc6) in complex with Hcn1 (Cdc26), showing that Cdc16/Cut9 is a contiguous TPR superhelix of 14 TPR units. A C-terminal block of TPR motifs interacts with Hcn1, whereas an N-terminal TPR block mediates Cdc16/Cut9 self-association through a homotypic interface. This dimer interface is structurally related to the N-terminal dimerization domain of Cdc27, demonstrating that both Cdc16/Cut9 and Cdc27 form homo-dimers through a conserved mechanism. The acetylated N-terminal Met residue of Hcn1 is enclosed within a chamber created from the Cut9 TPR superhelix. Thus, in complex with Cdc16/Cut9, the N-acetyl-Met residue of Hcn1, a putative degron for the Doa10 E3 ubiquitin ligase, is inaccessible for Doa10 recognition, protecting Hcn1/Cdc26 from ubiquitin-dependent degradation. This finding may provide a structural explanation for a mechanism to control the stoichiometry of proteins participating in multisubunit complexes.     

Repeatability of the duration of oestrus and breed differences in the relationship between druation of oestrus and ovulation rate of sheep.

The duration of oestrus and the time interval from removal of progestagen-impregnated pessaries to the onset and end of oestrus were examined in Texel, Finnish Landrace, Galway and Fingalway (Finnish Landrace X Galway) ewes. The differences among the breeds in the relationship between these variables and ovulation rate at the controlled oestrus were also investigated. Breed differences were significant for all traits except the interval from pessary withdrawal to the onset of oestrus. The relationship between ovulation rate and both the interval from pessary withdrawal to the onset of oestrus and the duration of oestrus differed significantly among the breeds. The repeatability of the duration of oestrus was significant for Texel and Rambouillet ewes (mean = 0.5) and for pooled data from ewe lambs of various breeds. It was concluded that, in view of the breed differences in the relationship between ovulation rate and duration of oestrus and other traits, generalizations should not be made from among-breed to within-breed relationships. The high repeatability for the duration of oestrus may mean substantial heritabilities for the physiological determinants of oestrus duration.     

Duration of oestrus, ovulation rate, time of ovulation and plasma LH, total oestrogen and progesterone in Galway adult ewes and ewe lambs

The mean duration of oestrus, ovulation rate, duration of the preovulatory LH discharge, time interval between sponge removal and beginning of the LH discharge, total LH discharged, maximum LH value observed and the concentration of progesterone in the peripheral plasma during the luteal phase of the oestrous cycle was similar in Galway adult ewes and 8-month-old ewe lambs after treatment with intravaginal sponges containing 30 mg cronolone for 12 days and injection of 500 i.u. PMSG. The interval between sponge removal and the onset of oestrus was shorter for adults than for ewe lambs; the interval between the onset of oestrus and the beginning of the LH discharge was longer in adults. During the period 12-36 h after sponge removal the mean plasma total oestrogen concentration was significantly higher in lambs than in adults. In a separate study of the time of ovulation in Galway ewe lambs given the same progestagen-PMSG treatment, ovulation did not occur in any lamb before 17 h after the onset of oestrus and the majority ovulated close to the end of oestrus.