The structure of Bacillus subtilis RecU Holliday junction resolvase and its role in substrate selection and sequence-specific cleavage

We have determined the structure of the enzyme RecU from Bacillus subtilis, that is the general Holliday junction resolving enzyme in Gram-positive bacteria. The enzyme fold reveals a striking similarity to a class of resolvase enzymes found in archaeal sources and members of the type II restriction endonuclease family to which they are related. The structure confirms the presence of active sites formed around clusters of acidic residues that we have also shown to bind divalent cations. Mutagenesis data presented here support the key role of certain residues. The RecU structure suggests a basis for Holliday junction selectivity and suggests how sequence-specific cleavage might be achieved. Models for a resolvase-DNA complex address how the enzyme might organize junctions into an approximately 4-fold symmetric form.     

Structure of TCTP reveals unexpected relationship with guanine nucleotide-free chaperones

The translationally controlled tumor-associated proteins (TCTPs) are a highly conserved and abundantly expressed family of eukaryotic proteins that are implicated in both cell growth and the human acute allergic response but whose intracellular biochemical function has remained elusive. We report here the solution structure of the TCTP from Schizosaccharomyces pombe, which, on the basis of sequence homology, defines the fold of the entire family. We show that TCTPs form a structural superfamily with the Mss4/Dss4 family of proteins, which bind to the GDP/GTP free form of Rab proteins (members of the Ras superfamily) and have been termed guanine nucleotide-free chaperones (GFCs). Mss4 also acts as a relatively inefficient guanine nucleotide exchange factor (GEF). We further show that the Rab protein binding site on Mss4 coincides with the region of highest sequence conservation in the TCTP family. This is the first link to any other family of proteins that has been established for the TCTP family and suggests the presence of a GFC/GEF at extremely high abundance in eukaryotic cells.     

Calcium influx is not required for thyrotropin-releasing hormone stimulation of prolactin release from GH3 cells

TRH stimulation of prolactin release from GH3 cells is dependent on Ca2+; however, whether TRH-induced influx of extracellular Ca2+ is required for stimulated secretion remains controversial. We studied prolactin release from cells incubated in medium containing 110 mM K+ and 2 mM EGTA which abolished the electrical and Ca2+ concentration gradients that usually promote Ca2+ influx. TRH caused prolactin release and 45Ca2+ efflux from cells incubated under these conditions. In static incubations, TRH stimulated prolactin secretion from 11.4 +/- 1.2 to 19 +/- 1.8 ng/ml in control incubations and from 3.2 +/- 0.6 to 6.2 +/- 0.8 ng/ml from cells incubated in medium with 120 mM K+ and 2 mM EGTA. We conclude that Ca2+ influx is not required for TRH stimulation of prolactin release from GH3 cells.     

Stimulation of anthocyanin synthesis in grape (<Emphasis Type="Italic">Vitis vinifera</Emphasis>) cell cultures by pulsed electric fields and ethephon

Anthocyanin from grape cell cultures can be used as a natural alternative to synthetic dyes; particularly due to their reported health-promoting properties. In this study, production of anthocyanin in cell suspension culture of Vitis vinifera was evaluated following treatment with either ethephon and/or pulsed electric fields (PEF). Overall, total production of anthocyanin increased in treated cells compared to untreated cells. Treatment of cell suspension with PEF at day 14 of culture resulted in 1.7-fold increase (1.42 mg/g DW) in anthocyanin content when compared to control cells; while, treatment with ethephon resulted in 2.3-fold increase (1.99 mg/g DW) in anthocyanin content. When cells were treated with both ethephon and PEF, 2.5-fold increase in anthocyanin content (2.2 mg/g DW) was observed. These findings demonstrate that PEF induces a defense response in plant cells, and it may also alter the dielectric properties of cells and/or cell membranes, and would serve as a viable elicitor of secondary metabolites in plant cell cultures.     

Effects of gastrin-releasing peptide 1-27 on taste responses in the rat

Gastrin-releasing-peptide(1-27) (GRP) has been implicated in the regulation of satiety and appetite in numerous paradigms. However, the specific site and mode of action of this gut-brain peptide has not been elucidated. The following experiment examined the effects of GRP on taste responses to sucrose in the behaving rat. A brief-exposure, multi-bottle gustometer was used to provide rats with momentary access to six different concentrations of sucrose in a single test session. This procedure has been used previously, resulting in monotonically increased licking behavior as concentrations of sucrose increase. Differing injection procedures were employed such that rats were tested immediately after i.p. injection or 5 min after i.p. injection of 5 nmol/kg body wt of GRP. Results indicate that GRP does reduce the oral reinforcing properties of sucrose, but the effect is transient, diminishing significantly within 5 min after injection.     

Characterization of the calcium response to thyrotropin-releasing hormone (TRH) in cells transfected with TRH receptor complementary DNA: importance of voltage-sensitive calcium channels.

TRH stimulates a biphasic increase in intracellular free calcium ion, [Ca2+]i. Cells stably transfected with TRH receptor cDNA were used to compare the response in lines with and without L type voltage-gated calcium channels. Rat pituitary GH-Y cells that do not normally express TRH receptors, rat glial C6 cells, and human epithelial Hela cells were transfected with mouse TRH receptor cDNA. All lines bound similar amounts of [3H][N3-Me-His2]TRH with identical affinities (dissociation constant = 1.5 nM). Both pituitary lines expressed L type voltage-gated calcium channels; depolarization with high K+ increased 45Ca2+ uptake 20- to 25-fold and [Ca2+]i 12- to 14-fold. C6 and Hela cells, in contrast, appeared to have no L channel activity. GH4C1 cells responded to TRH with a calcium spike (6-fold) followed by a sustained second phase. When TRH was added after 100 nM nimodipine, an L channel blocker, the initial calcium burst was unaffected but the second phase was abolished. GH-Y cells transfected with TRH receptor cDNA responded to TRH with a 6-fold [Ca2+]i spike followed by a plateau phase (>8 min) in which [Ca2+]i remained elevated or increased. Nimodipine did not alter the peak TRH response or resting [Ca2+]i but reduced the sustained phase, which was eliminated by chelation of extracellular Ca2+. In the transfected glial C6 and Hela cells without calcium channels, TRH evoked transient, monophasic 7- to 9-fold increases in [Ca2+]i, and [Ca2+]i returned to resting levels within 3 min. Thapsigargin stimulated a gradual, large increase in [Ca2+]i in transfected C6 cells, and subsequent addition of TRH caused no further rise. Removal of extracellular Ca2+ from transfected C6 cells shortened the [Ca2+]i responses to TRH, to endothelin 1, and to thapsigargin. The TRH responses were pertussis toxin-insensitive. In summary, TRH can generate a calcium spike in pituitary, C6, and Hela cells transfected with TRH receptor cDNA, but the plateau phase of the [Ca2+]i response is not observed when the receptor is expressed in a cell line without L channel activity.     

Conditioned suppression as a method of detecting taste thresholds in the rat

Taste thresholds of seven male Sprague—Dawley rats (meanage 10 weeks, mean weight 250 g) were determined for four basictaste qualities: sweet, sour, salty and bitter. The method ofconditioned suppression was employed. An apparatus capable ofpresenting any one of eight separate drinking tubes during atesting session was designed. Animals were reduced to 80–85%ad lib. body weight. They were then trained to lick a sippertube through a slot in the back of an experimental chamber forpellet reinforcements. Animals progressed through a series ofreinforcement schedules starting with a fixed ratio (FR) scheduleof five licks for each reinforcement. They advanced to a variableratio (VR) schedule of reinforcement and finally a variableinterval (VI) schedule with a mean of 17.5 s was used. Whileon the VI schedule animals were trained to suppress lickingwhen any tastant other than water was presented. The first lickon any tastant was followed 10 s later by a mild electric shockif a rat made more than 20 licks on the tube in the 10-s period.Less than 20 licks on a tastant tube resulted in no shock anda 5-s time out before proceeding to the next tube. Thresholdwas determined using a suppression ratio formula. Thresholdwas defined as the 0.33 suppression ratio. Results from thisexperiment reveal mean thresholds for the seven animals as:sucrose = 2.3 mM, NaCl = 0.63 mM, quinine HCl = 0.005 mM andcitric acid = 0.085 mM.     

Human calcitonin receptor is directly targeted to and retained in the basolateral surface of MDCK cells

The human calcitonin receptor (hCTR) is expressed in polarizedcells of the kidney, bone, and nervous system. In the kidney, hCTRs arefound in cells of the distal nephron to which blood-borne calcitoninhas access only at the basolateral surface. We expressed hCTR subtypes1 and 2 in Madin-Darby canine kidney (MDCK) cells to establish a cellmodel useful for delineating the molecular mechanisms underlying hCTRpolarity. Selective cell surface incubation demonstrated functionalpolarity of hCTRs by equilibrium binding or cross-linking ofradioiodinated salmon calcitonin(¹²⁵I-sCT) and cAMP accumulationstimulated by sCT. We estimated that at the steady state there are40-fold more hCTRs on the basolateral than on the apical side.Domain-selective cell surface biotinylation followed by immunoblottingof streptavidin-agarose-fractionated biotinylated glycoproteinsindependently confirmed the polarized distribution of FLAGepitope-tagged hCTR-2 in the basolateral domain. Confocal microscopy ofimmunostained receptors revealed that hCTRs are concentrated on alateral subdomain of the basolateral membrane. Cell surface arrivalassay of newly formed receptors demonstrated that direct delivery tothe basolateral domain is the mechanism by which hCTRs becomepolarized. Measurement of receptor turnover on the basolateral surfaceshowed that retention contributes to hCTR distribution at the steadystate.