Intrinsic Gating Properties of a Cloned G Protein-activated Inward Rectifier K^+ Channel

The voltage-, time-, and K^+-dependent properties of a G protein-activated inwardly rectifying K^+ channel (GIRK1/KGA/Kir3.1) cloned from rat atrium were studied in Xenopus oocytes under two-electrode voltage clamp. During maintained G protein activation and in the presence of high external K^+ (V_K = 0 mV), voltage jumps from V_K to negative membrane potentials activated inward GIRK1 K^+ currents with three distinct time-resolved current components. GIRK1 current activation consisted of an instantaneous component that was followed by two components with time constants T_f~50 ms and T_s~400 ms. These activation time constants were weakly voltage dependent, increasing approximately twofold with maximal hyperpolarization from V_K. Voltage-dependent GIRK1 availability, revealed by tail currents at -80 mV after long prepulses, was greatest at potentials negative to V_K and declined to a plateau of approximately half the maximal level at positive voltages. Voltage-dependent GIRK1 availability shifted with V_K and was half maximal at V_K -20 mV; the equivalent gating charge was ~1.6 e^-. The voltage-dependent gating parameters of GIRK1 did not significantly differ for G protein activation by three heterologously expressed signaling pathways: m2 muscarinic receptors, serotonin 1A receptors, or G protein β1y2 subunits. Voltage dependence was also unaffected by agonist concentration. These results indicate that the voltage-dependent gating properties of GIRK1 are not due to extrinsic factors such as agonist-receptor interactions and G protein-channel coupling, but instead are analogous to the intrinsic gating behaviors of other inwardly rectifying K^+ channels

Expression of an atrial G-protein-activated potassium channel in Xenopus oocytes

Injection of rat atrial RNA into Xenopus oocytes resulted in the expression of a guanine nucleotide binding (G) protein-activated K+ channel. Current through the channel could be activated by acetylcholine or, if RNA encoding a neuronal 5HT1A receptor was coinjected with atrial RNA, by serotonin (5HT). A 5HT-evoked current (I5HT) was observed in oocytes injected with ventricle RNA fractions (of 2.5-5.5 kb) and 5HT1A receptor RNA. I5HT displayed strong inward rectification with very little conductance above the K+ equilibrium potential, was highly selective for K+ over Na+, and was blocked by 5-300 µM Ba2+. I5HT was suppressed by intracellular injection of the nonhydrolyzable analog of GDP, guanosine 5'-[ß-thio]diphosphate, but not by treatment with pertussis toxin (PTX), suggesting coupling of the receptor to the G-protein-activated K+ channel via a PTX-insensitive G protein, possibly endogenously present in the oocyte. Coexpression of the subunit of a PTX-sensitive G protein, Gi2, rendered I5HT sensitive to PTX inhibition. Native oocytes displayed a constitutively active inwardly rectifying K+ current with a lower sensitivity to Ba2+ block; expression of a similar current was also directed by atrial or ventricle RNA of 1.5-3 kb. Xenopus oocytes may be employed for cloning of the G-protein-activated K+ channel cDNA and for studying the coupling between this channel and G proteins

A G protein-gated K channel is activated via beta 2-adrenergic receptors and G beta gamma subunits in Xenopus oocytes

In many tissues, inwardly rectifying K channels are coupled to seven- helix receptors via the Gi/Go family of heterotrimeric G proteins. This activation proceeds at least partially via G beta gamma subunits. These experiments test the hypothesis that G beta gamma subunits activate the channel even if released from other classes of heterotrimeric G proteins. The G protein-gated K channel from rat atrium, KGA/GIRK1, was expressed in Xenopus oocytes with various receptors and G proteins. The beta 2-adrenergic receptor (beta 2AR), a Gs-linked receptor, activated large KGA currents when the alpha subunit, G alpha s, was also overexpressed. Although G alpha s augmented the coupling between beta 2AR and KGA, G alpha s also inhibited the basal, agonist-independent activity of KGA. KGA currents stimulated via beta 2AR activated, deactivated, and desensitized more slowly than currents stimulated via Gi/Go-linked receptors. There was partial occlusion between currents stimulated via beta 2AR and the m2 muscarinic receptor (a Gi/Go-linked receptor), indicating some convergence in the mechanism of activation by these two receptors. Although stimulation of beta 2AR also activates adenylyl cyclase and protein kinase A, activation of KGA via beta 2AR is not mediated by this second messenger pathway, because direct elevation of intracellular cAMP levels had no effect on KGA currents. Experiments with other coexpressed G protein alpha and beta gamma subunits showed that (a) a constitutively active G alpha s mutant did not suppress basal KGA currents and was only partially as effective as wild type G alpha s in coupling beta 2AR to KGA, and (b) beta gamma subunits increased basal KGA currents. These results reinforce present concepts that beta gamma subunits activate KGA, and also suggest that beta gamma subunits may provide a link between KGA and receptors not previously known to couple to inward rectifiers

Poems

Poems include: The White Birch, by Lois Davidson and Death, by Nancy Clar

Baboon endogenous virus genome. I. Restriction enzyme map of the unintegrated DNA genome of a primate retrovirus

A detailed restriction map was deduced for the genome of an endogenous retrovirus of a higher primate, that of baboon. The cleavage sites for 12 restriction enzymes were mapped. The unintegrated linear viral DNA intermediate that is produced by infection of permissive cells with baboon endogenous virus was isolated. Hybridization with a strong-stop complementary DNA probe demonstrated presence of a terminal repetition in the linear viral DNA. The positions of restriction sites for two particular enzymes, SmaI and XhoI, near each end were consistent with this result and indicated that the length of the repetition is 0.55 +/- 0.01 kilobase. The linear viral DNA had a unique restriction map indicating that it is not a set of random circular permutations of the RNA genome. From hybridization with a 3'-specific probe, the DNA restriction map was aligned relative to the 5'-to-3' orientation of the viral RNA. We observed a minor heterogeneity in a BamHI recognition site 1.95 kilobases from the right end of the linear map

Pilot trial of paclitaxel-trastuzumab adjuvant therapy for early stage breast cancer: a trial of the ECOG-ACRIN cancer research group (E2198)

BACKGROUND: Blockade of human epidermal growth factor receptor type 2 (HER2) has dramatically improved outcome for patients with HER2-positive breast cancer. Trastuzumab, an anti-HER2 monoclonal antibody, has previously demonstrated improvement in overall survival (OS) in patients with metastatic and early stage HER2-positive breast cancer. However, trastuzumab can cause congestive heart failure (CHF) with an increased frequency for patients who have also received an anthracycline. The current trial was designed to evaluate the impact of the duration of trastuzumab on CHF. METHODS: E2198 included 227 eligible women with histologically confirmed stage II or IIIA HER2-positive breast cancer. The patients were randomised to receive 12 weeks of paclitaxel and trastuzumab followed by four cycles of doxorubicin and cyclophosphamide (abbreviated Arm) or the aforementioned treatment with additional 1 year of trastuzumab (conventional Arm). The primary end point was to evaluate the safety of this variable duration of trastuzumab therapy, particularly cardiac toxicity defined as CHF or left ventricular ejection fraction decrease >10%. Secondary end points included disease-free survival (DFS) and OS. RESULTS: Compared with 12-week treatment with trastuzumab, 1 year of trastuzumab-based therapy did not increase the frequency or severity of cardiac toxicity: three patients on the abbreviated Arm and four on the conventional Arm experienced CHF. The 5-year DFS was 76% and 73% for the abbreviated and conventional Arms, respectively, with a hazard ratio (HR) of 1.3 (95% CI: 0.8-2.1; P=0.3). There was also no statistically significance difference in OS (HR, 1.4; P=0.3). CONCLUSIONS: Compared with 12 weeks of treatment, 1 year of treatment with trastuzumab did not significantly increase the risk of cardiac toxicity. Although not powered for efficacy comparisons, the longer duration of trastuzumab therapy did not demonstrate a signal for marked superiority

Relationship between visual field loss and maximal combined electroretinographic responses in retinitis pigmentosa : comparison among genetically different types

1984年から1996年までに千葉大学眼科を受診した定型網膜色素変性症228例について常優26例, 常劣64例, 孤発例138例に分け動的量的視野および網膜電図を検討した。Goldmann視野におけるV-4イソプターでは5から10cm^2までと150から250cm^2までの2群に別れ, 1-4イソプターでは5cm^2以下の群のみ認められた。加齢及び遺伝形式による差異は認められなかった。網膜電図ではa, b波ともに正常対照群より減少してはいるものの, 遺伝形式による差異は認められなかった。また, 網膜電図で振幅を認められる割合はV-4イソプターおよびI-4イソプターの面積と相関していることが示唆されたが, 遺伝形式による差異は認められなかった。網膜電図の振幅の比であるb/a比は正常対象群に対して疾患群は減少していたが, 常備は特に他に比べ有意に減少していた。定型網膜色素変性症の網膜電図や視野の検討は数多くなされてきたが, b/a比について統計的考察がなされてきたことはない。a波およびb波は組織学的に発生起源が異なっており, b波はa波のインパルスによって二次的に引き起こされることは以前より知られてきている。網膜電図において常備のb/a比が有意な低下を示すことは, 網膜障害の機序が他と異なる可能性が示唆された。Analyses were performed on 228 Japanese patients with retinitis pigmentosa (RP) who were classified with autosomal dominant (ADRP, n=26), autosomal recessive (ARRP, n=64), and simplex (simplex RP, rc=138) inheritance. Visual fields were tested with Goldmann perimetry. Maximal combined responses of electroretinogram (ERG) with 20-Joule white flash stimulation were recorded after dark adaptation for 20 minutes. The visual field with the V-4 isopter demonstrated two unique groups, represented by dense areas between 5 and 10cm^2 and between 150 and 250cm^2, while only one unique group was observed within the 5cm^2 area with the 1-4 isopter. No age or inheritance type of effect was seen. A-and b-wave amplitudes were equally low in the 3 groups, as compared with normal subjects. The b/a ratio was significantly smaller in the ADRP group, compared with the others. The rate of detectable ERG responses decreased as the visual field became smaller. There was no inheritance effect. A lower b/a ratio in ADRP patients suggested that retinal functional abnormalities differed from ARRP and simplex RP patients

Proline-rich tyrosine kinase 2 mediates gonadotropin-releasing hormone signaling to a specific extracellularly regulated kinase-sensitive transcriptional locus in the luteinizing hormone beta-subunit gene

G protein-coupled receptor regulation of gene transcription primarily occurs through the phosphorylation of transcription factors by MAPKs. This requires transduction of an activating signal via scaffold proteins that can ultimately determine the outcome by binding signaling kinases and adapter proteins with effects on the target transcription factor and locus of activation. By investigating these mechanisms, we have elucidated how pituitary gonadotrope cells decode an input GnRH signal into coherent transcriptional output from the LH β-subunit gene promoter. We show that GnRH activates c-Src and multiple members of the MAPK family, c-Jun NH(2)-terminal kinase 1/2, p38MAPK, and ERK1/2. Using dominant-negative point mutations and chemical inhibitors, we identified that calcium-dependent proline-rich tyrosine kinase 2 specifically acts as a scaffold for a focal adhesion/cytoskeleton-dependent complex comprised of c-Src, Grb2, and mSos that translocates an ERK-activating signal to the nucleus. The locus of action of ERK was specifically mapped to early growth response-1 (Egr-1) DNA binding sites within the LH β-subunit gene proximal promoter, which was also activated by p38MAPK, but not c-Jun NH(2)-terminal kinase 1/2. Egr-1 was confirmed as the transcription factor target of ERK and p38MAPK by blockade of protein expression, transcriptional activity, and DNA binding. We have identified a novel GnRH-activated proline-rich tyrosine kinase 2-dependent ERK-mediated signal transduction pathway that specifically regulates Egr-1 activation of the LH β-subunit proximal gene promoter, and thus provide insight into the molecular mechanisms required for differential regulation of gonadotropin gene expression