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Type 1 diabetes mellitus (T1D) is a chronic disease with potentially severe complications, and β-cell deficiency underlies this disease. Despite active research, no therapy to date has been able to induce β-cell regeneration in humans. Here, we discover the β-cell regenerative effects of glucagon receptor antibody (anti-GcgR). Treatment with anti-GcgR in mouse models of β-cell deficiency leads to reversal of hyperglycemia, increase in plasma insulin levels, and restoration of β-cell mass. We demonstrate that both β-cell proliferation and α- to β-cell transdifferentiation contribute to anti-GcgR-induced β-cell regeneration. Interestingly, anti-GcgR-induced α-cell hyperplasia can be uncoupled from β-cell regeneration after antibody clearance from the body. Importantly, we are able to show that anti-GcgR-induced β-cell regeneration is also observed in non-human primates. Furthermore, anti-GcgR and anti-CD3 combination therapy reverses diabetes and increases β-cell mass in a mouse model of autoimmune diabetes.

Under homeostatic conditions, animals use well-defined hypothalamic neural circuits to help maintain stable body weight, by integrating metabolic and hormonal signals from the periphery to balance food consumption and energy expenditure. In stressed or disease conditions, however, animals use alternative neuronal pathways to adapt to the metabolic challenges of altered energy demand. Recent studies have identified brain areas outside the hypothalamus that are activated under these 'non-homeostatic' conditions, but the molecular nature of the peripheral signals and brain-localized receptors that activate these circuits remains elusive. Here we identify glial cell-derived neurotrophic factor (GDNF) receptor alpha-like (GFRAL) as a brainstem-restricted receptor for growth and differentiation factor 15 (GDF15). GDF15 regulates food intake, energy expenditure and body weight in response to metabolic and toxin-induced stresses; we show that Gfral knockout mice are hyperphagic under stressed conditions and are resistant to chemotherapy-induced anorexia and body weight loss. GDF15 activates GFRAL-expressing neurons localized exclusively in the area postrema and nucleus tractus solitarius of the mouse brainstem. It then triggers the activation of neurons localized within the parabrachial nucleus and central amygdala, which constitute part of the 'emergency circuit' that shapes feeding responses to stressful conditions. GDF15 levels increase in response to tissue stress and injury, and elevated levels are associated with body weight loss in numerous chronic human diseases. By isolating GFRAL as the receptor for GDF15-induced anorexia and weight loss, we identify a mechanistic basis for the non-homeostatic regulation of neural circuitry by a peripheral signal associated with tissue damage and stress. These findings provide opportunities to develop therapeutic agents for the treatment of disorders with altered energy demand.

JAK3 kinase plays a critical role in several cytokine signaling pathways involved in immune cell development and function. The studies presented in this report were undertaken to elucidate the kinetic mechanism of the JAK3 kinase domain, investigate the role of activation loop phosphorylation in regulating its catalytic activity, and examine its inhibition by the anti-rheumatoid arthritis drug, tofacitinib. Phosphorylation of two Tyr residues in JAK3's activation loop has been reported to impact its kinase activity. The recombinant JAK3 kinase domain used in our studies was heterogeneous in its activation loop phosphorylation, with the non-phosphorylated protein being the dominant species. Kinetic analysis revealed similar kinetic parameters for the heterogeneously phosphorylated JAK3, JAK3 mono-phosphorylated on Tyr 980, and the activation loop mutant YY980/981FF. Bisubstrate and product inhibition kinetic results were consistent with both sequential random and sequential ordered kinetic mechanisms. Solvent viscosometric experiments showed perturbation of k cat , suggesting the phosphoryl transfer step is not likely rate limiting. This was supported by results from quench-flow experiments, where a rapid burst of product formation was observed. Kinetic analysis of JAK3 inhibition by tofacitinib indicated inhibition is time dependent, characterized by on- and off-rate constants of 1.4 ± 0.1 μM −1 s −1 and 0.0016 ± 0.0005 s −1 , respectively.

The downstream promoter element (DPE) functions cooperatively with the initiator (Inr) for the binding of TFIID in the transcription of core promoters in the absence of a TATA box. We examined the properties of sequences that can function as a DPE as well as the range of promoters that use the DPE as a core promoter element. By using an in vitro transcription assay, we identified 17 new DPE-dependent promoters and found that all possessed identical spacing between the Inr and DPE. Moreover, mutational analysis indicated that the insertion or deletion of a single nucleotide between the Inr and DPE causes a reduction in transcriptional activity and TFIID binding. To explore the range of sequences that can function as a DPE, we constructed and analyzed randomized promoter libraries. These experiments yielded the DPE functional range set, which represents sequences that contribute to or are compatible with DPE function. We then analyzed the DPE functional range set in conjunction with a Drosophila core promoter database that we compiled from 205 promoters with accurately mapped start sites. Somewhat surprisingly, the DPE sequence motif is as common as the TATA box in Drosophila promoters. There is, in addition, a striking adherence of Inr sequences to the Inr consensus in DPE-containing promoters relative to DPE-less promoters. Furthermore, statistical and biochemical analyses indicated that a G nucleotide between the Inr and DPE contributes to transcription from DPE-containing promoters. Thus, these data reveal that the DPE exhibits a strict spacing requirement yet some sequence flexibility and appears to be as widely used as the TATA box in Drosophila.

The assembly of core histones and DNA into periodic nucleosome arrays is mediated by ACF, an ISWI-containing factor, and NAP-1, a core histone chaperone, in an ATP-dependent process. We describe the isolation of Drosophila acf1 cDNA, which encodes the p170 and p185 forms of the Acf1 protein in ACF. Acf1 is a novel protein that contains two PHD fingers, one bromodomain, and two new conserved regions. Human WSTF, which is encoded by one of multiple genes that is deleted in Williams syndrome individuals, is the only currently known mammalian protein with each of the conserved motifs in Acf1. Purification of the native form of Acf1 led to the isolation of ACF comprising Acf1 (both p170 and p185 forms) and ISWI. Native Acf1 did not copurify with components of NURF or CHRAC, which are other ISWI-containing complexes in Drosophila. Purified recombinant ACF, consisting of Acf1 (either p185 alone or both p170 and p185) and ISWI, catalyzes the deposition of histones into extended periodic nucleosome arrays. Notably, the Acf1 and ISWI subunits function synergistically in the assembly of chromatin. ISWI alone exhibits a weak activity that is approximately 3% that of ACF. These results indicate that both Acf1 and ISWI participate in the chromatin assembly process and suggest further that the Acf1 subunit confers additional functionality to the general 'motor' activity of ISWI.

The Janus kinases (JAKs) are involved in multiple signaling networks relevant to inflammatory diseases, and inhibition of one or more members of this class may modulate disease activity or progression. We optimized a new inhibitor scaffold, 3-amido-5-cyclopropylpyrrolopyrazines, to a potent example with reasonable kinome selectivity, including selectivity for JAK3 versus JAK1, and good biopharmaceutical properties. Evaluation of this analogue in cellular and in vivo models confirmed functional selectivity for modulation of a JAK3/JAK1-dependent IL-2 stimulated pathway over a JAK1/JAK2/Tyk2-dependent IL-6 stimulated pathway.

Dengue virus (DENV), an emerging pathogen from the Flaviviridae family with neither vaccine nor antiviral treatment available, causes a serious worldwide public health threat. In theory, there are several ways by which small molecules could inhibit the replication cycle of DENV. Here, we show that the nucleoside analogue beta- d -2′-ethynyl-7-deaza-adenosine inhibits representative strains of all four serotypes of DENV with an EC 50 around or below 1 μM. Using membrane-associated native replicase complex as well as recombinant RNA polymerase from each DENV serotype in enzymatic assays, we provide evidence that beta- d -2′-ethynyl-7-deaza-adenosine triphosphate (2′E-7D-ATP) targets viral replication at the polymerase active site by competing with the natural nucleotide substrate with an apparent K i of 0.060 ± 0.016 μM. In single-nucleotide incorporation experiments, the catalytic efficiency of 2′E-7D-ATP is 10-fold lower than for natural ATP, and the incorporated nucleotide analogue causes immediate chain termination. A combination of bioinformatics and site-directed mutagenesis demonstrates that 2′E-7D-ATP is equipotent across all serotypes because the nucleotide binding site residues are conserved in dengue virus. Overall, beta- d -2′-ethynyl-7-deaza-adenosine provides a promising scaffold for the development of inhibitors of dengue virus polymerase.

The relationship between nitric oxide synthase II (NOS II) inducibility and the metastatic ability of UV-2237 murine fibrosarcoma cells was determined. Highly metastatic cells survived to produce numerous lung metastases after i.v. injection in syngeneic C3H/HeN mice, whereas poorly metastatic cells did not. Highly metastatic clones exhibited higher levels of NOS II than did poorly metastatic clones in response to interleukin 1alpha and IFN-gamma stimulation. Furthermore, both poorly and highly metastatic clones contained an identical p53 mutation. Overexpression of NOS II in a highly metastatic clone by transfection with NOS II gene retarded tumor growth and completely suppressed metastasis. Our data indicate that a low to moderate level of NOS II expression directly correlates with metastatic ability of UV-2237 fibrosarcoma cells carrying mutant p53 and that a high level of nitric oxide production suppresses tumor growth and metastasis.