Activation of nuclear factor kappa B in mammary epithelium promotes milk loss during mammary development and infection

We investigated whether nuclear factor kappa B (NF‐κB), which exhibits a regulated pattern of activity during murine mammary gland development, plays an important role during lactation and involution, when milk production ceases and the gland undergoes apoptosis and re‐modeling. We generated a doxycycline inducible transgenic mouse model to activate NF‐κB specifically in the mammary epithelium through expression of a constitutively active form of IKK2, the upstream kinase in the classical NF‐κB signaling cascade. We found that activation of NF‐κB during involution resulted in a more rapid reduction in milk levels and increased cleavage of caspase‐3, an indicator of apoptosis. We also found that activation of NF‐κB during lactation with no additional involution signals had a similar effect. The observation that NF‐κB is a key regulator of milk production led us to investigate the role of NF‐κB during mastitis, an infection of the mammary gland in which milk loss is observed. Mammary gland injection of E. coli LPS resulted in activation of NF‐κB and milk loss during lactation. This milk loss was decreased by selective inhibition of NF‐κB in mammary epithelium. Together, our data reveal that activation of NF‐κB leads to milk clearance in the lactating mammary gland. Therefore, targeting of NF‐κB signaling may prove therapeutic during mastitis in humans and could be beneficial for the dairy industry, where such infections have a major economic impact. J. Cell. Physiol. 222:73–81, 2010. © 2009 Wiley‐Liss, Inc.     

Interleukin-32β Propagates Vascular Inflammation and Exacerbates Sepsis in a Mouse Model

Background

Inflammation is associated with most diseases, which makes understanding the mechanisms of inflammation vitally important.

Methodology/Principal Findings

Here, we demonstrate a critical function of interleukin-32β (IL-32β) in vascular inflammation. IL-32β is present in tissues from humans, but is absent in rodents. We found that the gene is highly expressed in endothelial cells. Three isoforms of IL-32, named IL-32α, β, and ε, were cloned from human endothelial cells, with IL-32β being the major isoform. Pro-inflammatory cytokines (TNFα and IL-1β) induced IL-32β expression through NF-κB. Conversely, IL-32β propagated vascular inflammation via induction of vascular cell adhesion molecules and inflammatory cytokines. Accordingly, IL-32β increased adhesion of inflammatory cells to activated endothelial cells, a paramount process in inflammation. These results illustrate a positive feedback regulation that intensifies and prolongs inflammation. Importantly, endothelial/hematopoietic expression of IL-32β in transgenic mice elevated inflammation and worsened sepsis. This was demonstrated by significant elevation of leukocyte infiltration and serum levels of TNFα and IL-1β, increased vascular permeability and lung damage, and accelerated animal death. Together, our results reveal an important function of IL-32 in vascular inflammation and sepsis development.

Conclusions/Significance

Our results reveal an important function of IL-32 in vascular inflammation and sepsis development.     

Classification and regression tree and spatial analyses reveal geographic heterogeneity in genome wide linkage study of Indian visceral leishmaniasis

Background

Genome wide linkage studies (GWLS) have provided evidence for loci controlling visceral leishmaniasis on Chromosomes 1p22, 6q27, 22q12 in Sudan and 6q27, 9p21, 17q11-q21 in Brazil. Genome wide studies from the major focus of disease in India have not previously been reported.

Methods and Findings

We undertook a GWLS in India in which a primary ∼10 cM (515 microsatellites) scan was carried out in 58 multicase pedigrees (74 nuclear families; 176 affected, 353 total individuals) and replication sought in 79 pedigrees (102 nuclear families; 218 affected, 473 total individuals). The primary scan provided evidence (≥2 adjacent markers allele-sharing LOD≥0.59; nominal P≤0.05) for linkage on Chromosomes 2, 5, 6, 7, 8, 10, 11, 20 and X, with peaks at 6p25.3-p24.3 and 8p23.1-p21.3 contributed to largely by 31 Hindu families and at Xq21.1-q26.1 by 27 Muslim families. Refined mapping confirmed linkage across all primary scan families at 2q12.2-q14.1 and 11q13.2-q23.3, but only 11q13.2-q23.3 replicated (combined LOD = 1.59; P = 0.0034). Linkage at 6p25.3-p24.3 and 8p23.1-p21.3, and at Xq21.1-q26.1, was confirmed by refined mapping for primary Hindu and Muslim families, respectively, but only Xq21.1-q26.1 replicated across all Muslim families (combined LOD 1.49; P = 0.0045). STRUCTURE and SMARTPCA did not identify population genetic substructure related to religious group. Classification and regression tree, and spatial interpolation, analyses confirm geographical heterogeneity for linkages at 6p25.3-p24.3, 8p23.1-p21.3 and Xq21.1-q26.1, with specific clusters of families contributing LOD scores of 2.13 (P = 0.0009), 1.75 (P = 0.002) and 1.84 (P = 0.001), respectively.

Conclusions

GWLS has identified novel loci that show geographical heterogeneity in their influence on susceptibility to VL in India.     

Challenges in deriving high-confidence protein identifications from data gathered by a HUPO plasma proteome collaborative study

The Human Proteome Organization (HUPO) recently completed the first large-scale collaborative study to characterize the human serum and plasma proteomes. The study was carried out in different locations and used diverse methods and instruments to compare and integrate tandem mass spectrometry (MS/MS) data on aliquots of pooled serum and plasma from healthy subjects. Liquid chromatography (LC)-MS/MS data sets from 18 laboratories were matched to the International Protein Index database, and an initial integration exercise resulted in 9,504 proteins identified with one or more peptides, and 3,020 proteins identified with two or more peptides. This article uses a rigorous statistical approach to take into account the length of coding regions in genes, and multiple hypothesis-testing techniques. On this basis, we now present a reduced set of 889 proteins identified with a confidence level of at least 95%. We also discuss the importance of such an integrated analysis in providing an accurate representation of a proteome as well as the value such data sets contain for the high-confidence identification of protein matches to novel exons, some of which may be localized in alternatively spliced forms of known plasma proteins and some in previously nonannotated gene sequences.     

Zinc induces ERK-dependent cell death through a specific Ras isoform

The effect of Zn on p53-independent cell death was examined in IIC9 embryonic fibroblasts. Despite the fact that these cells are p53-minus, Zn-mediated death occurs via an apoptotic mechanism. Death is facilitated by the presence of the Zn ionophore, pyrithione, indicating that intracellular Zn initiates the death response. Our investigations of the mechanism of Zn action demonstrate that Zn induces the death of IIC9 cells in a manner that is ERK-dependent. Expression of dn-(dominant negative)Ras attenuates ERK1/2 activation by Zn, and correspondingly reduces its cytotoxic effects. Raf-RBD pull-down experiments confirm that Zn treatment activates Ras and identified H-Ras as the specific isoform activated. This contrasts the activation of N-Ras that occurs when IIC9 cells are stimulated with thrombin. Thus, although the prolonged activation of the Ras/ERK pathway by Zn is similar to that seen when induced by mitogen, the distinguishing feature appears to be the isoform specificity of Ras activation. This work was supported by National Institute of Health Grants DK-52194 and AI-44458 (to J.A.C). P.L Blackwell was supported by an American Heart Association Grant in Aid 0555574Z (to C.K), K.J. Hughes was supported by the National Institute of Health Training Grant (GM008306) and Kimberly Creach was supported by St. Louis University Medical School Summer Research Fellowship.     

Hitting the SPOT: small-molecule macroarrays advance combinatorial synthesis

The small-molecule macroarray represents a new tool to accelerate combinational library synthesis and screening. This array platform originates from the SPOT-synthesis technique, or the spatially addressed synthesis of peptides on cellulose supports. Recent advances in the field have expanded this technique beyond peptidic systems into the realm of complex small-molecule synthesis. Small-molecule macroarrays offer some significant advantages over traditional combinatorial synthesis platforms--these focused, 50-200 compound arrays are straightforward to synthesize, inexpensive, and amenable to numerous screening applications where the array compounds are either bound to or cleaved from the planar support. Critical advances in the small-molecule macroarray technique are highlighted herein, including the use of microwave-assisted organic reactions, multicomponent reactions, and automated spotting methods to further accelerate and broaden macroarray technology.     

Purification and characterization of proteins,including procollagen type III,in salt extracts of fetal bovine skin

The nature of high-molecular-weight proteins in salt extracts of fetal bovine skin was investigated. A series of DEAE cellulose ion-exchange columns separated the mature collagen from the high molecular weight proteins and also separated the high molecular weight proteins from each other. The following proteins were isolated: (a) a very high molecular weight protein which appears to be aggregated mature collagen; (b) two high molecular weight proteins of slightly faster mobility on SDS polyacrylamide gels, one of which is collagen-like and one of which is not; and (c) a type III procollagen, purer than those previously reported in the literature. These latter three proteins were characterized by amino acid analysis, SDS polyacrylamide gel electrophoretic mobility, collagenase sensitivity, and CNBr peptide patterns from SDS-PAGE.     

Particle swarm optimization

Particle swarm optimization (PSO) has undergone many changes since its introduction in 1995. As researchers have learned about the technique, they have derived new versions, developed new applications, and published theoretical studies of the effects of the various parameters and aspects of the algorithm. This paper comprises a snapshot of particle swarming from the authors’ perspective, including variations in the algorithm, current and ongoing research, applications and open problems.