Isolation of lactobacilli with probiotic properties from the human stomach

Aims: Recent evidence suggests that the human gastric microbiota is much more diverse than previously thought. The aim of this study was to assess the potential for isolating lactobacilli from the human stomach. Methods and Results: Lactobacilli were selectively cultured from gastric biopsies from 12 patients undergoing routine endoscopy. Lactobacilli were present in four of 12 biopsies. We isolated, in total 10 different strains representing five species (Lactobacillus gasseri, L. fermentum, L. vaginalis, L. reuteri and L. salivarius). The 10 isolates varied greatly in their ability to inhibit the growth of two Gram‐positive bacteria and two Gram‐negative bacteria. Furthermore, the acid and bile resistance profiles of the 10 isolates spanned a wide range. Conclusions: Five different Lactobacillus species were cultured from human gastric biopsies for the first time. Significance and Impact of the Study: Diverse Lactobacillus species are more prevalent in the human stomach than previously recognized, representing an untapped source of bacteria with beneficial probiotic and/or biotechnological properties.     

Phosphorylation of the mitochondrial protein Sab by stress-activated protein kinase 3

Mitogen-activated protein kinases (MAPKs) transduce extracellular signals into responses such as growth, differentiation, and death through their phosphorylation of specific substrate proteins. Early studies showed the consensus sequence (Pro/X)-X-(Ser/Thr)-Pro to be phosphorylated by MAPKs. Docking domains such as the "kinase interaction motif" (KIM) also appear to be crucial for efficient substrate phosphorylation. Here, we show that stress-activated protein kinase-3 (SAPK3), a p38 MAPK subfamily member, localizes to the mitochondria. Activated SAPK3 phosphorylates the mitochondrial protein Sab, an in vitro substrate of c-Jun N-terminal kinase (JNK). Sab phosphorylation by SAPK3 was dependent on the most N-terminal KIM (KIM1) of Sab and occurred primarily on Ser321. This appeared to be dependent on the position of Ser321 within Sab and the sequence immediately surrounding it. Our results suggest that SAPK3 and JNK may share a common target at the mitochondria and provide new insights into the substrate recognition by SAPK3.     

Atomic resolution analysis of a 2:1 complex of CpG and acridine orange.

Cytidylyl-3', 5'-guanosine and acridine orange crystallize in a highly-ordered triclinic lattice which diffracts X-rays to 0.85 angstrom resolution. The crystal structure has been solved and refined to a residual factor of 9.5%. The two dinucleoside phosphate molecules form an antiparallel double helix with the acridine orange intercalated between them. The two base pairs of the double helical fragment have a twist angle of 10 degrees and it is found to have a C3' endo-(3', 5')-C2' endo mixed sugar puckering along the nucleotide backbone as has been observed for other simple intercalator complexes. Twenty-five water molecules have been located in the lattice together with a sodium ion. The intercalator double helical fragments form sheets which are held together by van der Waals interactions in one direction and hydrogen bonding interactions in the other. The crystal lattice contains aqueous channels in which sixteen water molecules are hydrogen bonded to the nucleotide, none to the intercalator, five water molecules are coordinated about the sodium ion and four water molecules bind solely to other water molecules. The bases in the base pairs have a dihedral angle of 7 to 8 degrees between them.     

Sequence similarity between alpha 2-macroglobulin from the horseshoe crab, Limulus polyphemus, and proteins of the alpha 2-macroglobulin family from mammals.

1. Purified alpha 2-macroglobulin (alpha 2M) from the American horseshoe crab, Limulus polyphemus was cleaved with trypsin and 20 of the tryptic peptides were sequenced and compared with the sequences of human alpha 2M, rat alpha 1M, alpha 2M, and alpha 1-inhibitor 3, and human complement proteins C3 and C4. 2. Ten of the peptides (233 residues), including that containing the thiol ester site, could be aligned unambiguously with stretches in mammalian alpha 2M, with a degree of identity greater than 30%. 3. The 12-residue thiol ester-containing peptide of Limulus alpha 2M showed 67% identity with the same stretch of human alpha 2M.     

A human memory T cell subset with stem cell-like properties

Immunological memory is thought to depend on a stem cell-like, self-renewing population of lymphocytes capable of differentiating into effector cells in response to antigen re-exposure. Here we describe a long-lived human memory T cell population that has an enhanced capacity for self-renewal and a multipotent ability to derive central memory, effector memory and effector T cells. These cells, specific to multiple viral and self-tumor antigens, were found within a CD45RO(-), CCR7(+), CD45RA(+), CD62L(+), CD27(+), CD28(+) and IL-7Rα(+) T cell compartment characteristic of naive T cells. However, they expressed large amounts of CD95, IL-2Rβ, CXCR3, and LFA-1, and showed numerous functional attributes distinctive of memory cells. Compared with known memory populations, these lymphocytes had increased proliferative capacity and more efficiently reconstituted immunodeficient hosts, and they mediated superior antitumor responses in a humanized mouse model. The identification of a human stem cell-like memory T cell population is of direct relevance to the design of vaccines and T cell therapies.     

Targeting the autolysis loop of urokinase-type plasminogen activator with conformation-specific monoclonal antibodies

Tight regulation of serine proteases is essential for their physiological function, and unbalanced states of protease activity have been implicated in a variety of human diseases. One key example is the presence of uPA (urokinase-type plasminogen activator) in different human cancer types, with high levels correlating with a poor prognosis. This observation has stimulated efforts into finding new principles for intervening with uPA's activity. In the present study we characterize the so-called autolysis loop in the catalytic domain of uPA as a potential inhibitory target. This loop was found to harbour the epitopes for three conformation-specific monoclonal antibodies, two with a preference for the zymogen form pro-uPA, and one with a preference for active uPA. All three antibodies were shown to have overlapping epitopes, with three common residues being crucial for all three antibodies, demonstrating a direct link between conformational changes of the autolysis loop and the creation of a catalytically mature active site. All three antibodies are potent inhibitors of uPA activity, the two pro-uPA-specific ones by inhibiting conversion of pro-uPA to active uPA and the active uPA-specific antibody by shielding the access of plasminogen to the active site. Furthermore, using immunofluorescence, the conformation-specific antibodies mAb-112 and mAb-12E6B10 enabled us to selectively stain pro-uPA or active uPA on the surface of cultured cells. Moreover, in various independent model systems, the antibodies inhibited tumour cell invasion and dissemination, providing evidence for the feasibility of pharmaceutical intervention with serine protease activity by targeting surface loops that undergo conformational changes during zymogen activation.     

Activity-based protein profiling implicates urokinase activation as a key step in human fibrosarcoma intravasation

Entry of malignant cells into the vasculature (i.e. intravasation) requires proteolytic remodeling of the extracellular matrix so that tumor cells may pass through the local stroma and penetrate the vessel wall. The circulatory system then provides a means of transporting tumor cells to distant sites where they extravasate and establish metastatic lesions. This study utilizes activity-based protein profiling to compare the active serine hydrolase repertoire in high intravasating (HT-hi/diss) and low intravasating (HT-lo/diss) variants of the human fibrosarcoma HT-1080 cell line to determine which enzyme(s) play a role in intravasation. Activity-based protein profiling revealed multiple serine hydrolases with altered activity between HT-hi/diss and HT-lo/diss cells, with the largest difference being the activity of urokinase-type plasminogen activator (uPA). Levels of inactive uPA zymogen were similar between the two cell variants, but only HT-hi/diss conditioned medium contained active uPA, suggesting that uPA activation may contribute to the enhanced intravasation of HT-hi/diss cells. To analyze the role of uPA activity specifically in the process of intravasation, we grafted cells from the two HT-1080 variants onto the chorioallantoic membrane of chick embryos and measured levels of tumor cell intravasation in the distal chorioallantoic membrane using quantitative human-specific Alu PCR. Inhibition of uPA activity with natural (plasminogen activator inhibitor-1) or synthetic (amiloride) inhibitors diminished HT-hi/diss Matrigel invasion in vitro and intravasation and metastasis in vivo. Additionally, treatment of HT-lo/diss tumors with exogenous active uPA increased the number of intravasated cells in vivo. These results indicate that active uPA promotes tumor cell intravasation and that uPA activation appears to be a key step in tumor progression.