Diacylglycerol kinase synthesized by commensal Lactobacillus reuteri diminishes protein kinase C phosphorylation and histamine-mediated signaling in the mammalian intestinal epithelium.

Lactobacillus reuteri 6475 (Lr) of the human microbiome synthesizes histamine and can suppress inflammation via type 2 histamine receptor (H2R) activation in the mammalian intestine. Gut microbes such as Lr promote H2R signaling and may suppress H1R proinflammatory signaling pathways in parallel by unknown mechanisms. In this study, we identified a soluble bacterial enzyme known as diacylglycerol kinase (Dgk) from Lr that is secreted into the extracellular milieu and presumably into the intestinal lumen. DgK diminishes diacylglycerol (DAG) quantities in mammalian cells by promoting its metabolic conversion and causing reduced protein kinase C phosphorylation (pPKC) as a net effect in mammalian cells. We demonstrated that histamine synthesized by gut microbes (Lr) activates both mammalian H1R and H2R, but Lr-derived Dgk suppresses the H1R signaling pathway. Phospho-PKC and IκBα were diminished within the intestinal epithelium of mice and humans treated by wild-type (WT) Lr, but pPKC and IκBα were not decreased in treatment with ΔdgkA Lr. Mucosal IL-6 and systemic interleukin (IL)-1α, eotaxin, and granulocyte colony-stimulating factor (G-CSF) were suppressed in WT Lr, but not in ΔdgkA Lr colonized mice. Collectively, the commensal microbe Lr may act as a "microbial antihistamine" by suppressing intestinal H1R-mediated proinflammatory responses via diminished pPKC-mediated mammalian cell signaling.

MRG15 activates the B-myb promoter through formation of a nuclear complex with the retinoblastoma protein and the novel protein PAM14.

The MORF4-Related Gene on chromosome 15 (MRG15) is a member of a novel family of genes originally identified in studies to reveal cell senescence-inducing factors. MRG15 contains several predicted protein motifs, including a nuclear localization signal, a helix-loop-helix region, a leucine zipper, and a chromodomain. These motifs are commonly associated with transcription factors, suggesting that MRG15 may likewise function as a transcriptional regulator. To examine the potential function(s) of MRG15, we sought to identify cellular factors associated with this MRG family member. In this regard, we have found that both the retinoblastoma tumor suppressor (Rb) and a novel nuclear protein PAM14 (Protein Associated with MRG, 14 kDa) specifically associate with MRG15. We have further demonstrated that these interactions require the helix-loop-helix and leucine zipper domains of MRG15. Interestingly we have found all three proteins present in a multiprotein complex, suggesting that at least some of their functions may be interdependent. Although the functions of PAM14 have yet to be elucidated, Rb has several well characterized activities, including repression of E2F-activated promoters such as that of B-myb. Significantly we have demonstrated that MRG15 blocks the Rb-induced repression of this promoter, leading to B-myb promoter activation. Collectively these results suggest that MRG15 regulates transcription through interactions with a cellular protein complex containing Rb and PAM14.

Characterization of a novel zinc finger gene with increased expression in nondividing normal human cells.

We report here the cloning and characterization of a novel KRAB zinc finger gene, ZFQR, which has eight tandemly repeated zinc fingers, a complete KRAB box at the N-terminal region, and a unique C-terminal sequence. It is expressed in a variety of human tissues, and mRNA levels are upregulated in nondividing senescent and quiescent human fibroblasts. Overexpression of the protein in quiescent cells stimulated with serum growth factors results in inhibition of entry into the cell cycle. The latter activity is lost when the N-terminal KRAB domain is deleted. The KRAB domain is also required for the transcriptional repression activity of ZFQR and in maintaining association of the protein with the nuclear matrix. The gene has been mapped to human chromosome 19q13.4. The association of ZFQR with the nuclear matrix, transcriptional repression activity, increased expression in senescent and quiescent cells, and the ability to inhibit quiescent cells stimulated with growth factors from entering the cell cycle suggests a role for ZFQR in the maintenance of the nondividing state of normal human cells.

Isolation of a paralog of the Doyne honeycomb retinal dystrophy gene from the multiple retinopathy critical region on 11q13.

A large number of extracellular matrix proteins have been found to contain variations of the epidermal growth factor (EGF) domain and have been implicated in functions as diverse as blood coagulation, activation of complement, and determination of cell fate during development. The gene for one such protein, S1-5, was identified from a subtractively enriched cDNA library from a patient with Werner syndrome and was shown to be preferentially expressed in senescent and quiescent fibroblasts. We have cloned and characterized, in human and mouse, a novel gene that shows significant homology to the gene for S1-5. We have determined that the encoded protein contains four EGF domains and six calcium-binding EGF domains. On the basis of its homology to known proteins, we have designated this gene EFEMP2 (Egf-containing fibulin-like extracellular matrix protein 2) and the gene for the S1-5 protein EFEMP1. Like EFEMP1, this novel gene is expressed in a wide range of adult and fetal tissues. In contrast to EFEMP1, however, EFEMP2 is not significantly overexpressed in senescent or quiescent fibroblasts, suggesting a diversity of function within this new EGF-like domain subfamily. We have mapped EFEMP2 to 11q13, in an area where several retinopathies have been genetically linked. Given that mutations in EFEMP1 have been recently described in patients with Doyne honeycomb retinal dystrophy, EFEMP2 becomes a good candidate for such disorders.

Rhythmic sensitization of nematocyst discharge in response to vibrational stimuli.

Sea anemones capture prey by discharging nematocysts and other cnidae. Discharge of microbasic p-mastigophore (mpm) nematocysts is regulated in part by hair bundle mechanoreceptors on tentacles arising from multicellular complexes consisting of supporting cells and a sensory neuron. Anemone hair bundles detect movements of prey and then sensitize cnidocytes (cnida-containing cells) to discharge mpm nematocysts in response to contact between the prey and tentacle. Data from a simple bioassay based on counting nematocysts discharged into test probes, indicate that approximately twice as many nematocysts discharge into test probes touched to tentacles after sensitization than before sensitization. We here report that sub-second bursts of vibrational stimuli at key frequencies (51, 55, 65, or 74 Hz; Watson GM, Mire P, Hudson RR. 1998. J Exp Zool 281:582-593) sensitize discharge for at least 90 sec. Very few complete cycles of vibration are sufficient to sensitize discharge. However, as the number of cycles of vibration is increased, discharge is sensitized in rhythmic patterns. Computer analysis of the data by fast Fourier transforms indicates discharge to vibrations at 65 Hz is sensitized every 6.75 cycles. At 51 Hz discharge is sensitized every 2.00 cycles. At 74 Hz, discharge is sensitized in a polyrhythm occurring every 4.26, 3.76, 2.46, and 2. 10 cycles, respectively. At 55 Hz, discharge is sensitized in a polyrhythm occurring every 6.09, 3.20, 2.91, and 2.0 cycles, respectively. Apparently, cells in the neuronal pathway interconnecting anemone hair bundles with cnidocytes count cycles of vibration and then sensitize discharge or not according to the tally. J. Exp. Zool. 286:262-269, 2000.

ATP enhances repair of hair bundles in sea anemones.

Hair bundle mechanoreceptors of sea anemones are similar to those of the acousticolateralis system of vertebrates (Watson, Mire and Hudson, 1997, Hear. Res. 107, 53-63). Anemone hair bundles are repaired by 'repair proteins' secreted following a complete loss of structural integrity and loss of function caused by 1 h exposure to calcium free seawater. Exogenously supplied repair proteins (RP) restore structural integrity to hair bundles and restore vibration sensitivity in 7-8 min (Watson, Mire and Hudson, 1998, Hear. Res. 115, 119-128). We here report that exogenously supplied ATP enhances the rate by which RP restore vibration sensitivity. A bimodal dose response to ATP indicates maximal enhancement at picomolar and micromolar concentrations of ATP. At these concentrations of ATP, vibration sensitivity is restored in 2 min. These data suggest that at least two ATPases exhibiting different binding affinities for ATP are involved in the repair process. Whereas the higher affinity site is specific for ATP, the lower affinity site does not discriminate between ATP and ADP. Nucleotidase cytochemistry localizes ATPase activity in isolated repair proteins. In the absence of exogenously added RP, sea anemones secrete and consume ATP during the 4 h recovery period after 1 h exposure to calcium free seawater. In the presence of exogenously added RP, ATP is secreted and then consumed within 10 min. Quinacrine cytochemistry localizes possible stores of ATP in the apical cytoplasm of sensory neurons located at the center of the hair bundle. According to our model, ATP is secreted by the sensory neuron after its hair bundle loses structural integrity. Hydrolysis of ATP by repair proteins is essential to the repair process.

Process mapping in screening mammography.

Successful screening mammography programs aim to screen large numbers of women efficiently and inexpensively. Development of an effective screening mammography program requires skilled personnel, solid infrastructure, and a robust computer system. A group of physicians, technologists, computer support personnel, and administrators carefully analyzed a growing screening mammography program as a series of steps, starting with the request for the examination and ending with the receipt of a hard-copy consultation. The analysis involved a detailed examination of every step and every possible outcome in the screening process. The information gained through process mapping may be used for identification of systemic and personnel problems, allocation of resources, modification of workplace architecture, and design of computer networks. Process mapping is helpful for those involved in designing and improving screening mammography programs. Viewing a process (i.e., obtaining a screening mammogram) as a series of steps may allow for the identification of inefficient components that may limit growth.

Loss of T-antigen sequences allows SV40-transformed human cells in crisis to acquire a senescent-like phenotype.

Normal human cells transfected with SV40 DNA exhibit an extended proliferative potential compared with controls, but they eventually enter a phase known as "crisis." During crisis, extensive cell death occurs and the cells exhibit some gene expression changes similar to senescent cells. This article presents results which indicate that crisis most likely depends on expression of the viral gene T-antigen. We have obtained a unique subpopulation of cells that have deleted the T-antigen gene and, rather than dying as cells do in crisis, remain viable and exhibit some senescent-like characteristics. We also found that the SV40 promoter is poorly expressed in senescent versus young cells. We hypothesize that decreased activity of the viral promoter may result in decreased expression of T-antigen, which is challenged by over-expression of the cell cycle inhibitors such as p21Sdi1. Conflicting signals to proceed/halt cells cycle progression result in the cell death associated with crisis.

Effect of aging on regulation of sdi-1 in rat hepatocytes.

We examined basal and EGF stimulated DNA synthesis as well as sdi-1 mRNA and protein in primary hepatocyte cultures, and basal levels of sdi-1 mRNA and protein in whole liver homogenates from 6 and 24 month old rats. Since EGF stimulated DNA synthesis decreases with age, it was hypothesized that basal and EGF stimulated levels of sdi-1 mRNA and protein, an inhibitor of DNA synthesis, might increase. Surprisingly, however both sdi-1 mRNA and protein actually decreased both in cells and homogenates of old rats. These results indicate that the age-related impairment in EGF stimulated DNA synthesis in hepatocytes appears to occur prior to or parallel with sdi-1 expression and cannot be explained on the basis of increased inhibition due to elevated levels of this protein.

Studies demonstrating the complexity of regulation and action of the growth inhibitory gene SDI1.

The identification of the DNA synthesis inhibitory gene SDI1 by investigators studying cell senescence, tumor suppression, cell cycle control and differentiation suggest a key regulatory role for this gene. To better understand the growth regulatory activity of this gene we proceeded with the experiments described here. The data demonstrate that SDI1 is an important downstream effector of p53, but here we report that it can also cause inhibition of DNA synthesis in several immortal human cell lines, independent of p53 or Rb status. Levels of SDI1 mRNA expression in immortal cells are consistently much lower than that of normal cells, indicating that immortalization is not compatible with high expression of SDI1. These results highlight the complex nature of regulation of this gene and its mode of action.

Exit from G0 and entry into the cell cycle of cells expressing p21Sdi1 antisense RNA.

p21Sdi1 (also known as Cip1 and Waf1), an inhibitor of DNA synthesis cloned from senescent human fibroblasts, is an inhibitor of G1 cyclin-dependent kinases (Cdks) in vitro and is transcriptionally regulated by wild-type p53. In addition, p21Sdi1 has been found to inhibit DNA replication by direct interaction with proliferating cell nuclear antigen. In this study we analyzed normal human fibroblast cells arrested in G0 and determined that an excess of p21Sdi1 was present after immunodepletion of various cyclins and Cdks, in contrast to mitogen-stimulated cells in early S phase. Expression of antisense p21Sdi1 RNA in G0-arrested cells resulted in induction of DNA synthesis as well as entry into mitosis. These results suggest that p21Sdi1 functions in G0 and early G1 and that decreased expression of the gene is necessary for cell cycle progression.

Cloning of senescent cell-derived inhibitors of DNA synthesis using an expression screen.

We here describe a rapid and simple expression screen method that has allowed us to isolate cDNAs coding for inhibitors of DNA synthesis from senescent human diploid fibroblasts. The assay involved transient transcriptional overexpression of a gene product encoded by a cDNA in a proliferating cell, on the assumption that this would be sufficient to block DNA synthesis in a short-term assay using tritiated thymidine autoradiography. Three cDNAs, referred to as senescent cell-derived inhibitors (sdi), that exhibit DNA synthesis-inhibitory activity when introduced into young cycling cells, were successfully identified. Expression of one of the cDNAs, sdi1, increased 10- to 20-fold in senescent compared with young cells and the increase in RNA closely paralleled the onset of the senescent phenotype and loss of cell proliferation. sdi1 expression was also increased in young cells made nondividing (quiescent) by deprivation of growth factors or contact inhibition. Following serum stimulation, RNA levels of sdi1 in quiescent cells were initially increased, but then declined to low levels just prior to the entry of the cells into S phase. In contrast, RNA levels of sdi1 in senescent cells failed to decline, suggesting a role for this gene in maintaining the senescent phenotype. The sdi1 gene has been mapped to the p arm of chromosome 6.