Colostrum whey down-regulates the expression of early and late inflammatory response genes induced by Escherichia coli and Salmonella enterica Typhimurium components in intestinal epithelial cells.

Pathogenic invasion by Escherichia coli and Salmonellae remains a constant threat to the integrity of the intestinal epithelium and can rapidly induce inflammatory responses. At birth, colostrum consumption exerts numerous beneficial effects on the properties of intestinal epithelial cells and protects the gastrointestinal tract of newborns from pathogenic invasion. The present study aimed to investigate the effect of colostrum on the early and late inflammatory responses induced by pathogens. The short-term (2 h) and long-term (24 h) effects of exposure to heat-killed (HK) E. coli and Salmonella enterica Typhimurium on gene expression in the porcine intestinal epithelial cell (IPEC-J2) model were first evaluated by microarray and quantitative PCR analyses. Luciferase assays were performed using a NF-κB-luc reporter construct to investigate the effect of colostrum whey treatment on the activation of NF-κB induced by HK bacteria. Luciferase assays were also performed using NF-κB-luc, IL-8-luc and IL-6-luc reporter constructs in human colon adenocarcinoma Caco-2/15 cells exposed to dose-response stimulations with HK bacteria and colostrum whey. Bovine colostrum whey treatment decreased the expression of early and late inflammatory genes induced by HK bacteria in IPEC-J2, as well as the transcriptional activation of NF-κB-luc induced by HK bacteria. Unlike that with colostrum whey, treatment with other milk fractions failed to decrease the activation of NF-κB-luc induced by HK bacteria. Lastly, the reduction of the HK bacteria-induced activation of NF-κB-luc, IL-8-luc and IL-6-luc by colostrum whey was dose dependent. The results of the present study indicate that bovine colostrum may protect and preserve the integrity of the intestinal mucosal barrier in the host by controlling the expression levels of early and late inflammatory genes following invasion by enteric pathogens.

Gummatous penile syphilis.

Syphilitic gumma involving the penis is a rare manifestation of tertiary syphilis. Only seventeen cases have been reported in the literature. It can mimic other diagnoses such as penile carcinoma. We report a case of a 56 year old male that had been sexually abstinent for over 10 years and presenting with a 4 cm painful penile lesion with clinically palpable bilateral inguinal nodes with no prior history of sexually transmitted diseases (STDs). A positron emission tomography-computed tomography scan identified the penile mass as being hypermetabolic and suspicious for penile carcinoma. Several inguinal and pelvic lymph nodes were also found to be suspicious for penile carcinoma. A penile biopsy was proposed and declined by the patient as he opted for a partial penectomy. The surgery was performed for diagnostic and palliative purposes. Histopathological studies revealed the presence of polymorphous, granulomatous, epitheloid inflammatory infiltrate with giant cells. Additional microbiologic testing confirmed the diagnosis of tertiary syphilis, presenting as gummatous syphillis associated with neurosyphilis. The patient was treated with intravenous penicillin and had adequate clinical clinical and serologic 12 months following treatment. Gummatous syphillis is a rare entity, but should be considered in the differential diagnosis of a penile lesion. To rule out this possibility, a biopsy should always be performed prior to invasive penis surgery.

Role of the three polyoma virus early proteins in tumorigenesis.

A modified polyoma virus genome which can encode the middle T protein but not the large or small T proteins transforms rat cells in culture with an efficiency about 20% that of the wild-type genome. Although middle T-transformed cells grow as tumors when transplanted into nude mice or syngeneic rats, the middle T gene alone is totally inactive when used in a more stringent and rigorous assay for tumorigenicity such as the injection of DNA into newborn rats. Thus, functions other than those expressed by middle T antigen are required for the elaboration of all the properties associated with tumorigenesis. To assess whether a complementary function could be exerted by the large or the small T antigen, we constructed plasmids containing two modified early regions which independently encoded middle T and one of the two other proteins. Both recombinants were tumorigenic in newborn rats. Cell lines derived by transfer of these plasmids under no special selective conditions did not acquire the property of growth in low-serum medium but exhibited the same tumorigenic properties as wild-type polyoma DNA-transformed cells. Furthermore, a recombinant which encoded the middle and small T antigens, but not the large T antigen, was tumorigenic in newborn rats. Although the small T antigen provides a complementary function for tumorigenicity, it cannot complement the middle T antigen for an efficient induction of transformation of cultured cells. This suggests that the complementary function exerted by the small T antigen is different from that of the N-terminal fragment of the large T protein.

Polyoma middle T antigen requires cooperation from another gene to express the malignant phenotype in vivo.

The oncogenic potential of polyomavirus in newborn hamsters can be expressed by a recombinant encoding only the middle T protein. However, polyoma middle T requires the cooperation from small T to induce tumors in newborn rats. Similar complementary functions such as cocarcinogens or tumor promotors can be exerted by the simian virus 40 T antigens as well as by one or several products of the early region 1A of adenovirus 2.

A cis-acting element in the promoter region of the murine c-myc gene is necessary for transcriptional block.

A block to elongation of transcription has been shown to occur within the first exon of the human and murine c-myc genes. The extent of this block was found to vary with the physiological state of cells, indicating that modulation of the transcriptional block can serve to control the expression of this gene. To determine which sequences are required in cis for the transcriptional block, we generated a series of constructs containing various portions of murine c-myc 5'-flanking and exon 1 sequences. We established populations of HeLa and CV-1 cells stably transfected with these constructs. The transcription start sites were determined by S1 nuclease mapping analysis, and the extent of transcriptional block was measured by nuclear run-on transcription assays. Our results demonstrate that at least two cis-acting elements are necessary for the transcriptional block. A 3' element was found to be located in the region where transcription stopped and showed features reminiscent of some termination sites found in procaryotes. A 5' element was positioned between the P1 and P2 (C. Asselin, A. Nepveu, and K. B. Marcu, Oncogene 4:549-558, 1989). Removal of the more 3' binding site abolished the transcriptional block.

Molecular requirements for transcriptional initiation of the murine c-myc gene.

We have identified sequences in the 5' flanking region of the murine c-myc gene's P1 and P2 transcription initiation sites which form specific complexes with nuclear factors of murine and human origin and are also required for normal P1 and P2 usage. Four nuclear factor binding sites were identified within 400 bp 5' of P1 (5'Mf, 5'Mg1, 5'Mg2, and 5'Mg3) and two others within 100 bp 5' of P2 (ME1a1 and ME1a2). The Sp1 transcription factor bound to 5'Mg1 and 5'Mg3 with high affinity and with low affinity to 5'Mg2, ME1a1 and ME1a2 which also bound with high affinity to other factors in crude nuclear extracts. Deletion mutagenesis of sequences 5' of the P1 initiation site revealed that 109 bp encompassing 5'Mg3 and a TATA sequence were sufficient for P1 usage. The ME1a1 binding site 5' of P2 was necessary for maximal P2 activity and the loss of this sequence resulted in enhanced P1 usage. These findings demonstrate that the P1 and P2 initiation sites are independently regulated and that the ME1a1 binding site plays a central role in the normal usage of the c-myc promoters.

Tissue-specific post-transcriptional regulation of c-myc expression in normal and H-2K/human c-myc transgenic mice.

We show that the steady-state levels of c-myc mRNAs vary considerably in different organs of normal adult mice, maximal expression being observed in lymphoid organs and minimal expression in liver and brain. Nuclear run-on analysis of c-myc gene transcription in adult liver and spleen reveals that the difference in c-myc gene expression in these two organs is due to differential post-transcriptional control. Moreover, these nuclear run-on assays indicate that no premature termination of c-myc gene transcription takes place in the nuclei of the three adult tissues analysed. In fetal liver development, we observe a decrease in c-myc mRNA, but this is not due to changes in transcriptional activity implicating post-transcriptional regulatory mechanisms. Our studies of c-myc gene expression in organs of H-2K/myc transgenic mice, harboring an H-2K promoter driven human c-myc gene, confirm that the in vivo c-myc regulation is mainly post-transcriptional and shows that sequences shared by the murine and human c-myc proto-oncogenes are involved in this control.

Signals from the AT2 (angiotensin type 2) receptor of angiotensin II inhibit p21ras and activate MAPK (mitogen-activated protein kinase) to induce morphological neuronal differentiation in NG108-15 cells.

In a previous study, we had shown that activation of the AT2 (angiotensin type 2) receptor of angiotensin II (Ang II) induced morphological differentiation of the neuronal cell line NG108-15. In the present study, we investigated the nature of the possible intracellular mediators involved in the AT2 effect. We found that stimulation of AT2 receptors in NG108-15 cells resulted in time-dependent modulation of tyrosine phosphorylation of a number of cytoplasmic proteins. Stimulation of NG108-15 cells with Ang II induced a decrease in GTP-bound p21ras but a sustained increase in the activity of p42mapk and p44mapk as well as neurite outgrowth. Similarly, neurite elongation, increased polymerized tubulin levels, and increased mitogen-activated protein kinase (MAPK) activity were also observed in a stably transfected NG108-15 cell line expressing the dominant-negative mutant of p21ras, RasN17. These results support the observation that inhibition of p21ras did not impair the effect of Ang II on its ability to stimulate MAPK activity. While 10 microM of the MEK inhibitor, PD98059, only moderately affected elongation, 50 microM PD98059 completely blocked the Ang II- and the RasN17-mediated induction of neurite outgrowth. These results demonstrate that some of the events associated with the AT2 receptor-induced neuronal morphological differentiation of NG108-15 cells not only include inhibition of p21ras but an increase in MAPK activity as well, which is essential for neurite outgrowth.

Introns enable the polyomavirus middle and small T antigens to stimulate the growth of primary rat embryo fibroblasts.

We constructed spliceable vectors that separately encode polyomavirus MT and ST. The addition of an intron enables MT to transform and to immortalize more efficiently and ST to transiently stimulate the growth of primary rat embryo fibroblasts.

Regulation of c-myc expression by sodium butyrate in the colon carcinoma cell line Caco-2.

The human colon carcinoma cell line Caco-2 spontaneously undergoes enterocytic differentiation in culture. We used sodium butyrate to modify differentiation and growth properties of this cell line and considered c-myc expression as a potential target. Degradation of normal c-myc mRNAs with a half-life of 20 min is not coupled to translation in this cell line, as determined by cycloheximide treatment. We show that butyrate reduces c-myc mRNA levels after a 30 min delay. Butyrate does not affect c-myc expression at the level of transcriptional initiation or elongation, as determined by run-on analysis, but at a post-transcriptional level. Cycloheximide blocks butyrate-dependent reduction of c-myc mRNA levels. Cross-linking experiments show that a 34 kDa protein binds specifically to the c-myc AU-rich instability determinant found in the 3'-untranslated region (ARE). Binding of this protein to the ARE is not modulated by butyrate or cycloheximide. These experiments suggest that butyrate induces a factor involved in c-myc mRNA degradation that differs from the known ARE-associated proteins. Post-transcriptional modification of gene expression could be one of the major targets for this anti-proliferative agent.

Activation of haptoglobin gene expression by cAMP involves CCAAT/enhancer-binding protein isoforms in intestinal epithelial cells.

CCAAT/enhancer-binding protein (C/EBP) isoforms are expressed in rodent intestine and in the rat intestinal epithelial cell line IEC-6 but their role remains to be determined. Treatment of IEC-6 cells with the adenylate cyclase activator forskolin led to coordinate induction of C/EBP isoforms alpha, beta and delta at the mRNA and protein levels. Transient transfection assays showed that their expression is controlled at the transcriptional level. Forskolin treatment induced haptoglobin mRNA levels. Electrophoretic mobility shift and supershift assays demonstrated an increase in DNA-binding activities of the three C/EBP isoforms to the haptoA and haptoC C/EBP DNA-binding sites of the proximal haptoglobin promoter. Site-specific mutations of both sites led to a decrease in transcriptional induction by forskolin, suggesting that C/EBP isoforms are involved in the cAMP-dependent regulation of the acute-phase protein gene haptoglobin in intestinal epithelial cells.

Effect of human recombinant leptin on lipid handling by fully differentiated Caco-2 cells.

It has been established that leptin displays a number of effects on peripheral tissues. We have investigated the effect of the hormone on lipid synthesis, apolipoprotein biogenesis and lipoprotein secretion in Caco-2 cells. Immunocytochemistry revealed the presence of leptin receptors (Ob-Rb) on the basolateral membrane. Incubation of cells with 200 nM leptin resulted in a decreased export of triglycerides in the basolateral medium without affecting monoglyceride, diglyceride and cholesterol ester lipid classes. It also significantly reduced the output of de novo-synthesized apolipoprotein (Apo)B-100 and ApoB-48 as well as that of newly formed chylomicrons and of low-density lipoproteins. It also enhanced that of ApoA-I, ApoA-IV and ApoE. Our results support the hypothesis that leptin can affect energy balance at the gut level by reducing lipid release into the circulation.

New hemodynamic approach to angiogenesis: color and pulsed Doppler ultrasonography.

RATIONALE AND OBJECTIVES: To determine the capacity of color and spectral Doppler ultrasonography (US) to quantify angiogenesis in vivo and to characterize low-resistance intratumor blood flow. METHODS: Thirty-two tumors, xenografted into mice, were studied with Doppler US. The number of intratumor vessels visualized with color Doppler US was compared with the density of microvessels and the number of vessels >100 microm determined by histologic examination. The resistance index and the peak systolic velocities were evaluated. RESULTS: The number of intratumor vessels visualized by color Doppler US was correlated with the number of vessels >100 microm (P<0.001) determined histologically. When vessel density was >30, intratumor vessels were always detected by color Dopper US. The resistance index and peak systolic velocities were significantly lower in intratumor than in peritumor vessels. CONCLUSIONS: Color Doppler US evaluated tumor angiogenesis accurately. Spectral analysis confirmed the low resistance of intratumor blood flow.

CCAAT/enhancer binding proteins beta and delta regulate alpha1-acid glycoprotein gene expression in rat intestinal epithelial cells.

Isoforms of CCAAT/enhancer binding protein (C/EBP) are expressed in rodent intestine as well as in the rat intestinal epithelial cell line IEC-6. However, no specific roles have been attributed to these isoforms in intestinal epithelial cells. To determine whether C/EBP family members could be implicated in the regulation of acute-phase response gene expression in intestinal epithelial cells, we have studied the effect of glucocorticoids on expression of the alpha1-acid glycoprotein gene and C/EBP isoforms in IEC-6 cells. Glucocorticoids induced alpha1-acid glycoprotein gene expression in these cells. This induction coincided with an increase of DNA-binding capacity of both C/EBPbeta and C/EBPdelta to the B1 and B2 C/EBP-interacting sites localized in the rat AGP promoter. Transforming growth factor beta, (TGFbeta), a cytokine involved in the transcriptional regulation of several acute-phase plasma proteins, antagonized the glucocorticoid-dependent induction of alpha1-acid glycoprotein gene expression. In parallel, TGFbeta downregulated the DNA-binding capacities of both the C/EBPbeta and C/EBPdelta isoforms. Mutations of the B1 or the B2 C/EBP-interacting site strongly reduced the responsiveness of the alpha1-acid glycoprotein promoter to glucocorticoids and TGFbeta. These results demonstrate a functional role for C/EBPbeta and C/EBPdelta in rat intestinal epithelial cells and suggest that these isoforms represent important modulators of the acute-phase response and of glucocorticoid, as well as TGFbeta, responsiveness.

Higher circulating 4-hydroxynonenal-protein thioether adducts correlate with more severe diastolic dysfunction in spontaneously hypertensive rats.

OBJECTIVE: Accumulating evidence supports a role of 4-hydroxynonenal (4HNE) in oxidative-stress related diseases, but its specific contribution to disease development remains to be clarified. Further to our finding of high circulating 4HNE-protein thioether adducts (4HNE-P) in spontaneously hypertensive rats (SHRs), we aimed at correlating 4HNE-P with cardiac function and testing the impact of antioxidant therapy. MATERIALS AND METHODS: The lipoperoxidation inhibitor probucol (10 mg/kg/day) or vehicle (corn oil) were administered daily (i.p.) for 4 weeks in 18-week-old SHRs (9 rats/group). Cardiac functions were assessed by echocardiography and 4HNE-P by gas chromatography/mass spectrometry. RESULTS: Diastolic dysfunction worsened in SHRs receiving vehicle as reflected by changes (P < 0.05) in indexes of left ventricular relaxation (increased isovolumic relaxation time) and compliance (increased E-wave deceleration rate; EDR). Higher circulating 4HNE-P correlated with diastolic dysfunction (EDR: R(2) = 0.518; P < 0.001) and heart rate (R(2) = 0.225; P < 0.05). Probucol prevented the deterioration of diastolic function, while lowering the mean and median of circulating 4HNE-P by 21% and 35%, respectively. CONCLUSION: Collectively, these results support a role for 4HNE in the pathophysiological events linked to disease progression in SHRs.

The accuracy of the Cartier tibial guide in knee arthroplasty.

The authors reviewed, radiologically, the postoperative position of tibial component in 51 knee arthroplasties performed between 1987 and 1989 with Cartier's extramedullary tibial guide. The results compared favourably with those of other studies. A variation of 1.9 degrees on anteroposterior roentgenograms and 2.4 degrees on lateral roentgenograms was obtained by five orthopedic surgeons, suggesting that, for knee arthroplasties, the guide of Cartier is a useful tool and produces reliable results.

Sequences from polyomavirus and simian virus 40 large T genes capable of immortalizing primary rat embryo fibroblasts.

We developed a procedure to evaluate quantitatively the capacity of subgenomic fragments from polyomavirus and simian virus 40 (SV40) to promote the establishment of primary cells in culture. The large T antigen from both of these viruses can immortalize primary rat embryo fibroblasts. Both antigens have amino-terminal domains that retain biological activity after deletion of other parts of the polypeptide chain. However, this activity varies considerably among various mutants, presumably because of alterations in the stability or conformation of the truncated polypeptides. The polyomavirus middle T gene alone immortalizes at a low efficiency, which indicates that this oncogene can have both immortalization and transformation potentials depending on the assay system chosen. We generated deletions in the polyomavirus and SV40 large T genes to localize more precisely the functional domains of the proteins involved in the immortalization process. Our results show that the region of the SV40 large T antigen involved in immortalization is localized within the first 137 amino acid residues. This region is encoded by the first large T exon and a small portion from the second exon which includes the SV40 large T nuclear location signal. The polyomavirus sequence involved in immortalization comprises a region from the second large T exon, mapping between nucleotides 1016 and 1213, which shares no homology with SV40 and is thought to be of cellular origin. We suggest that this region of the polyomavirus large T gene functions either as a nuclear location signal or as part of the large T protein sequence involved in DNA binding.