A Mouse Model of Targeted Musashi1 Expression in Whole Intestinal Epithelium Suggests Regulatory Roles in Cell Cycle and Stemness.

The intestinal epithelium is very peculiar for its continuous cell renewal, fuelled by multipotent stem cells localized within the crypts of Lieberkühn. Several lines of evidence have established the evolutionary conserved RNA-binding protein Musashi1 as a marker of adult stem cells, including those of the intestinal epithelium, and revealed its roles in stem cell self-renewal and cell fate determination. Previous studies from our laboratories have shown that Musashi1 controls stem cell-like features in medulloblastoma, glioblastoma, and breast cancer cells, and has pro-proliferative and pro-tumorigenic properties in intestinal epithelial progenitor cells in vitro. To undertake a detailed study of Musashi1's function in the intestinal epithelium in vivo, we have generated a mouse model, referred to as v-Msi, overexpressing Musashi1 specifically in the entire intestinal epithelium. Compared with wild type litters, v-Msi1 mice exhibited increased intestinal crypt size accompanied by enhanced proliferation. Comparative transcriptomics by RNA-seq revealed Musashi1's association with gut stem cell signature, cell cycle, DNA replication, and drug metabolism. Finally, we identified and validated three novel mRNA targets that are stabilized by Musashi1, Ccnd1 (Cyclin D1), Cdk6, and Sox4. In conclusion, the targeted expression of Musashi1 in the intestinal epithelium in vivo increases the cell proliferation rate and strongly suggests its action on stem cells activity. This is due to the modulation of a complex network of gene functions and pathways including drug metabolism, cell cycle, and DNA synthesis and repair.

The anatomy of non-carious cervical lesions.

OBJECTIVE: The term "non-carious cervical lesion" (NCCL) describes a dental hard tissue defect of unknown origin. Two very distinct variations are known: wedge-shaped and saucer-shaped lesions. Reasons for occurrence of two forms might include different contributing factors. METHODS: Forty-two teeth, 19 wedge-shaped and 23 saucer-shaped lesions, were analysed by light and confocal laser scanning microscopy (CLSM) to investigate presence of calculus and organic matter, surface structure of the lesion, borders of the lesion, and potential fractures in the dental hard tissues. RESULTS: One hundred percent of the wedge-shaped teeth showed evidence of additional abrasion (incisal/occlusal surface) but only 70% of the saucer-shaped teeth. In most teeth, the edge was rounded. Tiny grooves parallel to the cemento-enamel junction (CEJ) were present in 11% of the wedge-shaped and in 39% of the saucer-shaped lesions. Seventy-nine percent wedge-shaped and 52% saucer-shaped lesions had some sort of apposition. Eighty-eight percent of all teeth had dead tracts, 62% of which were located directly next to the defect (in the lesion). In 48%, sclerotic dentin was present right next to the defect (in the lesion). Tertiary dentin was visible in 60%. Not a single fracture was detected. CONCLUSION: Different characteristics associated with each type of cervical lesion support the theory of different aetiology or at least of differing contributions from different factors that participate in the development of NCCLs. CLINICAL RELEVANCE: Only knowledge of the correct aetiology of NCCLs will allow the best treatment and prevention for such lesions.

[Diagnosis of diseases of the large salivary glands of the head by ultrasound, sialography and CT-sialography. A comparison of methods]. / Die Diagnostik der Erkrankungen der grossen Kopfspeicheldrüsen durch Sonographie, Sialographie und CT-Sialographie. Ein Methodenvergleich.

In the present study 162 patients with clinically suspected diseases of the major salivary glands were examined via sonography (n = 162), sialography (n = 111) and CT-sialography (n = 49). The reliability of the three radiological procedures was assessed in diagnosing sialoadenitis, sialolithiasis and glandular and extraglandular tumors. Forty-seven patients were examined with all three methods, 64 patients with sonography and sialography, 2 patients with sonography and CT-sialography and 49 patients with only sonography. The results were compared retrospectively with histologically (70%), cytologically (26%) and clinically proven diagnoses. A sialoadenitis was diagnosed via sonography at a sensitivity of 58%. Sialography frequently produced a false diagnosis of "glandular tumor", which resulted in a comparatively lower sensitivity of 54%. This finding contrasted with the experiences of other authors. A glandular tumor was correctly diagnosed by all three methods and had approximately the same sensitivity (sonography 89%, sialograph 91% and CT-sialography 92%). The correct diagnosis of salivary gland tumors was found by sonography and CT-sialography in 76% of cases and by sialography in 83% of cases. CT-sialography was clearly the superior diagnostic method for detecting extraglandular tumors. Sonography proved its worth as the fundamental procedure for special diagnostic testings of the salivary glands. Sialography is necessary for obtaining important additional information, especially in cases with suspect glandular tumors. CT-sialography is indispensable in the diagnosis of tumors, especially if a malignant, extraglandular or medially localized process is suspected.

Cigarette smoke elicits leukocyte adhesion to endothelium in hamsters: inhibition by CuZn-SOD.

Although cigarette smoking has been identified as a major risk factor for cardiovascular diseases, the underlying pathomechanism is largely unknown. Using a dorsal skinfold chamber model in Syrian golden hamsters for intravital microscopy on striated muscle microcirculation, we investigated whether cigarette smoke (CS) affects the adhesion of circulating leukocytes to the endothelium, a constant feature of early atherogenesis and a hallmark of ischemia-reperfusion injury. Awake hamsters were exposed for 5 min to the mainstream smoke of one cigarette (2R1 research cigarette), inducing nicotine, cotinine, and carboxyhemoglobin plasma levels comparable to levels found in human smokers. In control animals (n = 7), CS exposure elicited the rolling and subsequent adhesion of fluorescently stained leukocytes to the endothelium of arterioles and postcapillary venules. Leukocyte/endothelium interaction was preceded by an early rise in xanthine oxidase activity and intravascular hemolysis. Leukocyte adhesion and xanthine oxidase (XO) activation were significantly attenuated in hamsters pretreated with superoxide dismutase (5 mg/kg, 10 min prior to CS, n = 7), suggesting a key role of superoxide in this event. These in vivo results suggest a novel pathomechanism of CS-induced cardiovascular pathology.

Involvement of 5-lipoxygenase products in cigarette smoke-induced leukocyte/endothelium interaction in hamsters.

Although cigarette smoke (CS) has been identified as an independent risk factor for atherogenesis and sudden cardiac death, the underlying pathomechanism has not been clarified. A common factor of both atherogenesis and ischemia/reperfusion damage associated with myocardial infarction is the adhesion of circulating leukocytes to the vascular endothelium. Searching for the mechanism by which CS exerts its deleterious effects on the cardiovascular system, we used a dorsal skinfold chamber model in hamsters for intravital microscopy to examine the effect of CS on the interaction of fluorescently stained leukocytes with the microvascular endothelium in striated muscle. Exposure of awake animals (n = 7) for 5 minutes to the mainstream smoke of one cigarette (2R1 research cigarette) elicited rolling and subsequent adhesion of circulating leukocytes to the endothelium of both arterioles and postcapillary venules with a maximum 30 minutes after CS-exposure. In order to test a putative mediator role of the chemotactic and adhesion-promoting leukotrienes in this event, we pretreated another group of 7 animals with MK-886, a potent and specific inhibitor of leukotriene biosynthesis (20 mumol/kg body weight, iv, 30 minutes prior to CS exposure). While no inhibitory effect was seen on CS-induced leukocyte rolling along the microvascular endothelium. MK-886 pretreatment significantly attenuated leukocyte adhesion to arterioles (5.2 +/- 13.7 cells/mm2 vs. 54.1 +/- 54.8 in control animals. P < 0.01) and venules (37.0 +/- 33.6 cells/mm2 vs. 161.6 +/- 91.1 in control animals, P < 0.01), 30 minutes after CS exposure, suggesting a key mediator role of leukotrienes in this event. We propose that the stimulation of leukocyte/endothelium interaction by CS may provide the pathophysiologic basis for--or at least contribute to--its deleterious effects on cardiovascular mortality and morbidity. The identification of the mediator role of leukotrienes in this event may open the way to novel pharmacologic and dietary approaches for the prophylaxis of CS-induced cardiovascular pathology.

Acute pancreatitis: induced by heroin intoxication?

Hyperamylasemia has been reported in heroin addicts and ascribed to an increase of salivary isoamylase. Ours is the first report on acute pancreatitis in a heroin user. All prognostic parameters indicating a severe course of the disease were present, and computed tomography showed an edematous pancreatitis, but the acute pancreatitis took a benign clinical course.

Use of column switching for the determination of niacinamide in compound feed.

A high-performance liquid chromatographic method is described for the determination of niacinamide in compound feed. The niacinamide is extracted with 0.2 M hydrochloric acid in order to suppress the hydrolysis of the niacinamide to nicotinic acid. The chromatography is carried out with the aid of column switching. An RP-18 column is used for the preseparation, and the analytical separation takes place in a cation-exchange column. With the proposed method, the limit of determination is about 2 ppm of niacinamide.

Thymidine triphosphate synthesis in senescent WI38 cells. Relationship to loss of replicative capacity.

The effect of in vitro age on thymidine triphosphate (TTP) synthesis was assessed in WI38 cultures according to the following measurements: (1) thymidine kinase activity of broken cell preparations; (2) in situ incorporation of [3H]thymidine into acid-soluble material; and (3) total intracellular TTP content as determined by an enzymatic assay. All three parameters were maximal in exponentially proliferating populations and minimal in quiescent monolayers; no significant differences between young and old cultures were observed despite the reduced replicative capacity of the latter. The addition of serum to density-arrested cultures induced both TTP synthesis and DNA replication after a lag of approx. 12 h; although a greater percentage of young cells initiated replication as compared with old, pool sizes expanded to a similar extent in both populations. Pool expansion did not require entry into S phase; the pool sizes of control and cytosyl arabinoside-treated cultures were comparable. These findings suggest that senescent cells retain the ability to synthesize TTP, even though they are incapable of replicating DNA. Because TTP synthesis is a cell cycle-dependent event that normally begins in late G1, senescent cells might be blocked in the latter portion of the prereplicative phase and not in G0 as are quiescent cells.

Trypsinization frequency and loss of proliferative capacity in WI-38 cells.

The effect of the trypsinization procedure employed in routine serial subcultivation on the proliferative life span of WI-38 cells was studied by varying the frequency of trypsinization and by using a nonenzymatic subcultivation technique. The results indicate that recurrent trypsinization does not contribute to loss of proliferative capacity and may, in fact, delay this loss to some degree.

Hydrocortisone effects on cell proliferation: specificity of response among various cell types.

The effect of 14 micrometer hydrocortisone (HC) on the proliferative activity of various vertebrate cell lines has been measured. Such activity in human deploid cell line WI-38 was enhanced by HC addition whereas in a number of other vertebrate cell lines under identical conditions HC was inhibitory to growth. We examined the effects of HC on normal human diploid cells other than WI-38; some were stimulated, some inhibited and some were unresponsive. The results suggest that cells derived from different human tissues retain in vitro some aspects of their cell-specific hormonal responses.