Second harmonic imaging of intrinsic signals in muscle fibers in situ

We use second harmonic generation (SHG) imaging to study and quantify a strong intrinsic SHG signal in skeletal muscle fiber preparations and single isolated myofibrils. The intrinsic signal follows the striation pattern of the muscle cells and is positioned at the sarcomeric location of the myosin filaments. Interestingly, the signal is enhanced at the region where the myosin heads are located on the myosin filaments. As the intrinsic signal reflects the subcellular structure in an accurate way, SHG can be used for noninvasive high resolution structural imaging without exogenous labels in living muscle cells. This may be very important for detecting changes in myofibrillar organization occurring under pathophysiological conditions, e.g., in cardiac and skeletal myopathies. Due to the strong dependency of SHG on orientation and symmetries of the tissue, it may allow the study of dynamic interactions between the contractile proteins actin and myosin during force production and muscle shortening. Furthermore, SHG imaging can be combined with other nonlinear microscopical techniques, such as laser scanning multiphoton fluorescence microscopy, to simultaneously measure other dynamic cellular processes, representing a complementary method and extending the range of nonlinear microscopical methods.     

Possible Relation of p53 and mdm-2 Oncoprotein Expression in Thyroid Carcinoma: A Molecular-Pathological and Immunohistochemical Study on Paraffin-Embedded Tissue

Routinely processed tissues from a series of benign and malignant thyroid lesions were immunohistochemically investigated with antibodies against p53 and mdm-2. p53 was immunolocalized in <10% of nuclei in 2/80 nodular goiters, 2/60 follicular adenomas, 26/68 follicular carcinomas, 7/40 papillary carcinomas, 3/10 “insular” carcinomas, and 10/31 anaplastic carcinomas. More than 10% positively stained nuclei were found in 2 widely invasive follicular, 2 insular, and 15 anaplastic carcinomas. All p53-positive cases showed a concomitant immunohistochemical mdm-2 expression; an immunohistochemical colocalization on serial section was demonstrated in 12 anaplastic carcinomas. Screening by polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis of these 12 cases revealed no relevant mutations in the coding regions of exons 2–11 of the p53 gene. Additionally, 1 follicular adenoma, 6 follicular carcinomas (4 minimally and 2 widely invasive), 1 papillary, and 2 poorly differentiated insular carcinomas were mdm-2 positive without immunohistochemically detectable p53 expression. These results provide evidence that wild-type p53 expression in thyroid carcinomas may be associated with mdm-2 induced formation of stable complexes. However, the role of p53 mutations and p53 protein inactivation owing to other factors (e.g., mdm-2) in the progression of thyroid carcinomas is still poorly understood.     

Potentiation of CCNU activity by misonidazole in metastases

The KHT sarcoma is a model system in which metastases can be studied in multiple organs without prior clonal selection. The present series of experiments were designed to evaluate and compare the extent of potentiation of chemotherapeutic agent activity by radiosensitizers when KHT sarcoma cells were grown in different anatomical sites. In these studies the effect of combining misonidazole (MISO) and 1-(2-chloroethyl)-3-cyclohexyl-l-nitrosourea (CCNU) on KHT lung nodules and ovarian metastases was determined. Ovarian metastases were a consequence of the direct spread of tumor cells growing as lung nodules. Once established, KHT cells in the lungs and ovaries grew with a similar doubling time (1–2 days). Response of tumors at either site to chemotherapy in the presence or absence of a sensitizer was assessed using an in vivo to in vitro excision assay. MISO (1·0 mmol/kg) was administered simultaneously with a range of CCNU doses and survival of clonogenic tumor cells was measured 24 h after treatment. The results demonstrate that the addition of MISO to CCNU treatment potentiated the action of this chemotherapeutic agent at both tumor sites although greater cell kill enhancement occurred in the ovarian metastases. In the lung nodules, when combined with CCNU, a 1·0 mmol/kg dose of MISO was found to yield a dose modifying factor (DMF) of 1·3. The same combination resulted in a DMF of 1·8 in the ovarian metastases. This difference in DMF was not a result of an intrinsic difference in sensitivity to CCNU since cells grown at either site gave rise to the same dose response curve. Rather the difference in dose modification by MISO appears to be a consequence of the larger fraction of radiobiologically hypoxic cells in the ovarian tumors (50 per cent) than in the lung nodules (5 per cent) at the time of treatment. These findings suggest the use of drug-sensitizer combinations to treat disseminated disease and provide further evidence for the requirement of hypoxia in chemopotentiation by radiosensitizers.     

Cell-matrix interactions during development and apoptosis of the mouse mammary gland in vivo.

Epithelial cell survival is dependent on extracellular signals provided by both soluble factors and by adhesion. In the mammary gland, extensive apoptosis of epithelial cells occurs rapidly when lactation ceases, but the mechanism of apoptosis induction is not known. In tissue culture, mammary epithelial cells require laminin as a survival ligand and specific beta1 integrins are necessary to suppress apoptosis. To explore the possibility that dynamic changes in cell-matrix interactions contribute to the onset of apoptosis during mammary involution in vivo, a detailed immunohistochemical analysis of the expression of integrin subunits and their extracellular matrix ligands during mouse mammary gland development has been performed. The kinetics of apoptosis were determined by using tissue samples obtained from virgin, pregnant, lactating, and involuting gland. The maximal elevation of apoptosis occurred within 24 hr of weaning as determined by histologic analysis and caspase-3 staining. A wide variety of laminin subunits, together with nidogen-1 and -2, and perlecan were identified within the basement membrane region of epithelial ducts, lobules, and alveoli in both human and mouse mammary gland. However, no change in the distribution of any of the basement membrane proteins or their cognate integrin receptors was observed during the transition from lactation to apoptosis. Instead, we discovered that altered ligand-binding conformation of the beta1 integrin to a nonbinding state coincided with the immediate onset of mammary apoptosis. This finding may provide a novel dynamic mechanism for inhibiting the transduction of extracellular matrix survival signals, thereby contributing to the onset of apoptosis in a developmental context in vivo.     

Noise-induced cell death in the mouse medial geniculate body and primary auditory cortex

Noise-induced effects within the inner ear have been well investigated for several years. However, this peripheral damage cannot fully explain the audiological symptoms in noise-induced hearing loss (NIHL), e.g. tinnitus, recruitment, reduced speech intelligibility, hyperacusis. There are few reports on central noise effects. Noise can induce an apoptosis of neuronal tissue within the lower auditory pathway. Higher auditory structures (e.g. medial geniculate body, auditory cortex) are characterized by metabolic changes after noise exposure. However, little is known about the microstructural changes of the higher auditory pathway after noise exposure. The present paper was therefore aimed at investigating the cell density in the medial geniculate body (MGB) and the primary auditory cortex (AI) after noise exposure. Normal hearing mice were exposed to noise (10 kHz center frequency at 115 dB SPL for 3 h) at the age of 21 days under anesthesia (Ketamin/Rompun, 10:1). After 1 week, auditory brainstem response recordings (ABR) were performed in noise exposed and normal hearing animals. After fixation, the brain was microdissected and stained (Kluever-Barrera). The cell density in the MGB subdivisions and the AI were determined by counting the cells within a grid. Noise-exposed animals showed a significant ABR threshold shift over the whole frequency range. Cell density was significantly reduced in all subdivisions of the MGB and in layers IV-VI of AI. The present findings demonstrate a significant noise-induced change of the neuronal cytoarchitecture in central key areas of auditory processing. These changes could contribute to the complex psychoacoustic symptoms after NIHL.     

P-selectin glycoprotein ligand-1 mediates rolling of mouse bone marrow-derived mast cells on P-selectin but not efficiently on E-selectin

It has been shown recently that mast cells play an essential role as a source of tumor necrosis factor-α production during neutrophil recruitment to sites of bacterial infection. Increased numbers of mast cells are indeed noted at sites of wound healing and inflammation. These cells are either recruited from the bone marrow or proliferate locally under cytokine stimulation. Little is known about how mast cell progenitors extravasate into tissue. Using antibody-like fusion proteins of mouse E-selectin and P-selectin, we have analyzed the ability of immature mouse bone marrow-derived mast cells (BMMC) to interact with the endothelial selectins. The P-selectin glycoprotein ligand-1 (PSGL-1) was affinity-isolated from detergent extracts of surface biotinylated BMMC with both selectin-IgG fusion proteins. However, only P-selectin-IgG, but not E-selectin-IgG showed significant interaction with intact BMMC as tested by flow cytometry and cell attachment assays with the immobilized fusion proteins under flow and non-flow conditions at physiological shear stress. Thus, in spite of carrying the necessary carbohydrate modifications which enable solubilized PSGL-1 to bind avidly to E-selectin, PSGL-1 on the surface of BMMC is presented in a way that prevents it from interacting efficiently with E-selectin. Affinity-purified rabbit antibodies against mouse PSGL-1 almost completely blocked the interaction of BMMC with P-selectin-IgG in flow cytometry as well as in cell adhesion assays under static and under flow conditions. Our data reveal that PSGL-1 is the major binding site for P-selectin on mouse BMMC progenitors, but does not support efficient interactions with E-selectin.     

Serum neopterin for early assessment of severity of severe acute respiratory syndrome

Neopterin and C-reactive protein (CRP) concentrations were determined in serum samples from 129 severe acute respiratory syndrome (SARS) patients and 156 healthy blood donors. In the patients with confirmed SARS, an early neopterin elevation was detected already at the day of onset of symptoms and rose to a maximum level of 45.0 nmol/L 3 days after the onset. All SARS patients had elevated neopterin concentrations (>10 nmol/L) within 9 days after the onset. The mean neopterin concentrations were 34.2 nmol/L in acute sera of SARS patients, 5.1 nmol/L in convalescent sera, and 6.7 nmol/L in healthy controls. In contrast, the mean CRP concentrations in both acute and convalescent sera of SARS patients were in the normal range (<10 mg/L). Serum neopterin level in SARS patients was associated with fever period and thus the clinical progression of the disease, while there was no significant correlation between the CRP level and the fever period. Serum neopterin may allow early assessment of the severity of SARS. The decrease of neopterin level was found after steroid treatment, which indicates that blood samples should be collected before steroid treatment for the neopterin measurement.     

Retinoblastoma: revisiting the model prototype of inherited cancer

Hereditary retinoblastoma is an autosomal dominant disorder caused by mutations in the RB1 gene. Analysis of this rare condition has helped to elucidate the mechanisms underlying hereditary cancer predisposition in general. As identification of RB1 gene mutations has become a part of clinical management of patients with retinoblastoma, there is now a wealth of data. In this article, we summarize the current knowledge on the relations between the genotype and phenotypic expression. Moreover, detailed analysis of genotype-phenotype relations shows that hereditary retinoblastoma has features of a complex trait.     

Glomerular capillary morphology in normal humans

Knowledge of quantitative glomerular structure in normal man is limited. At the time of renal transplantation, 28 living-related normal kidney donors had renal biopsies performed. Tissue was processed for light and electron microscopy using standard techniques. Standard stereologic techniques were used to determine mean glomerular volume, capillary length density, fractional volumes of mesangium and capillary lumen, surface densities of the peripheral capillary, mesangial-epithelium interface, and the lumenal-mesangial interface. Volume of total mesangium/glomerulus, capillary lumenal volume/glomerulus, peripheral capillary filtration surface/glomerulus, capillary mesangial-epithelial surface/glomerulus, capillary lumenal-mesangial surface/glomerulus, capillary length/glomerulus, and average capillary diameter were calculated. There were no significant differences between males and females in any structural parameter. There were no significant correlations between age or body surface area and any structural parameter. In comparison with these normal values, previously reported diabetic patients with clinical nephropathy demonstrate markedly expanded mesangial volume/glomerulus and a diminished filtration surface/glomerulus. The availability of these normal values will allow comparison with functional data in normal humans and greater understanding of human glomerular disease.