Larynx morphology and sound production in three species of Testudinidae.

Although the ability to vocalize is widespread among tortoises, the mechanisms of sound production in chelonians remain undescribed. In this study, we analyze the morphology and histology of the larynx of three species of Testudinidae (Testudo hermanni, T. graeca, and T. marginata) in order to ascertain the presence of vibrating acoustic structure, and based on our findings we propose a general model for phonation in tortoises. The structure of the larynx of the three tortoises analyzed is simple: three cartilages (the cricoid and two arytenoids) form the skeleton of the larynx, while two pairs of muscles (the dilators and constrictors) control the widening and closing of the glottis. The larynx is supported in the oral cavity by the hyoid cartilage, which in tortoises assumes the same functions of the thyroid cartilage of mammals. Two bands of elastic fibers are inserted in the lateral walls of the larynx just upstream of the glottis, and can be stretched away from the hyoid by the movements of the arytenoids. Their position and structure suggest that these bands are capable of vibrating during exhalation, and therefore may be considered vocal cords. The cricoid of T. marginata and T. graeca hold two diverticula, not previously reported, which might function as a low-frequency resonating chamber, improving the harmonic structure of tortoise calls. The structure of the larynx is compared with that of other vertebrates and the relationships between morphology and phonation are discussed. This is the first detailed description of anatomical structures possibly devoted to vocalization in chelonians.

Expression of natriuretic peptides, nitric oxide synthase, and guanylate cyclase activity in frog mesonephros during the annual cycle.

Natriuretic peptides (NPs), a family of structurally related hormones and nitric oxide (NO), generated by nitric oxide synthase (NOS), are believed to be involved in the regulation of fluid balance and sodium homeostasis. Differential expression and regulation of these factors depend on both physiological and pathological conditions. Both NPs and NO act in target organs through the activation of guanylate cyclase (GC) and the generation of guanosine 3',5'-cyclic monophosphate (cGMP), which is considered a common messenger for the action of these factors. The present study was designed to investigate--by histochemical methods--the expression of some NPs (proANP and ANP) and isoforms of NOS (neuronal NOS, nNOS, and inducible NOS, iNOS) in the mesonephros of Rana esculenta in different periods of the year including hibernation, to evaluate possible seasonal changes in their expression. We also studied the enzyme activity of NOS-related nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) and of GC. The experiments were performed on pieces of kidney of R. esculenta collected in their natural environment during active and hibernating life. The study was carried out using immunohistochemical techniques to demonstrate proANP, ANP, and some NOS isoforms. Antigen capture by enzyme linked immunosorbent assay (ELISA) was also performed to determine the presence of NPs in the frog kidney extract. Enzyme histochemistry was used to demonstrate the NOS-related NADPHd activity at light microscopy; GC activity was visualized at the electron microscope, using cerium as capture agent. The application of the immunohistochemical techniques demonstrated that frog mesonephros tubules express different patterns of distribution and/or expression of ANP and NOS during the annual cycle. Comparing the results obtained on active and hibernating frogs has provided interesting data; the NOS/NADPHd and GC activities showed some variations as well. Furthermore, the presence of NPs in the frog kidney extract was evidenced by dose-dependent response in the ELISA. The data suggest that both ANP and NO are intra-renal paracrine and/or autocrine factors which may modulate the adaptations of frog renal functions to seasonal changes through the action of the cGMP generated from GC activity.

Stem cell recruitment and liver de-differentiation in MMTV-neu (ErbB-2) transgenic mice.

The liver of tumor-bearing hosts manifests fetal phenotypes. We investigated the expression of differentiation markers on the liver in MMTV-neu (ErbB-2) transgenic mice, in the period from incipient neoangiogenesis to lung metastatization. We report AFP expression by hepatocytes in all lobular zones, CD34 cell arrest and subsequent hemopoiesis in periportal and mid-zone areas, oval-like cells (CD34+, CK19+, AFP+) and ductular reaction in portal tracts, portal CK19+ and GGT+ hepatoblast-like cells, and midzonal large dysplastic hepatocytes. We hypothesize that CD34 cells are recruited by the tumor from the marrow for angiogenic purposes and that their differentiation in the liver is influenced by altered liver microenvironment(s). AFP may act as a growth factor and biological response modifier for these cells and for the tumor. Dysplasia might be enhanced by metabolic stress. We conclude that the liver differentiation potential is lobular-zone-dependent and that the risk for eventually developing a pre-malignant lesion is not negligible.

Quantification of the DHFR gene in blast cells of leukaemia patients by fluorescence in situ hybridisation.

The fluorescence signal intensity of the DHFR gene was analysed in lymphocytes from 15 normal donors, in MTX-resistant HeLa cells (carrying DHFR gene amplification) and in bone marrow blasts from 16 patients with acute leukaemia (AL) by in situ hybridisation. Our aim was to verify if DHFR gene amplification may be responsible for increased enzyme activity in leukemic cells. The results obtained with a fluorescence in situ hybridisation method were quantified using the Scion image software program and compared with cytochemical and cytophotometric data relating to DHFR activity. In AL a heterogeneous hybridisation pattern was generally observed at the single cell level. However, leukemic lymphoblasts showed higher fluorescence signal intensity of the DHFR gene as compared with normal lymphocytes, and leukemic myeloblasts a much higher signal than lymphoblasts. HeLa cells showed the highest fluorescence signal intensity. In all samples enzyme activity behaved in parallel. These results indicate that the increased expression of DHFR in leukemic blasts is due to a gene amplification. The high levels in AML can explain the MTX natural resistance.