Analysis of telomeric repeats and telomerase activity in human colon carcinoma cells with gene amplification

COLO320DM and COLO320HSR are cell lines derived from a human malignant neuroendocrine colon carcinoma. Both lines have a 30–40-fold amplification of a large DNA domain containing the MYC oncogene. By using fluorescence in situ hybridization techniques with a MYC probe, we could demonstrate that MYC amplicons are contained in a large marker chromosome in COLO320HSR cells, in double minutes (dmin) of COLO320DM cells, and in the interstitial regions of 3–4 additional chromosomes in both cell lines. Amplicons in homogeneous staining regions (HSRs) comprise normal MYC genes, while dmin chromosomes contain PVT/MYC chimeras. Although both cell lines showed similar levels of telomerase activity, the telomere length and telomere distribution in chromosomal termini were considerably lower in COLO320DM than in COLO320HSR cells. This indicates that the average telomere length in cancer cells is regulated no only by the rates of telomerase activity but also by some other non-enzymatic mechanisms.     

Differential diagnosis of Taenia saginata and Taenia solium infection by PCR

We have designed species-specific oligonucleotides which permit the differential detection of two species of cestodes, Taenia saginata and Taenia solium. The oligonucleotides contain sequences established for two previously reported, noncoding DNA fragments cloned from a genomic library of T. saginata. The first, which is T. saginata specific (fragment HDP1), is a repetitive sequence with a 53-bp monomeric unit repeated 24 times in direct tandem along the 1, 272-bp fragment. From this sequence the two oligonucleotides that were selected (oligonucleotides PTs4F1 and PTs4R1) specifically amplified genomic DNA (gDNA) from T. saginata but not T. solium or other related cestodes and had a sensitivity down to 10 pg of T. saginata gDNA. The second DNA fragment (fragment HDP2; 3,954 bp) hybridized to both T. saginata and T. solium DNAs and was not a repetitive sequence. Three oligonucleotides (oligonucleotides PTs7S35F1, PTs7S35F2, and PTs7S35R1) designed from the sequence of HDP2 allowed the differential amplification of gDNAs from T. saginata, T. solium, and Echinococcus granulosus in a multiplex PCR, which exhibits a sensitivity of 10 pg.     

Histamine liberation and membrane fluidisation of mast cells exposed to the beta-adrenoceptor blocking drug propranolol

Propranolol liberates histamine from isolated mast cells and decreases the uptake of extracellular histamine in a dose-dependent way. Histamine liberation due to propranolol is accompanied by calcium displacement from intracellular storage sites. The significant increase in membrane fluidity due to propranolol is temperature dependent. The perturbation of membranes is most probably the explanation of propranolol's interaction with isolated rat mast cells which results in altered histamine transportation.     

Lectins in the unicellular Tetrahymena. II. Impact of nutrition and sugar treatment on anti-lectin binding

Tetrahymena pyriformis GL cells showed partly intracytoplasmic, partly intramembraneous accumulation of lectin-like proteins in conditions of starvation, as demonstrated cytochemically by reaction with antibodies to pea, lens, bean, Datura, and snail lectin. The lectins binding to simple sugars tended to accumulate in the membrane, whereas those capable of interacting with hexosamines in the cytoplasm. While the fluorescence pattern of lectin localization was generally homogeneous in normally nourished cells, it assumed a variegated appearance in starved cells, owing to patching of the membrane, and spot-like accumulation of lectins in the cytoplasm, presumably within membranes of endocytosed vesicles. Snail lectin was an exception, since its distribution remained homogeneous also in conditions of starvation.     

Different patterns of the L-histidine decarboxylase (HDC) gene expression in mice resistant and susceptible to experimental cutaneous leishmaniasis

Objective and design: In the present study the experimental murine Leishmania major (L. major) infection model was used to investigate the role of histamine biosynthesis in cutaneous leishmaniasis.Subjects, treatment and methods: A novel RNase Protection Assay (RPA) was developed and applied for the assessment of L-histidine decarboxylase (HDC) gene expression in organs of resistant C57BL/6 and susceptible BALB/c mice after infection with L. major.Results: In the acute phase of infection a rapid but transient induction of HDC expression was observed in the infected lymph nodes of both strains correlating both temporally and spatially with parasite spread. The signal was present in the draining popliteal lymph nodes of both hosts, however, only susceptible mice known to be unable to control parasite dissemination showed induction of HDC in their distant periaortic lymph nodes as well. During the chronic phase of infection only the heavily parasitized organs of BALB/c mice showed high HDC gene expression.Conclusions: These data suggest that expression of the histamine-producing enzyme HDC in the decisive acute phase of leishmaniasis is not coupled with development of either appropriate Th1 or inadequate Th2 responses to L. major. We hypothesize however, that during the chronic phase of infection elevated HDC levels, possibly of mast cell origin, are associated with Th2-dominated responses and serious disease development.Received 12 May 2003; returned for revision 3 July 2003; returned for final revision 11 September 2003; accepted by M. Parnham 24 September 2003     

Induction of micronucleated cells in the shed skin of salamanders (Ambystoma sp.) treated with colchicine or cyclophosphamide

The micronucleus (MN) assay can be used to detect the genotoxic effects of chemical agents in virtually any cell that divides frequently. Salamanders (Ambystoma sp.) are amphibians that can be easily maintained and bred in the laboratory and spontaneously shed their skin every 2.5-4 days. In this present study, we have evaluated the usefulness of this shed skin for the MN assay. We exposed salamanders to different concentrations of both the aneugen colchicine (COL) and the clastogen cyclophosphamide (CP) and we determined the frequency of micronucleated cells (MNCs) in their sheds. Fragments of shed skin were placed on clean slides, fixed, stained, observed with a light microscope, and the number of MNCs was counted. The MNC frequency was increased significantly by all doses of COL and CP tested, administered either as single or repeated exposures. The presence of MNCs in the shed skin and the speed of sloughing lead us to propose that the sheds of Ambystoma sp., or other amphibians that slough their skin, are suitable alternative models for detecting genotoxic exposures relevant to aquatic environments.     

Development of the human elbow joint

Many studies have been published on the development of the human elbow joint, but authors disagree on its morphogenetic timetable. Most discrepancies center on the cavitation of the elbow joint (including the humeroradial, humeroulnar, and superior radioulnar joints), and the organization of the tunnel of the ulnar nerve. We summarize our observations on the development of the elbow joint in 49 serially sectioned human embryonic (n = 28) and fetal (n = 21) upper limbs. During week 12, ossification begins in the epiphyses of the elements comprising the elbow joint. At the end of the embryonic period, the shallow groove between the posterior aspect of the medial epicondyle and the olecranon process, begins to be visible. The elbow joint cavity appears in O'Rahilly stage 21 (51 days) at the level of the humeroulnar and humeroradial interzones. Formation of the cavity begins at the medialmost portion of the humeroradial interzone and the lateralmost portion of the humeroulnar interzone. The annular ligament begins to develop in O'Rahilly stage 21 (51 days), and the superior radioulnar joint cavity appears between this ligament and the lateral aspect of the head of the radius during O'Rahilly stage 23 (56 days). We established the morphogenetic timetable of the human elbow joint.     

Topographic studies on medial reticular nucleus.

Several distinct classes of neurons have been identified in the medial reticular nucleus of the medulla and pons and in proximity thereto. Neurons projecting down the spinal cord comprised the principal class with two subclasses according as the neurons did or did not receive monosynaptic inputs from the fastigial nuclei of the cerebellum. Two other classes were recognized accordings as they projected to the cerebellum or rostrally to the mesencephalon. Topographic planar maps giving the location of these neurons have been constructed by exploring the nucleus with series of microelectrode tracks in parasagittal or in transverse planes. The different classes of neurons were not arranged in large discrete nuclei. In part they appeared to be randomly distributed, but many colonies of one or another class of neurons could be recognized with 3-11 neurons in zones with dimensions of a millimeter or so. Because of the limitations of sampling by microelectrode tracks at spacings of 0.5 mm, single colonies might have an actual population of 100 or more. Many of the class of neurons projecting to the cerebellum were in the region of the perihypoglossal nucleus. However, almost as many were located deep in the medial reticular nucleus. None was found at the pontine level. Reticulospinal neurons with fast axonal conduction velocities tended to be located dorsally to those with slow velocities. Correlation with the findings of Ito et al. leads to the conjecture that the neurons with fast axons are excitatory, while those with slow axons are primary inhibitory neurons. There is a brief reference to the problems raised by the admixture of the various neuronal classes, there being discrete colonies immersed in a scattered arrangement of all classes.