Substance P analogues function as bombesin receptor antagonists and inhibit small cell lung cancer clonal growth

Human small cell lung cancer (SCLC) produces and secretes BN/GRP (bombesin/gastrin releasing peptide). Because BN stimulates the growth of SCLC cells and these cells have receptors for BN-like peptides, it is important to define agents which disrupt this self-promoting autocrine growth cycle. Here, substance P analogues were evaluated as BN receptor antagonists using SCLC cell lines. (D-Arg¹, D-Pro², D-Trp^7,9, Leu¹¹) substance P [(APTTL)SP] was one of the more potent analogues tested in inhibiting BN-like peptide receptor binding with an IC₅₀ value of 1 μM. Micromolar concentrations of (APTTL)SP antagonized BN receptor mediated elevation of cytosolic Ca²⁺ levels and decreased the colony formation in soft agarose. These data suggest that SP analogues function as SCLC BN receptor antagonists and may be useful in disrupting the autocrine growth function of BN-like peptides.     

Immunotoxins containing recombinant anti-CTLA-4 single-chain fragment variable antibodies and saporin: in vitro results and in vivo effects in an acute rejection model

Immunotoxins containing recombinant human-derived single-chain fragment variable (scFv) reagents (83 and 40) against CTLA-4 (CD152) linked to saporin, a ribosome-inactivating protein, were prepared and tested on CD3/CD28-activated T lymphocytes, MLRs, CTLA-4-positive cell lines, and hemopoietic precursors. Immunotoxins induced apoptosis in activated T lymphocytes and were able to specifically inhibit MLR between T lymphocytes and dendritic cells. The 83-saporin immunotoxin also inhibited the T cell activation in an MLR between T lymphocytes and an EBV-positive lymphoblastoid B cell line. Toxicity tests on hemopoietic precursors showed little or no effects in inhibiting colonies' growth. As the 83 scFv Ab was reactive also with activated mouse T lymphocytes, 83-saporin was tested in a model of tumor rejection consisting of C57BL/6 mice bearing a murine H.end endothelioma cell line, derived from DBA/2 mice. The lymphoid infiltration due to the presence of the tumor was reduced to a high extent, demonstrating that the immunotoxin was actually available and active in vivo. Thus, taking the results altogether, this study might represent a new breakthrough for immunotherapy, showing the possibility of targeting CTLA-4 to kill activated T cells, using conjugates containing scFv Abs and type 1 ribosome-inactivating protein.     

Tick-borne diseases in ruminants of Central and Southern Italy: epidemiology and case reports

Sera and blood from cattle and sheep were examined for the presence of Babesia and Theileria spp by microscopy and serology at the Parasitology Department of the Istituto Zooprofilattico Sperimentale of Abruzzo and Molise (IZSAM). Of the 47 bovine herds (323 animals) tested, 15 were found positive for Babesia bigemina and 1 for Babesia bovis. Two outbreaks occurred, one caused by B. bigemina and one by B. bovis. The B. bigemina outbreak occurred in Abruzzo and has been followed for two years. The isolate of B. bigemina was very pathogenic leading to the death of two cows out of 57. The vector responsible of the transmission appeared to be Rhipicephalus bursa. Parasites were observed in the erythrocytes for 30 days whereas sera were positive to indirect immunofluorescence (IFA) for at least one year. The B. bovis outbreak occurred in the province of Mantova (Northern Italy) in a group of 70 beef cattle imported from France. The infection resulted in the death of 5 animals and severe illness in another 6. In contrast with what occurred for Babesia infection, no clinical cases were recorded in cattle when species of Theileria were detected by microscopy. Of the 24 bovine herds (252 animals) tested for Theileria, 21 were found positive for the T. "sergenti"/buffeli/orientalis group. Single and mixed infection of T. "sergenti" and T. buffeli/orientalis were detected in herds of cross-bred cattle from Abruzzo and Marche. The parasites were identified by using a polymerase chain reaction which amplified DNA encoding p32/34. Most of the collected ticks (90%) were adults of R. bursa whereas the others were adults of Hyalomma detritum. During the period the animals have been observed (18 months), no clinical cases have been recorded and no associations have been found between blood abnormalities and animals found infected with Theileria. Prevalences of subclinically infected carriers increased from February till December (95.4%) even if the animals were indoors and no ticks were present. The prevalence then dropped dramatically six months later (76.7%). In calves less than 1 year old, the prevalence of infection significantly (p<0.05) increased with age, however intraerythrocytic stages of Theileria were found in the blood of three newborn calves (<7 days of age). Of the 18 ovine flocks tested for Babesia spp. (150 animals examined), 1 was positive for B. ovis and 2 for B. motasi. B. motasi infection was not associated with symptoms, while an outbreak of babesiosis caused by B. ovis occurred in Abruzzo. The infection resulted in the death of 3 animals (0.75% of the flock), two rams (20% of the total number) and a ewe, and severe illness in another 5 ewes (2% of the flock). Specimens of R. bursa and R. turanicus were collected from the infected animals. Of the 18 flocks (150 animals) examined, 12 were microscopically positive for Theileria spp. No clinical cases were recorded and identification at species level was not possible on the basis of morphological criteria. The prevalence distribution of infected herds and infected animals within herds and flocks have been calculated by a Monte Carlo simulation model, running 10,000 iterations. The most likely levels of prevalence of infected herds and infected animals within herds found for the species observed were as follows: 20% for B. bigemina with a prevalence within herd of 27%, 11% for B. bovis (18% within herd), 10% for Babesia ovis (19% within herd), 10% for B. motasi (17.5% within herd), 63% for Theileria in cattle (66% within herd) and 51% for Theileria in sheep (55% within herd).     

Stereospecific synthesis of "para-hydroxymexiletine" and sodium channel blocking activity evaluation

Both enantiomers of "para-hydroxymexiletine" (PHM), one of the main metabolites of mexiletine, were synthesized and fully characterized. Properties of (R)- and (S)-PHM, in terms of blocking potency and stereoselectivity on frog skeletal muscle Na(+) channels, were evaluated. The presence of a hydroxy group on the aryloxy moiety in the 4-position, as in PHM, reduced potency with respect to mexiletine in reducing I(Na max). However, PHM showed clear use-dependent behavior similar to that of mexiletine and, in contrast with what is observed with the parent compound, maintained its stereoselectivity during the use-dependent block. Chirality 16:72-78, 2004.     

Boar spermatozoa encapsulated in barium alginate membranes: a microdensitometric evaluation of some enzymatic activities during storage at 18 degrees C

The customary dilution of boar semen for subsequent artificial insemination (AI) procedures damages the cell membrane of spermatozoa, resulting in a loss of enzymes and other cytoplasmic contents and acrosomal reactions. We encapsulated non-diluted boar semen in barium alginate membranes to optimize AI procedures and to improve the functional integrity of spermatozoal membranes during storage. The percentage of non-reacted acrosomes (NRA) and measurements of enzyme leakage (cytochrome c oxidase (COX), lactate dehydrogenase (LDH), and glucose-6-phosphate dehydrogenase (G6PDH)) were used as indices of the functional status of diluted, unencapsulated and encapsulated spermatozoa, stored for 72 h at 18 degrees C. Enzymatic activity was assessed in situ by microdensitometry, and non-reacted acrosomes were microscopically determined by staining. The percentage of acrosome integrity and the intracellular enzymatic activities during storage were different for unencapsulated and encapsulated semen. Semen dilution caused a rapid decline in enzymatic activities and concomitant acrosomal reactions. Encapsulated spermatozoa had significantly higher acrosome integrity (77% versus 55%; P < 0.01 after 72 h) and an overall higher in situ enzymatic activity. For cytochrome c oxidase and lactate dehydrogenase the greatest differences between encapsulated and unencapsulated spermatozoa were present after 72 h whereas for glucose-6-phosphate dehydrogenase significant differences were found within 24h of storage. The encapsulation process maintains a better preservation environment for boar spermatozoa and could be a promising, innovative technique to improve storage of these cells.     

Molecular basis of bitter taste

The sense of taste responds to a large variety of stimuli through specific transduction mechanisms. The molecular events in the perception of bitter taste are believed to start with the binding of specific water-soluble molecules to G-protein-coupled receptors encoded by the type 2 family of taste receptor genes and expressed at the surface of taste receptor cells. Recent advances in the identification and cloning of the complete repertoire of genes of this family in humans and rodents provide an opportunity to address unresolved questions in bitter taste. The functional characterization of some of the receptors that these genes encode suggests that it will be possible to understand more precisely their specific functions.