Binding of an Indenoisoquinoline to the Topoisomerase-DNA Complex Induces Reduction of Linker Mobility and Strengthening of Protein-DNA Interaction

Long-duration comparative molecular dynamics simulations of the DNA-topoisomerase binary and DNA-topoisomerase-indenoisoquinoline ternary complexes have been carried out. The analyses demonstrated the role of the drug in conformationally stabilizing the protein-DNA interaction. In detail, the protein lips, clamping the DNA substrate, interact more tightly in the ternary complex than in the binary one. The drug also reduces the conformational space sampled by the protein linker domain through an increased interaction with the helix bundle proximal to the active site. A similar alteration of linker domain dynamics has been observed in a precedent work for topotecan but the molecular mechanisms were different if compared to those described in this work. Finally, the indenoisoquinoline keeps Lys532 far from the DNA, making it unable to participate in the religation reaction, indicating that both short- and long-range interactions contribute to the drug poisoning effect.     

Immunohistochemical and molecular analysis of caveolin-1 expression in canine mammary tumors

Caveolin-1 (Cav-1) is a structural protein present in invaginations of the cell membrane. In human breast cancer, the cav-1 gene is believed to be a tumor suppressor gene associated with inhibition of tumor metastasis. However, little is known about its expression, regulation and function in canine mammary tumors. Expression levels of cav-1 were investigated using real-time PCR and immunohistochemical detection with an anti-human Cav-1 antibody. Gene expression stability of different samples was analyzed using the geNorm software. Mammary tumors from 51 female dogs were compared to normal mammary tissue from 10 female dogs. Malignant mammary cells showed a loss of Cav-1 expression by quantitative RT-PCR and weak Cav-1 staining by immunohistochemistry compared to normal mammary gland tissue. There was a significant relationship between outcome and immunostaining as well as with tumor size, indicating that caveolin subexpression has a positive predictive value and is related to higher survival and smaller tumor size. Our findings indicate that Cav-1 is a potential prognostic marker for canine mammary tumors.     

The short spermatodesm of Arge pagana (Hymenoptera: symphyta)

In the seminal vesicle of the 'symphyta'Arge pagana the spermatozoa are stored in motile spermatodesm bundles, maintained by an anterior cap of extracellular material. This cap consists of a denser cortex and of an internal matrix, where part of the sperm heads are embedded. The number of spermatozoa per bundle is variable. The spermatozoa are short, only 30microm long, with a head region of about 23microm, and a very short flagellum of about 7microm. The head includes the acrosome, with a perforatorium, and the nucleus. The flagellum consists of an axoneme, with a 9+9+2 microtubule pattern, a centriolar adjunct, two mitochondrial derivatives and two accessory bodies. The mitochondrial derivatives are very slender and of different lengths. The longer begins at the base of the nucleus, while the shorter one starts just below the base of the centriolar adjunct. This latter is asymmetric and appears at the nuclear base, extending parallel to the axoneme up to the anterior end of the smaller mitochondrial derivative. The short spermatodesmata and the small mitochondrial derivatives characterize the A. pagana sperm. In addition, the centriolar adjunct asymmetry and the occurrence of spermatodesm bundles might be considered plesiomorphic states present in the basal Tenthredinoidea.     

Anti-HIV-1 response elicited in rabbits by anti-idiotype monoclonal antibodies mimicking the CD4-binding site

Antibodies against conserved epitopes on HIV-1 envelope glycoproteins (Env), such as the gp120 CD4-binding site (CD4bs), could contribute to protection against HIV-1. Env-based immunogens inducing such a response could be a major component of future anti-HIV-1 strategies. In this proof-of-concept study we describe the generation of two anti-idiotype (AI) murine antibodies mimicking the CD4bs epitope. Sera were collected from long-term non-progressor patients to obtain CD4bs-directed IgG, through sequential purification steps. The purified IgG were then used as Fab fragments to immunize mice for hybridoma generation. Two hybridomas (P1 and P2), reacting only against the CD4bs-directed IgG, were identified and characterized. The P1 and P2 antibodies were shown to recognize the idiotype of the broadly neutralizing anti-CD4bs human mAb b12. Both P1 and P2 Fabs were able to induce a strong anti-gp120 response in rabbits. Moreover, the rabbits' sera were shown to neutralize two sensitive tier 1 strains of HIV-1 in an Env-pseudotype neutralization assay. In particular, 3/5 rabbits in the P1 group and 1/5 in the P2 group showed greater than 80% neutralizing activity against the HXB2 pseudovirus. Two rabbits also neutralized the pseudovirus HIV-MN. Overall, these data describe the first anti-idiotypic vaccine approach performed to generate antibodies to the CD4bs of the HIV-1 gp120. Although future studies will be necessary to improve strength and breadth of the elicited neutralizing response, this proof-of-concept study documents that immunogens designed on the idiotype of broadly neutralizing Abs are feasible and could help in the design of future anti-HIV strategies.     

A closer look at prion strains

Prions are infectious proteins that are responsible for transmissible spongiform encephalopathies (TSEs) and consist primarily of scrapie prion protein (PrP^Sc), a pathogenic isoform of the host-encoded cellular prion protein (PrP^C). The absence of nucleic acids as essential components of the infectious prions is the most striking feature associated to these diseases. Additionally, different prion strains have been isolated from animal diseases despite the lack of DNA or RNA molecules. Mounting evidence suggests that prion-strain-specific features segregate with different PrP^Sc conformational and aggregation states.

Strains are of practical relevance in prion diseases as they can drastically differ in many aspects, such as incubation period, PrP^Sc biochemical profile (e.g., electrophoretic mobility and glycoform ratio) and distribution of brain lesions. Importantly, such different features are maintained after inoculation of a prion strain into genetically identical hosts and are relatively stable across serial passages.

This review focuses on the characterization of prion strains and on the wide range of important implications that the study of prion strains involves.     

MCS/SEL/BAS program--an overlapping clustering method with examples from mating type interactions of ciliated protozoa

The MCS/SEL/BAS program provides a method for group recognition,based on a criterion of homegeneity within the groups. The basicaim of this clustering method is not to ‘force’data into a number of separate groups, as it allows the possibilitythat a given element in the data set can be assigned to morethan one group. Moreover, a parsimonious path through the groupsis sought by selecting groups on the basis of two suitably chosen,peak-ordered criteria. This selection continues until a coveringof the data set is obtained (i. e., until each element in thedata set is assigned to at least one group). Then relationshipsoccurring among the set of selected groups are investigatedby means of two coefficients, called overlapping and cohesioncoefficient, respectively. The utility of this program has beendemonstrated here in elaborating large sets of data derivedfrom mating type interactions of ciliates, but it can be usedalso for analyzing data derived from a wide spectrum of compatibilityphenomena exhibited by other living organisms. Algorithms ofthis program are written in BASIC and formulated in a conversationalmode for processing on a Macintosh. A computer program (MCS/SEL/BAS)is available from G.Mancini upon request. Received on September 18, 1990; accepted on January 21, 1991