Reinforcement of bio-apatite by zinc substitution in the incisor tooth of a prawn.

Various material-strengthening strategies have evolved in the cuticle and the feeding tools of arthropods. Of particular interest is the crustacean mandible, which is frequently reinforced with calcium phosphate, giving a minerology similar to that of human bones and teeth. We report here a biological strengthening method of apatite by Zn substitution, found in the incisor teeth of the freshwater prawn Macrobrachium rosenbergii. Nanoindentation measurements show a clear positive correlation between the Zn/Ca ratio and the stiffness and hardness of the composite. In the incisor, Zn-substituted apatite forms an internal vertical axis, extending from the sharp outer edges of the tooth to its basal segment. The substitution level in this zone (up to 40%) is very high compared with the levels achieved in synthetic ceramics (<20%). Finite element simulation suggests that the high-Zn axis acts as a unique internal load transfer element, directing stress from the biting cusps to the more compliant underlying layers. In light of the considerable research being invested in developing synthetic calcium phosphate derivatives for human bone and tooth grafts, the innovative mineralogy of the M. rosenbergii incisor may inspire beneficial biomimetic applications. STATEMENT OF SIGNIFICANCE: The controlled incorporation of impurities into biominerals is a widespread phenomenon in biomineralization that may pave the way to new classes of biomimetic materials. The present study reveals a biogenic mineral of zinc-substituted apatite found in the incisor teeth of a prawn. A clear correlation between zinc substitution level and stiffness and hardness, suggests that zinc substitution serves to mechanically reinforce the bioapatite. The spatial arrangement of the high-zinc apatite unveils a material-level adaptation strategy for tooth fortification, in which the rigid high-Zn structure servs as an internal load-transfer element that transmits the stress directly from the tooth's sharp cusps to the more compliant underlying layers.

Identification and Characterization of an Insulin-Like Receptor Involved in Crustacean Reproduction.

Sexual differentiation and maintenance of masculinity in crustaceans has been suggested as being regulated by a single androgenic gland (AG) insulin-like peptide (IAG). However, downstream elements involved in the signaling cascade remain unknown. Here we identified and characterized a gene encoding an insulin-like receptor in the prawn Macrobrachium rosenbergii (Mr-IR), the first such gene detected in a decapod crustacean. In mining for IRs and other insulin signaling-related genes, we constructed a comprehensive M. rosenbergii transcriptomic library from multiple sources. In parallel we sequenced the complete Mr-IR cDNA, confirmed in the wide transcriptomic library. Mr-IR expression was detected in most tissues in both males and females, including the AG and gonads. To study Mr-IR function, we performed long-term RNA interference (RNAi) silencing in young male prawns. Although having no effect on growth, Mr-IR silencing advanced the appearance of a male-specific secondary trait. The most noted effects of Mr-IR silencing were hypertrophy of the AG and the associated increased production of Mr-IAG, with an unusual abundance of immature sperm cells being seen in the distal sperm duct. A ligand blot assay using de novo recombinant Mr-IAG confirmed the existence of a ligand-receptor interaction. Whereas these results suggest a role for Mr-IR in the regulation of the AG, we did not see any sexual shift after silencing of Mr-IR, as occurred when the ligand-encoding Mr-IAG gene was silenced. This suggests that sexual differentiation in crustaceans involve more than a single Mr-IAG receptor, emphasizing the complexity of sexual differentiation and maintenance.

Isolation and characterization of a female-specific DNA marker in the giant freshwater prawn Macrobrachium rosenbergii.

In this study, a female-specific DNA marker in the freshwater prawn Macrobrachium rosenbergii was identified through amplified fragment length polymorphism (AFLP). The AFLP-derived sequence-characterized amplified region (SCAR) marker was tested in over 200 individuals, giving reproducible sex identification. Further molecular characterization of the sex-marker's genomic region (∼ 3 kb long) revealed the presence of tandem and inverted repeats. The ∼ 3-kb sequence was identified both in male and female prawns, but with subtle differences: a deletion of 3 bp (present in female prawn but absent in male prawn) identified upstream of the SCAR marker sequence and two female-specific single-nucleotide polymorphisms, both indicating that male prawns are homozygous, whereas female prawns are heterozygous in this locus. Fluorescent in situ hybridization showed the ∼ 3-kb sequence to be unique: to the best of our knowledge, this is the first report of a unique sex-specific sequence observed in situ in crustaceans. The sex-specific marker identified in M. rosenbergii may have considerable applied merit for crustacean culture in that it will enable the determination of genetic sex at early developmental stages when phenotypic differences are not identifiable.

Tyrphostin AG-555 inhibits early and late stages of Moloney murine leukemia virus replication cycle.

We have previously shown that certain tyrphostin derivatives, known as protein tyrosine blockers, inhibited Moloney murine leukemia virus (Mo-MuLV) replication in acutely and chronically infected NIH/3T3 cells, without affecting cell viability or growth. In our present work, we examined the stages in the viral life cycle that are affected by tyrphostin AG-555. We found that this drug inhibited the integration of the viral DNA into the host genome in acutely infected cells. This compound also reduced the level of viral RNA and specifically inhibited viral protein synthesis in NIH/3T3/Mo-MuLV chronically infected cells while no effect on the cellular beta-actin was observed. Since tyrphostin AG-555 inhibited both the early stages (integration process) and the late stages (viral protein synthesis) in the virus life cycle, it offers a potential advantage over other compounds which affect only one stage in the viral life cycle. Therefore, tyrphostin AG-555 may be considered as a potent antiretroviral drug.

Isolation of an 11-kDa protein associated with the topoisomerase I activity from equine infectious anemia virus.

We have previously demonstrated the presence of topoisomerase I (topo I) activity in purified retroviral particles (i.e., human immunodeficiency virus type 1, equine infectious anemia virus-EIAV and moloney murine leukemia virus). In our present work, an attempt was made to determine the nature and origin of the protein that is associated with this activity. For that purpose we have isolated the topo I activity from equine infectious anemia virus cores and showed that a major protein band of an 11 kDa is present in the topo I active fractions. It was able to form a DNA-protein cleavable complex, which is one of the characteristics of topoisomerases. This protein was recognized by anti-EIAV p11 nucleocapsid protein (NC) antibodies that can also specifically remove the topo I activity from the purified topo I active fractions. Therefore, our present findings, which are compatible with our previous data concerning the HIV NC protein, suggest that the 11 kDa protein which is associated with the topo I activity in EIAV is the nucleocapsid protein.

DNA binding properties of the zinc-bound and zinc-free HIV nucleocapsid protein: supercoiled DNA unwinding and DNA-protein cleavable complex formation.

The HIV nucleocapsid (NC) protein contains, as those of other retroviruses, two Cys-His arrays which function as zinc finger binding domains. The nucleic acid binding properties of retroviral NC have been previously demonstrated. In this study, we characterized the DNA binding ability of the zinc-bound and zinc-free forms of HIV NC. We found that in addition to binding single-stranded DNA, both forms bind and unwind supercoiled plasmid DNA. The binding ability of the zinc-bound form was higher than the zinc-free form. In addition we showed the formation of NC protein-DNA cleavable complex which is the result of a presumably covalent bond formed between the protein and the phosphate moiety of the DNA backbone. The NC unwinding activity and the protein-DNA cleavable complex formation resembles the first step of the relaxing mechanism of DNA topoisomerase. Our results shed light on the possibility of a novel physiological function for the HIV NC protein in the viral life cycle.

Inhibition of topoisomerase I activity by tyrphostin derivatives, protein tyrosine kinase blockers: mechanism of action.

DNA topoisomerase I (topo I) is a member of a group of essential nuclear enzymes which control and modify the topological state of DNA and is recognized as the target for anticancer drugs. During the course of the catalytic activity of topo I, a covalent bond is formed between a tyrosine group at the active site of the enzyme and a 3' phosphate group along the DNA backbone. This chemical reaction resembles the protein kinase-mediated tyrosine phosphorylation process. We assumed, therefore, that tyrphostins, potent and selective blockers of protein tyrosine kinases, might affect topo I activity. We found that of three derivatives of tyrphostins (AG-555, AG-18, and AG-213) that inhibited topo I activity in an in vitro assay, AG-555 was the most active. Examination of the mechanism by which these compounds act as topo I inhibitors revealed that AG-555 blocked the binding of this enzyme to the DNA due to its interaction with the topo I enzyme. We showed that its mode of action differed from that observed for camptothecin, a known topo I inhibitor. However, AG-555 did not affect the activity of other major DNA binding enzymes (i.e., DNA ligase, DNA polymerase I, and reverse transcriptase). This study suggests that tyrphostins may serve as a new class of topo I inhibitors, and these results also present additional explanations for their antiproliferative effect.

Inhibition of retrovirus-induced disease in mice by camptothecin.

We have previously shown that noncytotoxic doses of camptothecin (CPT), a topoisomerase I-specific antagonist, inhibit retrovirus replication in acutely and chronically infected cells. To evaluate the efficacy of CPT as an antiretroviral drug in vivo, we injected newborn BALB/c mice with Moloney murine leukemia virus and adult NFS mice with Friend spleen focus-forming virus. The Moloney murine leukemia virus-injected mice developed lymphoma, and the Friend spleen focus-forming virus-injected mice developed erythroleukemia. CPT, administrated together with the virus or 1 or 2 days after virus injection, prevented the onset of the disease in both cases. We showed that repeated CPT treatments increased the effectiveness of the drug when administrated 3 days after virus injection. This ability of CPT to inhibit retrovirus-induced disease in vivo without causing any apparent toxic side effects suggests its application as a legitimate remedy for the treatment of retroviral diseases.

Oligoclonality of Moloney leukemias.

Induction of leukemia by non-transforming retroviruses results in the appearance of various hematopoietic tumors. It is believed that these tumors are monoclonal. In this work, the clonal nature of Moloney leukemia virus (MoLV)-induced tumors was studied. Two genetic parameters were used in order to identify leukemic clones: the pattern of the proviral integration sites and the rearrangement of the T-cell receptor complex (TCR). In more than 60% of the mice, different leukemic clones populated tumors developed in different organs of the same animal. Genotypic analysis of cell lines derived from a leukemic organ revealed that the tumor is composed of more than one clone. Phenotypic analysis of subclones which were derived from a monoclonal cell line showed variability in the expression of the Thy 1.2 and MHC antigens. The results indicate that MoLV-induced tumors are of oligoclonal nature. Each leukemic organ contains a mixture of leukemic clones, of which one is dominant.

Elevated activities of protein kinase C and tyrosine kinase correlate to leukemic cell aggressiveness.

We report a linkage between cell aggressiveness, protein kinase C (PKC) activity, tyrosine kinase (PTK) activity and serum requirement. We used 2 leukemic cell lines induced by Moloney murine leukemia virus (MLV). One line was highly aggressive (BS-24-1) and required low serum concentrations (3%) for optimal growth in comparison to the less aggressive line (RO2T) that needed 10% serum for optimal growth. The more malignant cells exhibited higher PKC and PTK activity. This activity was independent of serum concentration between 0.01-10%. In contrast, the weakly malignant cells need a high serum concentration (10%) for optimal PKC or PTK activity. Immunoblot analysis revealed a higher level of PKC protein in the BS-24-1 cells than in the RO2T cells. Serum induction of PKC activity did not change the amount of PKC protein in the cytosol or the membrane fractions, indicating post-translational mechanism regulation of PKC. We suggest that the aggressiveness of BS-24-1 resulted from its ability to become independent of growth regulation by serum factors, via autocrine stimulation of PKC and PTK.

Human melanoma cell lines established from metastases of a patient with a completely regressed primary site.

Two unique human melanoma cell lines were established from each of two metastases, with collections separated by a 1-year interval, in a patient with a spontaneously completely regressed primary cutaneous malignant melanoma. These cell lines were distinct, and under culture, they had characteristic features that correlated with those shown by the original tumors from which they were derived. Cells derived from the second metastasis were more aggressive and had a higher proliferative growth rate, serious chromosomal abnormalities, a greater capacity to form colonies on agar, and a lesser dependence on serum-derived growth factors. This study of malignant melanoma cell lines covered the range from the stage of complete spontaneous regression of the primary lesion through the development of the first metastasis (from which the cell line designated L1M1 was established) to the second metastasis (discovered 1 year later, from which the cell line G1M2 was established). These cell lines grow continuously in the laboratory and can be carried for an unlimited number of passages. They afford an opportunity to investigate and compare the malignant pattern and behavior of human malignant melanoma originating after a completely spontaneously regressed primary lesion.

Competition among leukemic cells.

We investigated competition among leukemic cells induced by Moloney murine leukemia virus (MoLV) in order to understand the mechanisms involved in the generation of leukemia. We used six leukemic cells lines from Balb/C mice infected with MoLV. Each line had a unique genetic marker which enabled us to trace it in mixtures of cells either in vivo or in vitro. The markers were a unique rearrangement of T-cell receptors, the integration sites of the retrovirus and rearrangements in the Pim-1 oncogene. A mixture of two cell lines (1:1) injected into intact Balb/C mice usually produced a monoclonal tumor originating from one cell line. In most cases, the cell lines that were aggressive in vivo were also dominant in mixing experiments in vitro. In some lines, we could correlate the aggressiveness of the tumor to its superior growth rate and lower requirement for serum factors in vitro. In others, this correlation did not hold, and we assumed that host factors like the immune system contribute to the malignant potential of the leukemic cell.

Characterization of mouse thymocyte subpopulations by the enzymatic marker 20-alpha-hydroxysteroid dehydrogenase: differential responses to IL-1 and IL-2.

The responses of thymocytes to Concanavalin A (Con A), and interleukin 1 (IL-1), interleukin 2 (IL-2) and phorbol myristate acetate (PMA) were investigated. The enzyme 20-alpha-hydroxysteroid dehydrogenase (20 alpha SDH) was used as a marker to distinguish between various populations of activated thymocytes. Thymocytes that were selected in Con A + pure or crude IL-2 expressed high 20 alpha SDH activity, while those that were selected in Con A + recombinant IL-1 (rIL-1) or crude IL-1, or Con A + PMA expressed low 20 alpha SDH activity. Both groups proliferate in response to Con A and had IL-2 receptors. After selection, the enzymatic phenotype was stable even if the cells were transferred from Con A + IL-2 to Con A + PMA (or IL-1) or vice versa. A third group was selected from thymocytes that were cultured in PMA + T cell growth factor (TCGF). This group expressed low levels of 20 alpha SDH, had IL-2 receptors, but did not respond to Con A. This paper demonstrates that 20 alpha SDH can be used as an enzymatic marker to distinguish between subpopulations of activated T cells, which have not been previously detected by the conventional surface markers.