Mapping of Mhc class I and class II regions to different linkage groups in the zebrafish, Danio rerio

 The mammalian major histocompatibility complex (Mhc) consists of three closely linked regions, I, II, and III, occupying a single chromosomal segment. The class I loci in region I and the class II loci in region II are related in their structure, function, and evolution. Region III, which is intercalated between regions I and II, contains loci unrelated to the class I and II loci, and to one another. There are indications that a similar Mhc organization exists in birds and amphibians. Here, we demonstrate that in the zebrafish (Danio rerio), a representative of the teleost fishes, the class II loci are divided between two linkage groups which are distinct from the linkage group containing the class I loci. The β₂-microglobulin-encoding gene is loosely linked to one of the class II loci. The gene coding for complement factor B, which is one of the region III genes in mammals, is linked neither to the class I nor to the class II loci in the zebrafish. These results, combined with preliminary data suggesting that the class I and class II regions in another order of teleost fish are also in different linkage groups, indicate that close linkage of the two regions is not necessary either for regulation of expression or for co-evolution of the class I and class II loci. They also raise the question of whether linkage of the class I and class II loci in tetrapods is a primitive or derived character. Received: 16 December 1996 / Revised: 6 February 1997     

A comparative study on efficiency of adult fibroblasts and amniotic fluid-derived stem cells as donor cells for production of hand-made cloned buffalo (Bubalus bubalis) embryos

The efficiency of two cell types, namely adult fibroblasts, and amniotic fluid stem (AFS) cells as nuclear donor cells for somatic cell nuclear transfer by hand-made cloning in buffalo (Bubalus bubalis) was compared. The in vitro expanded buffalo adult fibroblast cells showed a typical “S” shape growth curve with a doubling time of 40.8 h and stained positive for vimentin. The in vitro cultured undifferentiated AFS cells showed a doubling time of 33.2 h and stained positive for alkaline phosphatase, these cells were also found positive for undifferentiated embryonic stem cell markers like OCT-4, NANOG and SOX-2, which accentuate their pluripotent property. Further, when AFS cells were exposed to corresponding induction conditions, these cells differentiated into osteogenic, adipogenic and chondrogenic lineages which was confirmed through alizaran, oil red O and alcian blue staining, respectively. Cultured adult fibroblasts and AFS cells of passages 10–15 and 8–12, respectively, were used as nuclear donors. A total of 94 embryos were reconstructed using adult fibroblast as donor cells with cleavage and blastocyst production rate of 62.8 ± 1.8 and 19.1 ± 1.5, respectively. An overall cleavage and blastocyst formation rate of 71.1 ± 1.2 and 29.9 ± 2.2 was obtained when 97 embryos were reconstructed using AFS cells as donor cells. There were no significant differences (P > 0.05) in reconstructed efficiency between the cloned embryos derived from two donor cells, whereas the results showed that there were significant differences (P < 0.05) in cleavage and blastocyst rates between the cloned embryos derived from two donor cell groups. Average total cell numbers for blastocyst generated using AFS cells (172.4 ± 5.8) was significantly (P < 0.05) higher than from adult fibroblasts (148.2 ± 6.1). This study suggests that the in vitro developmental potential of the cloned embryos derived from AFS cells were higher than that of the cloned embryos derived from adult fibroblasts in buffalo hand-made cloning.     

A histological and immunohistochemical study of the effects of N-acetyl cysteine on retinopathy of prematurity by modifying insulin-like growth factor-1

Retinopathy of prematurity (ROP) is a vasoproliferative disorder that occurs in premature infants and may lead to permanent visual impairment. We investigated both the possible protective role of N-acetyl cysteine (NAC) for preventing ROP and the role of IGF-1 in the disorder. Forty-five newborn rats were divided into three groups. Group 1 was raised in room air as controls. Group 2 was exposed to 60% oxygen for 14 days after birth, then transferred to room air. Group 3 was exposed to the same conditions as group 2, but received intraperitoneal injections of NAC on postnatal days 7–17. After 35 days, both eyes of all rats were processed for histology. Some sections were stained with hematoxylin and eosin to assess structural changes and other sections were immunostained to determine the location of IGF-1. Frozen sections also were prepared and stained for adenosine triphosphatase to detect retinal blood vessels. Compared to the controls, more blood vessels, many of which were abnormal, and increased IGF-1 expression were observed in group 2. In group 3, abnormal blood vessels and IGF-1 expression were less evident. NAC appeared to be an effective vascular-protective agent for ROP by decreasing IGF-1 expression.     

Purification and characterization of acid phosphatase-1 from Drosophila melanogaster

Acid phosphatase-1 (orthophosphoric monoester phosphohydrolase, acid optimum, EC 3.1.3.2), the major phosphatase in adult Drosophila melanogaster, has been purified to apparent homogeneity. The final product is a glycoprotein homodimer with a subunit molecular weight of about 50,000, as measured by its electrophoretic mobility in denaturing conditions on polyacrylamide gels containing sodium dodecyl sulfate. It has a turnover number of 1720 1-naphthyl phosphate molecules hydrolyzed/s by each acid phosphatase-1 molecule at 37 degrees C, pH 5.0. An average fly contains about 5 ng of enzyme. Pure acid phosphatase-1 displays heterogeneity in isoelectric focusing, with a major band at pH 5.3. The enzyme hydrolyzes a wide variety of phosphate monoesters, including AMP, glucose 6-phosphate, ATP, choline phosphate, or phosphoproteins. The maximum reaction rates are different for all substrates, and some substrates appear to inhibit the reaction at high substrate concentrations. The Michaelis constants for 1-naphthyl phosphate and p-nitrophenyl phosphate are 79 microM and 68 microM, respectively, at pH 5.0 and 37 degrees C. The optimum pH level for 1-naphthyl phosphate is 4.5. Acid phosphatase-1 is inhibited by L(+)-tartrate (but not D(-)-tartrate), phosphate, and fluoride. The reaction rate increases 2.1-fold for every 10 degrees C rise in temperature. Above 48 degrees C, the rate of thermal denaturation is greater than the rate of the enzyme reaction.     

Functional characterization and genetic mapping of alk8

The novel type I TGFβ family member receptor alk8 is expressed both maternally and zygotically. Functional characterization of alk8 was performed using microinjection studies of constitutively active (CA), kinase modified/dominant negative (DN), and truncated alk8 mRNAs. CA Alk8 expression produces ventralized embryos while DN Alk8 expression results in dorsalized phenotypes. Truncated alk8 expressing embryos display a subtle dorsalized phenotype closely resembling that of the identified zebrafish dorsalized mutant, lost-a-fin (laf). Single-strand conformation polymorphism (SSCP) analysis was used to map alk8 to zebrafish LG02 in a region demonstrating significant conserved synteny to Hsa2, and which contains the human alk2 gene, ACVRI. Altogether, these functional, gene mapping and phylogenetic analyses suggest that alk8 may be the zebrafish orthologue to human ACVRI (alk2), and therefore extend previous studies of Alk2 conducted in Xenopus.     

Differential responsiveness to immunoablative therapy in refractory rheumatoid arthritis is associated with level and avidity of anti-cyclic citrullinated protein autoantibodies: a case study

In order to identify pathogenic correlates of refractory rheumatoid arthritis (RA), antibodies against anti-cyclic citrullinated protein (ACPAs) were investigated in RA patients in whom the dysregulated immune system had been ablated by high-dose chemotherapy (HDC) and autologous haematopoietic stem cell transplantation (HSCT). Six patients with refractory RA were extensively characterized in terms of levels of total immunoglobulins, RA-specific autoantibodies (ACPAs and rheumatoid factor) and antibodies against rubella, tetanus toxoid (TT) and phosphorylcholine before and after HDC plus HSCT. Additionally, the avidity of ACPAs was measured before and after treatment and compared with the avidity of TT antibodies following repeated immunizations. Synovial biopsies were obtained by arthroscopy before HDC plus HSCT, and analyzed by immunohistochemistry. In the three patients with clinically long-lasting responses to HDC plus HSCT (median 423 days), significant reductions in ACPA-IgG levels after therapy were observed (median level dropped from 215 to 34 arbitrary units/ml; P = 0.05). In contrast, stable ACPA-IgG levels were observed in three patients who relapsed shortly after HDC plus HSCT (median of 67 days). Clinical responders had ACPA-IgG of lower avidity (r = 0.75; P = 0.08) and higher degree of inflammation histologically (r = 0.73; P = 0.09). Relapse (after 38 to 530 days) in all patients was preceded by rising levels of low avidity ACPA-IgG (after 30 to 388 days), in contrast to the stable titres of high avidity TT antibodies. In conclusion, humoral autoimmune responses were differentially modulated by immunoablative therapy in patients with synovial inflammation and low avidity ACPA-IgG autoantibodies as compared with patients with high levels of high avidity ACPA-IgG. The distinct clinical disease course after immunoablative therapy based on levels and avidity of ACPA-IgG indicates that refractory RA is not a single disease entity.     

Evolutionary developmental biology and genomics

Reciprocal questions often frame studies of the evolution of developmental mechanisms. How can species share similar developmental genetic toolkits but still generate diverse life forms? Conversely, how can similar forms develop from different toolkits? Genomics bridges the gap between evolutionary and developmental biology, and can help answer these evo-devo questions in several ways. First, it informs us about historical relationships, thus orienting the direction of evolutionary diversification. Second, genomics lists all toolkit components, thereby revealing contraction and expansion of the genome and suggesting mechanisms for evolution of both developmental functions and genome architecture. Finally, comparative genomics helps us to identify conserved non-coding elements and their relationship to genome architecture and development.     

Desferrioxamine inhibits protein tyrosine nitration: Mechanisms and implications

Tissues are exposed to exogenous and endogenous nitrogen dioxide (()NO(2)), which is the terminal agent in protein tyrosine nitration. Besides iron chelation, the hydroxamic acid (HA) desferrioxamine (DFO) shows multiple functionalities including nitration inhibition. To investigate mechanisms whereby DFO affects 3-nitrotyrosine (3-NT) formation, we utilized gas-phase ()NO(2) exposures, to limit introduction of other reactive species, and a lung surface model wherein red cell membranes (RCM) were immobilized under a defined aqueous film. When RCM were exposed to ()NO(2) covered by +/- DFO: (i) DFO inhibited 3-NT formation more effectively than other HA and non-HA chelators; (ii) 3-NT inhibition occurred at very low[DFO] for prolonged times; and (iii) 3-NT formation was iron independent but inhibition required DFO present. DFO poorly reacted with ()NO(2) compared to ascorbate, assessed via ()NO(2) reactive absorption and aqueous-phase oxidation rates, yet limited 3-NT formation at far lower concentrations. DFO also inhibited nitration under aqueous bulk-phase conditions, and inhibited 3-NT generated by active myeloperoxidase "bound" to RCM. Per the above and kinetic analyses suggesting preferential DFO versus ()NO(2) reaction within membranes, we conclude that DFO inhibits 3-NT formation predominantly by facile repair of the tyrosyl radical intermediate, which prevents ()NO(2) addition, and thus nitration, and potentially influences biochemical functionalities.