Proliferative activity in the cerebellar external granular layer evaluated by bromodeoxyuridine labeling

We have optimised an indirect immunoperoxidase technique demonstrating bromodeoxyuridine (BrdU) incorporation into dividing cells for cerebellar tissue sections of four-day-old rats injected with this marker. This permits confident identification of granule-cell precursors engaged in DNA synthesis in the external granular layer of the developing cerebellum. Preservation of BrdU immunoreactivity is attained using methanol/acetic acid fixation and different pretreatments before immunostaining, while unlabeled nuclei can be recognized clearly after Feulgen or hematoxylin counterstaining. We established conditions to ensure satisfactory BrdU uptake without affecting cell-cycle progression during the postlabeling time period. The dose of BrdU employed provides saturation S-phase labeling from at least 1 h after BrdU delivery. Various kinetic parameters and phase durations have been determined in experiments involving a single injection or cumulative labeling sequences, and the cycle time was calculated based on two models of generative behavior: steady-state and exponential growth. The working hypothesis of steadystate kinetics can be adopted successfully if the existence of neuroblasts with different proliferation rates is taken into account.     

Induction of mixed function oxidase (MFO) system in two species of antarctic fish from Terra Nova Bay (Ross Sea)

Summary The aim of this study was to investigate the detoxicant Mixed Function Oxidase system in two species of Antarctic fish, C. hamatus and P. bernacchii, collected during the Antarctic summer of 1989–1990 at the Italian Scientific Station at Terra Nova Bay (Ross Sea). Several specimens were induced by injection of phenobarbital (PB), 3-methylcholanthrene (3-MC) and PCBs in the caudal vein. The results show significant differences between the two species. In C. hamatus, basal activity of benzo(a)pyrene monooxygenase (BPMO) was among the lowest measured even in fish of temperate seas, and in P. bernacchii it was 20 times lower. The values of regenerating activities (NADPH-cytochrome c reductase (NADPH-CYTCRED), NADH-cytochrome c reductase (NADH-CYTCRED) and NADH-ferricyanide reductase (NADH-FERRIRED) suggest that the two species use different electron donor molecules. Injection of chemicals in the caudal vein did not provoke induction of MFO activity in P. bernacchii. In C. hamatus, phenobarbital and PB-type inducers did not cause induction but there was a statistically significant response to 3-MC.     

The use of non destructive biomarkers in the study of marine mammals

Marine mammals have been subject to heavy anthropogenic pressure by direct killing and chemical pollution all over the world. Most studies of contamination and biomarker responses in marine mammals have been conducted using animals killed by hunting out of a total of 12 cetacean species studied, 45 of the specimens were obtained by sacrificing the animal; out of a total of eight pinniped species studied, 40 of the specimens were obtained by killing . The development of a series of non destructive techniques to evaluate biomarker responses and residue levels is recommended for the hazard assessment and conservation of endangered species of marine mammals. Here we review the current status of the non destructive biomarker approach in marine mammals, describing the biological materials available for non destructive tests in stranded brain, liver, blood, skin, subcutaneous blubber, muscle and fur and free ranging animals blood, skin biopsy, fur and faeces and the respective biomarker techniques mixed function oxidase activity and DNA damage in skin biopsy samples; porphyrins in faeces and fur; esterases, porphyrins, clinical biochemical parameter, vitamin A and micronuclei in blood samples . Residue analysis can be carried out in the various biological materials. We also report the results of applying this methodological approach to cetaceans minke whale Balaenoptera acutorostrata, fin whale-- Balaenoptera physalus, beluga whale-- Delphinapterus leucas, short finned pilot whale-- Globicephala macrorhynchus, harbour porpoise -- Phocoena phocoena, Rissos dolphin-- Risso s Grampus griseus, Dall s porpoise-- Phocoenoides dalli dalli, melon headed whale-- Peponocephala electra, bottlenose dolphin -- Tursiops truncatus, striped dolphin-- Stenella coeruleoalba, spinner dolphin-- Stenella longirostris, killer whale-- Orcinus orca and pinnipeds northern fur seal- Callorhinus ursinus, hooded seal-- Cystophora cristata, grey seal-- Halichoerus grypus, harbour seal-- Phoca vitulina, ringed seal-- Phoca hispida, harp seal-- Phoca groenlandica, ribbon seal-- Phoca fasciata, largha seal- Phoca largha, southern sea lion-- Otaria flavescens in field studies for prognostic and diagnostic purposes.     

Study of the respiratory sensation on high-altitude andeans

The respiratory sensation and some routine cardiorespiratory parameters were studied on native Highlanders from the Argentine Andes and on Lowlanders from Europe, already tested during previous high altitude expeditions. The tests were performed at various altitude levels from 2688m e.i., the village altitude for Highlanders, to 5600m during an expedition to Mt. Aconcagua (6990m). At rest, the perception of 4 external inspiratory resistive loads (ranged between 2.5 and 13 cm.H2O.L-1.s) can allow us to fix by discrimination the sensitivity index P(A) independently of response bias (B) according to Sensory Decision Theory (SDT). The Andean highlanders did not experience the respiratory sensation at the same limits as the European lowlanders well adaptated to high altitude. At higher altitudes than their village altitude, their respiratory sensation presented a lower threshold of perception and a weaker discrimination which might be partly explained by the evolution of some parameters of their cardio-respiratory function when altitude increased. Indeed, in response to high altitude hypoxia (5600m), they increased their respiratory frequency and not their minuteventilation or mouth pressure. This chosen ventilatory pattern was opposite to the one chosen by the Lowlanders and did not allow for sufficient adaptation to a more important altitude hypoxia than that of their village altitude. In conclusion, the Andean highlanders wellbeing adapted to their village altitude, exhibited a difficult acclimatization to higher altitudes which might be due to the characteristics of their respiratory sensation. These results might explain their weak physical performances during ascent to the Mt. Aconcagua summit in spite of special training.     

Variant forms of a group I intron in nuclear small-subunit rRNA genes of the marine red alga Porphyra spiralis var. amplifolia

A group IC1 intron occurs in nuclear small-subunit (18S) ribosomal RNA (SSU rRNA) genes of the marine red alga Porphyra spiralis var. amplifolia. This intron occurs at the same position as the self- splicing group IC1 introns in nuclear SSU rDNAs of the fungus Pneumocystis carinii and in the green alga Chlorella ellipsoidea and shares sequence identity with the Pneumocystis carinii intron in domains L1, P1, P2, and L2, outside the conserved core. Three size variants, differing in amount of sequence in L1, exist and are differentially distributed in geographically distinct populations. Preliminary data suggest that the largest variant can self-splice in vitro. Short open reading frames are present but do not correspond to known genes. Repeated nucleotide motifs, reminiscent of duplicated target sites of transposons or Alu elements, are associated with the intron and with one of the variant forms of L1. Insertions are present in nuclear SSU rDNAs of several other Porphyra species and of the red alga Bangia atropurpurea; insertionless rDNA variants also occur in several Porphyra species. Our observations are most readily explained by intron mobility, although it remains unclear how transfer could have been mediated between genomes of organisms as ecologically diverse as marine red algae, freshwater green algae, and a mammalian-pathogenic fungus.      

Interleukin-23 is critical for full-blown expression of a non-autoimmune destructive arthritis and regulates interleukin-17A and RORγt in γδ T cells

Introduction

A protein analysis using a mass spectrometry indicated that there are serum proteins showing significant quantitative changes after the administration of infliximab. Among them, connective tissue growth factor (CTGF) seems to be related to the pathogenesis of rheumatoid arthritis (RA). Therefore, this study was conducted to investigate how CTGF is associated with the disease progression of RA.

Methods

Serum samples were collected from RA patients in active or inactive disease stages, and before or after treatments with infliximab. CTGF production was evaluated by ELISA, RT-PCR, indirect immunofluorescence microscopy, and immunoblotting. Osteoclastogenesis was evaluated using tartrate-resistant acid phosphatase (TRAP) staining, a bone resorption assay and osteoclasts specific catalytic enzymes productions.

Results

The serum concentrations of CTGF in RA were greater than in normal healthy controls and disease controls. Interestingly, those were significantly higher in active RA patients compared to inactive RA patients. Furthermore, the CTGF levels significantly were decreased by infliximab concomitant with the disease amelioration. In addition, tumour necrosis factor (TNF)α can induce the CTGF production from synovial fibroblasts even though TNFα can oppositely inhibit the production of CTGF from chondrocytes. CTGF promoted the induction of the quantitative and qualitative activities of osteoclasts in combination with M-CSF and receptor activator of NF-κB ligand (RANKL). In addition, we newly found integrin αVβ3 on the osteoclasts as a CTGF receptor.

Conclusions

These results indicate that aberrant CTGF production induced by TNFα plays a central role for the abnormal osteoclastic activation in RA patients. Restoration of aberrant CTGF production may contribute to the inhibition of articular destruction in infliximab treatment.