Laboratory culture of Chironomus tentans for use in toxicity testing: optimum initial egg-stocking densities

The midge Chironomus tentans Fabricius is a commonly used freshwater invertebrate in sediment toxicity tests. Rigorous laboratory culturing techniques are needed to provide organisms of uniform quality and known age for use in testing and for the continuation of the culture itself. This study was conducted to determine the effect of initial culture stocking density on: (1) post-hatch (larval) dry weight, body length and head-capsule width at 10 and 20 days; (2) time to emergence; (3) number and sex of emergent adults; (4) number of larvae and pupae at test termination (day 42 post hatch); and (5) adult dry weight. Three egg stocking densities were used 690 (1.1 eggs cm^–2), 1043 (1.7 eggs cm^–2) and 1463 (2.4 eggs cm^–2). Mean weight of larvae at 10 days in high density tanks (0.13 mg/organism) was significantly higher (P=0.003) than both the medium and low density tanks (0.10 and 0.09 mg/organism, respectively). No significant differences between the three stocking densities were observed for the body length or head-capsule width at either 10 or 20 days post-hatch. Although not statistically significant, larval dry weight decreased with increased stocking density at day 20. A significantly (P=0.02) greater number of females (173±28) emerged from the low stocking density compared to both the medium and high stocking densities (123±45 and 118±54, respectively). Peak adult emergence for the low and medium stocking densities occurred between days 22 and 25 post-hatch, whereas peak adult emergence occurred between days 30 and 33 for the high stocking density. Survival relative to the initial number of eggs stocked was significantly greater (P=0.007) in the low density treatment compared to that in either the medium or the high density treatments. Mean adult weight exhibited an inverse relationship with initial stocking densities. At test end, there was not a significant difference in the mean number of organisms surviving and emerging in the three density levels. The central tendency for number of organisms surviving for all three treatments was 504 organisms per tank (0.82 organisms cm^–2). The results of this experiment suggest that an optimal egg stocking density of 1.0 egg cm^–2 (600 eggs/tank) be used with the feeding rate identified. This would ensure uniform larvae at the appropriate developmental stage (2nd–3rd instar) needed for toxicological research/testing (e.g. 10 days post-hatch), as well as producing sufficient emergence of males and females for future culture establishment.     

CELL KINETICS OF A CHONDROSARCOMA

The cell kinetics of the transplantable DC-II mouse chondrosarcoma have been studied by the pulse labelled mitoses method. The analysis gave the following estimates for the phases of the cell cycle: G, 10-5 hr; S, 9-5 hr; G₂, 4 hr with an intermitotic time of 23-5 hr. Consideration of the overall growth of the tumour indicated that the growth fraction and cell loss factor both had values of about 0–5. The results are compared with cell kinetic data from sarcomas and other cartilage tissues.     

A new manual power grip

A manual power grip for holding a cylindrical object using a relaxed index finger is described and analysed. In a group of 21 young adult students it provided greater grip strength than the conventional oblique power grip. It allowed a significant increase in the range of adduction-abduction at the wrist. In recent years it appears to have been empirically used by a number of top class racquet players and is the basis of the modern assault rifle grip. A new design of strain gauge dynamometer, in the shape of a cylinder, which permits the testing of a number of grip types and wrist positions is described. Standard grip testing protocol is used with this device which also allows the concomitant use of a goniometer for the assessment of wrist mobility.     

Chemical synthesis, characterization and activity of RK-1, a novel alpha-defensin-related peptide.

The 32-residue peptide, RK-1, a novel kidney-derived three disulfide-bonded member of the antimicrobial alpha-defensin family, was synthesized by the continuous flow Fmoc-solid phase method. The crude, cleaved and S-reduced linear peptide was both efficiently folded and oxidized in an acidic solution of aqueous dimethyl sulfoxide. Following purification of the resulting product, it was shown by a variety of analytical techniques, including matrix assisted laser desorption time of flight mass spectrometry, to possess a very high degree of purity. The disulfide bond pairing of the synthetic peptide was determined by 1H-NMR spectroscopy and confirmed to be a Cys1-Cys6, Cys2-Cys4, Cys3-Cys5 arrangement similar to other mammalian alpha-defensin peptides. The synthetic RK-1 was also shown to inhibit the growth of Escherichia coli type strain NCTC 10418.     

The molecular characterisation of HPRT CHERMSIDE and HPRT COORPAROO: two Lesch-Nyhan patients with reduced amounts of mRNA.

A complete deficiency of the purine salvage enzyme, hypoxanthine phosphoribosyltransferase (HPRT; EC 2.4.2.8), in man results in the Lesch-Nyhan (LN) syndrome. Two unrelated patients with the full LN syndrome showed no evidence of a major alteration to the gene encoding HPRT (HPRT) by restriction endonuclease analysis, but exhibited negligible levels of HPRT mRNA on Northern blots. DNA from these patients was characterised further. Amplification, by the polymerase chain reaction (PCR), of individual HPRT-exon fragments from genomic DNA followed by nucleotide (nt) sequence analysis using automated technology, revealed single-base mutations in each patient. One patient has an insertion of a T within exon-2, which places a stop codon in frame, presumably resulting in premature termination of translation of the HPRT mRNA. The other patient has a G----A base substitution at the 5' end of intron-6, at the junction of exon-6 and intron-6. Although dot blot analysis indicated negligible HPRT mRNA in lymphoblast cells from both patients, we were successful in amplifying HPRT cDNA using PCR. Direct nt sequence analysis of the amplified cDNA confirmed the insertion of a T in exon-2 in the one patient and revealed a complete deletion of exon-6 in the other patient, the latter event presumably arising due to aberrant splicing of primary message. Both mutations were also confirmed by hybridisation of amplified genomic DNA with allele-specific oligodeoxyribonucleotide probes. This study illustrates two approaches for analysing DNA mutations at the molecular level and demonstrates the power of PCR technology in the study of genetic diseases.(ABSTRACT TRUNCATED AT 250 WORDS)     

ras p21 and other Gn proteins are detected in mammalian cell lines by [gamma-35S]GTP gamma S binding

The presence of guanine nucleotide binding proteins in mouse and human cell lines was investigated using [gamma-35S]GTP gamma S and [gamma-32P]GTP. Cell lysate polypeptides were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred to nitrocellulose. Incubation of the nitrocellulose blots with [gamma-35S]GTP gamma S identified 9 distinct GTP-binding polypeptides in all lysates. One of these is the ras oncogene product, p21, as demonstrated by subsequent immunochemical staining of the nitrocellulose blots. We have shown that this procedure provides a sensitive method for detection of p21 in culture cell lines.