A new method for the concentration of Cryptosporidium oocysts from water

A novel method for the concentration of Cryptosporidium oocysts from water has been developed, based upon the precipitation of calcium carbonate. A 10 1 water sample is treated by adding solutions of calcium chloride and sodium bicarbonate and raising the pH value to 10 with sodium hydroxide. Crystals of calcium carbonate form and enmesh particles in the Cryptosporidium oocyst size range. The crystals are allowed to settle, the supernatant fluid is discarded and the calcium carbonate precipitate dissolved in sulphamic acid. The sample can be concentrated further by centrifugation. Recoveries of oocysts from seeded samples of deionized, tap and river water were in excess of 68%.     

An integrated genetic linkage map for eucalypts using RFLP,RAPD and isozyme markers

An integrated genetic linkage map for E. nitens was constructed in an outbred three-generation pedigree. Analysis of 210 RFLP, 125 RAPD and 4 isozyme loci resulted in 330 markers linked in 12 linkage groups covering 1462 cM (n=11 in eucalypts). The 12th linkage group is comprised of only 5 markers and will probably coalesce with another linkage group when further linked loci are located. Co-dominant RFLP loci segregating in both parents were used to integrate linkages identified in the male and female parents. Differences in recombination frequencies in the two parents were observed for a number of pairs of loci, and duplication of sequences was identified both within and between linkage groups. The markers were distributed randomly across the genome except for the RFLPs in linkage group 10 and for some loci showing segregation distortion, which were clustered into three regions of the map. The use of a large number of co-dominant RFLP loci in this map enables it to be used in other pedigrees of E. nitens and forms a basis for the detection and location of QTL in E. nitens and other eucalypt species.     

Cellular Events of Wrinkled Blastula Formation and the Influence of the Fertilization Envelope on Wrinkling in the Seastar Patiriella exigua

Like many echinoderms, the seastar, Patiriella exigua has a wrinkled blastula rather than the smooth-walled blastula typical of most phyla. The cellular events of wrinkled blastula formation in P. exigua were documented using light, confocal and electron microscopy. Wrinkled blastulae have a highly infolded epithelium. Prior to wrinkling, the blastomeres are cuboidal with lipid droplets and yolk granules distributed throughout their cytoplasm. During wrinkling, the cells become columnar and the lipid and yolk reserves become redistributed to the basal and apical ends of the cells, respectively. Gastrulae have a tall columnar epithelium, with a basal accumulation of lipid. Interdigitation of numerous cell projections, including short lateral processes, basal lamellipodia and apical filopodia, assists in maintaining epithelial integrity during wrinkling. Apical filopodia have not been observed in other echinoderm embryos. Although 1 M urea caused elevation of the fertilization envelope, the embryos did not expand into the newly-created space. This is suggested to be due to the adhesive properties of the hyaline layer. Embryos removed from their envelope were enlarged with shallower and fewer wrinkles compared with controls. It appears that the integrity of the hyaline layer and fertilization envelope both influence the compact wrinkled profile of P. exigua blastulae.     

Variation within and among the chloroplast genomes ofMelaleuca alternifolia andM. linariifolia (Myrtaceae)

Melaleuca alternifolia andM. linariifolia are commercially important Australian species harvested for their essential oils. Both species have relatively narrow and disjunct distributions on the central coast of eastern Australia. Variation in the chloroplast genome was assessed for eight individuals from each of twelve populations, representing the species' geographic range. Low nucleotide diversity withinM. alternifolia contrasted with high nucleotide diversity inM. linariifolia. CpDNA data are consistent with the southern population ofM. alternifolia being a hybrid population withM. linariifolia. The two species are sympatric in this region. Variation inM. linariifolia was geographically structured, with northern populations differing from southern populations by seven restriction site mutations, five length mutations and an inversion. There was no evidence of hybridisation of the cp genome of northernM. linariifolia with the partially sympatric speciesM. trichostachya. Intra- and interspecific variation in the chloroplast genomes ofM. alternifolia, M. linariifolia, andM. trichostachya indicate considerable potential for the use of intraspecific cpDNA studies in examining phylogenetic relationships in melaleucas.     

Chemically crosslinked protein dimers: stability and denaturation effects.

Nine single substitution cysteine mutants of staphylococcal nuclease (nuclease) were preferentially crosslinked at the introduced cysteine residues using three different bifunctional crosslinking reagents; 1,6-bismaleimidohexane (BMH), 1,3-dibromo-2-propanol (DBP), and the chemical warfare agent, mustard gas (bis(2-chloroethyl)sulfide; mustard). BMH and mustard gas are highly specific reagents for cysteine residues, whereas DBP is not as specific. Guanidine hydrochloride (GuHCl) denaturations of the resulting dimeric proteins exhibited biphasic unfolding behavior that did not fit the two-state model of unfolding. The monofunctional reagent, epsilon-maleimidocaproic acid (MCA), was used as a control for the effects of alkylation. Proteins modified with MCA unfolded normally, showing that this unusual unfolding behavior is due to crosslinking. The data obtained from these crosslinked dimers was fitted to a three-state thermodynamic model of two successive transitions in which the individual subunits cooperatively unfold. These two unfolding transitions were very different from the unfolding of the monomeric protein. These differences in unfolding behavior can be attributed in large part to changes in the denatured state. In addition to GuHCl titrations, the crosslinked dimers were also thermally unfolded. In contrast to the GuHCl denaturations, analysis of this data fit a two-state model well, but with greatly elevated van't Hoff enthalpies in many cases. However, clear correlations between the thermal and GuHCl denaturations exist, and the differences in thermal unfolding can be rationalized by postulating interactions of the denatured crosslinked proteins.     

Immunological effects of cancer chemotherapy in malignant melanoma

Summary The effects on the immune system of highdose cyclical combination chemotherapy were studied in nine patients with advanced malignant melanoma. Chemotherapy consisted of monthly cycles of dimethyl triazeno imidazole carboxamide 150 mg/m² i.v. daily from days 1–5, cyclophosphamide 1000 mg/m² i.v. on day 5, and vincristine 1.4 mg/m² i.v. on day 5.Immunological testing was carried out prior to treatment and at weekly intervals during the first month.B, T and non-B, non-T cell numbers all tended to fall early in the cycle as did the phytohaemagglutinin(PHA)-induced transformation and PHA-induced cytotoxicity to chicken red cells. Although PHA-induced transformation and cytotoxicity usually returned to normal by day 29, B and T cell numbers often remained subnormal. In contrast, levels for antibody-dependent, cell-mediated cytotoxicity (ADCC) were relatively stable throughout the cycle. Two patients with subsequent tumour response to therapy had rebound supranormal PHA transformations between weeks 1 and 3 of the first cycle. No other changes correlated with prognosis in individual patients.Analysis of the temporal relationships between PHA transformation, PHA-induced cytotoxicity, and ADCC supported the concept that the three assays reflect the function of separate mononuclear cell subpopulations.The stability of ADCC is of particular interest in view of other work suggesting that this function may be important in immune responses to tumours, including melanoma.Work was supported by grants from the National Health and Medical Research Council and the Western Australian Arthritis and Rheumatism Foundation, and the Cancer Council of Western Australia     

A new method for the isolation of renal basement membranes

A method is described for the isolation of basement membranes from rabbit renal cortex in which the detergent $\text{N-}\text{lauroyl}$ sarcosine is used as the disruptive agent. The isolated membranes have been compared with membranes prepared using ultrasonication and they were comparable both in terms of purity and gross chemical composition. Glomerular and tubular basement membranes were isolated by first separating glomeruli from tubules by density gradient centrifugation followed by detergent treatment of the separated tissues.The detergent method has the advantage that the basement membranes retained their native structure to a large degree, whereas sonicated membranes were severely fragmented. Collagen fibres were a significant contaminant in both preparations and were revealed more clearly by negative staining than by examination of thin sections. Studies with the detergent-treated membrane revealed that a few proteins, which seemed to be membrane components, were extracted with 1 M NaCl and that these proteins were lost from the basement membranes during sonication used in the conventional isolation procedure.