Molecular characterization of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency: identification of a lys329 to glu mutation in the MCAD gene,and expression of inactive mutant enzyme protein in E. coli

Summary A series of experiments has established the molecular defect in the medium-chain acyl-coenzyme A (CoA) dehydrogenase (MCAD) gene in a family with MCAD deficiency. Demonstration of intra-mitochondrial mature MCAD indistinguishable in size (42.5-kDa) from control MCAD, and of mRNA with the correct size of 2.4 kb, indicated a point-mutation in the coding region of the MCAD gene to be disease-causing. Consequently, cloning and DNA sequencing of polymerase chain reaction (PCR) amplified complementary DNA (cDNA) from messenger RNA of fibroblasts from the patient and family members were performed. All clones sequenced from the patient exhibited a single base substitution from adenine (A) to guanine (G) at position 985 in the MCAD cDNA as the only consistent base-variation compared with control cDNA. In contrast, the parents contained cDNA with the normal and the mutated sequence, revealing their obligate carrier status. Allelic homozygosity in the patient and heterozygosity for the mutation in the parents were established by a modified PCR reaction, introducing a cleavage site for the restriction endonuclease NcoI into amplified genomic DNA containing G⁹⁸⁵. The same assay consistently revealed A⁹⁸⁵ in genomic DNA from 26 control individuals. The A to G mutation was introduced into an E. coli expression vector producing mutant MCAD, which was demonstrated to be inactive, probably because of the inability to form active tetrameric MCAD. All the experiments are consistent with the contention that the G⁹⁸⁵ mutation, resulting in a lysine to glutamate shift at position 329 in the MCAD polypeptide chain, is the genetic cause of MCAD deficiency in this family. We found the same mutation in homozygous form in 11 out of 12 other patients with verified MCAD deficiency.     

Osteogenesis imperfecta due to recurrent point mutations at CpG dinucleotides in the COL1A1 gene of type I collagen

Summary Most individuals with osteogenesis imperfecta (OI) are heterozygous for dominant mutations in one of the genes that encode the chains of type I collagen. Each of the more than 30 mutations characterized to date has been unique to the affected member (s) of the family. We have determined that two individuals with a progressive deforming variety of OI, OI type III, have the same new dominant mutation [1(I)gly154 to arg] and that two unrelated infants with perinatal lethal OI, OI type II, share a second new dominant muation [1(I)gly1003 to ser]. These mutations occurred at CpG dinucleotides, in a manner consistent with deamination of a methylated cytosine residue, and raise the possibility that CpG dinucleotides are common sites of recurrent mutations in collagen genes. Further, these findings confirm that the OI type-III phenotype, previously thought to be inherited in an autosomal recessive manner, can result from new dominant mutations in the COL1A1 gene of type-I collagen.     

Correction of the published sequence for the human proteolipid protein gene

Summary In the human proteolipid protein gene, the base sequence of the intronic region 5 to exon 6 was found to be 5-ctctttcattttcctgcag-3 and not 5-ctctttt-cattttcctgcag-3 as previously reported.     

Cytogenetic and molecular characterization of a newly established neuroblastoma cell line LS

Summary A new human neuroblastoma cell line (LS) that originated from an abdominal tumor of a 16-month-old girl is presented; it was classified, according to Evans, as being stage III. Morphological (dense-core particles) and biochemical characteristics (dopamine--hydroxylase, acetylcholinesterase, neuron-specific-enolase) confirmed the diagnosis. In addition to a slightly variable modal chromosome number of 48 or 49 (because of marker-chromosomes and autosomal trisomies), cytogenetic analysis revealed two constantly appearing chromosomes with homogeneously stained regions (HSR's). The karyo-type remained constant over 50 passages in vitro [49,XX, –12,+der5, + 17,+mar1,+mar2]. Double minutes were a rare phenomenon and appeared only in a few metaphases. In situ hybridization showed that some of the HSR's consisted of amplified N-myc copies. The distribution of the N-myc copies according to in situ hybridization signals along the HSR's was compared with the data of Southern and Northern blotting analyses.     

Bacterial superantigens as anti-tumour agents: induction of tumour cytotoxicity in human lymphocytes by staphylococcal enterotoxin A

Summary Activation of lymphocytes by interleukin-2 (IL-2) induces lymphokine-activated killer (LAK) cells that show promising effects on tumour growth in clinical trials. We examined the effect of the superantigen staphylococcal enterotoxin A (SEA) on anti-tumour activity of freshly prepared human lymphocytes. Picomolar amounts of SEA rapidly induced cytotoxic activity against K562 and Raji cells as well as some natural-killer(NK)-resistant tumour cell lines. Cytotoxic activity was not dependent on target cell expression of either major histocompatibility complex (MHC) class I or II antigens as shown using mutated cell lines. Cell-sorting experiments showed that the activity was expressed by NK (CD5^–CD56⁺) as well as T (CD5⁺) cells, although the former contained the majority of cytotoxic activity. NK cells could not be directly activated by SEA. In contrast, SEA activated purified T cells to the same extent as in bulk cultures. It is suggested that SEA activation of NK cells is secondary to that brought about by lymphokines produced by T cells. Activation of LAK cells with SEA was comparable in magnitude as well as target cell spectrum to that of IL-2. In addition to the LAK-like cytotoxic activity induced by SEA, a superimposed cytotoxicity towards target cells expressing MHC class II antigens coated with SEA was observed. This staphylococcal-enterotoxin-dependent cell-mediated cytotoxicity (SDCC) was exclusively mediated by T cells. It is well established that MHC class II antigens function as receptors for staphylococcal enterotoxins on mammalian cells and that the complex between MHC class II antigen and — SEA apparently functions as a target structure for activated T cells with target cell lysis as a consequence. Activation of T lymphocytes with IL-2 also resulted in the capability to mediate SDCC. Staphylococcal enterotoxins represent a novel way of inducing anti-tumour activity in human lymphocytes, which could be of value in therapeutic applications.     

Induction of lymphokine-activated killer activity in rat splenocyte cultures: The importance of 2-mercaptoethanol and indomethacin

Summary The role of 2-mercaptoethanol and indomethacin in the induction of lymphokine-activated killer (LAK) activity by interleukin-2 (IL-2) in rat splenocyte cultures was investigated. Spleens from 4-month-old male rats of five different strains were tested. Splenocytes were cultured for 3–5 days in the presence of IL-2 (1000 U/ml) and LAK activity was assessed by 4-h⁵¹Cr release assays with P815 and YAC-1 cells as targets. LAK activity could be induced by IL-2 in splenocytes from all rat strains, but only when 2-mercaptoethanol was present in the culture medium. Optimal LAK activity was induced when the 2-mercaptoethanol concentration in splenocyte cultures was at least 5 µM. Different rat strains showed differences in levels of in vitro induction of LAK activity. In the presence of 2-mercaptoethanol the level of LAK activity induced by IL-2 was high in BN and Lewis rats, intermediate in Wistar and Wag rats, and low in DZB rats. In the absence of 2-mercaptoethanol no or minimal LAK activity was induced. Furthermore we observed that addition of 50 µm indomethacin to the culture medium in the presence of 2-mercaptoethanol augmented the induction of LAK activity to some extent. In the absence of 2-mercaptoethanol, addition of indomethacin resulted only in low levels or no induction of LAK activity. We conclude that for optimal induction of LAK activity by IL-2 in rat splenocyte cultures 2-mercaptoethanol is essential, while indomethacin can only marginally further improve this induction.     

Acetylene inhibition technique underestimates in situ denitrification rates in intact cores of freshwater sediment

Abstract Denitrification in intact sediment cores was measured by the acetylene inhibition technique and compared with the nitrate flux between water and sediment. Less than half of the nitrate-N consumed by the sediment could be recovered as nitrous oxide-N. The low recovery rate of nitrous oxide from intact sediment cores indicated losses of nitrous oxide by diffusion down to nitrate-free sediment layers, where reduction of nitrous oxide may take place. In sediment slurries 100% of nitrate-N could be recovered as nitrous oxide-N as long as the nitrate concentration in the liquid phase was above 10 μM. Nitrous oxide added to nitrate-free sediment slurries was reduced regardless of whether acetylene was present or not. Therefore denitrification may be significantly underestimated by this method.     

Diversity of extinct and living actinistian fishes (Sarcopterygii)

Synopsis A total of 121 actinistian species belonging to 47 genera and 17 undetermined actinistians is reported from the literature. There are 69 valid species with fair assessment of their phylogenetic position; 21 valid species with poor assessment of their phylogenetic position; 31 actinistian incertae sedis; and 18 taxa that had been identified incorrectly as actinistians or are nomen nuda. The fossil record of the actinistians covers a history of approximately 380 million years. The greatest diversity occurred during the Scythian (Early Triassic).     

Experiences with reducing point sources of phosphorus to lakes

Experiences over the last 25 years have demonstrated that eutrophication can be reversed, and that phosphorus is most often the nutrient through which control should be exerted.The reduction of the external load of phosphorus to a eutrophic lake is a necessary condition for lake restoration, but may not in itself be sufficient. Three main response patterns to a reduction in external load are identified. These include reduction in lake phosphorus that leads to sufficient reduction in chlorophyll to change the trophic category, to make the lakes less eutrophic or have small or no effect. The factors that determine the actual response are discussed.It is clear that interventions to restore eutrophic lakes have not always given the results expected. Limnological studies are necessary for well-grounded predictions.     

Aspects of the life history of Cercopithifilaria johnstoni (Nematoda:Filarioidea)

and 1988. Aspects of the life history of Cercopithifilaria johnstoni (Nematoda:Filarioidea). International Journal for Parasitology 18: 1087–1092. Cercopithifilaria johnstoni (Nematoda:Filarioidea) occurs in the subcutaneous connective tissues of a spectrum of native murid and marsupial hosts in Eastern Australia. Life cycle studies revealed that: (i) microfilaria occur in lymphatic capillaries and extravascular connective tissue of the dermis (= ‘skin-inhabiting’), (ii) ixodid ticks, particularly Ixodes trichosuri, are intermediate hosts in nature, (iii) development from microfilariae to infective third-stage larva occurs only while the tick is off the host, that is, during ecdysis from larva to nymph or from nymph to adult. Transmission of C. johnstoni in a wild population of bush rats (Rattus fuscipes) occurred in summer and winter, and was associated with peaks in the number of larval and/or nymphal stages of ticks on rats. C. johnstoni was transmitted experimentally to bandicoots (Isoodon macrourus, Perameles nasuta), bush rats and laboratory rats (R. norvegicus), indirectly by subcutaneous inoculation of third-stage larvae and directly by tick feeding. The prepatent period was approximately 3 months and the longest duration of microfilariae in the ‘ skin’ was more than 25 months. Dermal and ocular lesions were observed in R. fuscipes. The host-parasite relationship has the potential for development as an inexpensive and practical model for human onchocerciasis.