A high proportion of ADA point mutations associated with a specific alanine-to-valine substitution.

In 15%-20% of children with severe combined immunodeficiency (SCID), the underlying defect is adenosine deaminase (ADA) deficiency. The overall goal of our research has been to identify the precise molecular defects in patients with ADA-deficient SCID. In this study, we focused on a patient whom we found to have normal sized ADA mRNA by Northern analysis and an intact ADA structural gene by Southern analysis. By cloning and sequencing this patient's ADA cDNA, we found a C-to-T point mutation in exon 11. This resulted in the amino acid substitution of a valine for an alanine at position 329 of the ADA protein. Sequence analysis revealed that this mutation created a new BalI restriction site. Using Southern analyses, we were able to directly screen individuals to determine the frequency of this mutation. By combining data on eight families followed at our institution with data on five other families reported in the literature, we established that five of 13 patients (seven of 22 alleles) with known or suspected point mutations have this defect. This mutation was found to be associated with three different ADA haplotypes. This argues against a founder effect and suggests that the mutation is very old. In summary, a conservative amino acid substitution is found in a high proportion of patients with ADA deficiency; this can easily be detected by Southern analysis.     

The respiratory burst oxidase of human neutrophils. Further studies of the purified enzyme

A superoxide-forming oxidase from activated human neutrophil membranes was solubilized by two slightly different methods, then purified by "dye-affinity" chromatography. Kinetic studies of the purified preparations gave Vmax values of 5-10 mumol of O-2/min/mg of protein, and Km values for NADH and NADPH that were in reasonable agreement with values determined previously using particulate and crude solubilized preparations of the respiratory burst oxidase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed prominent bands at 67, 48, and 32 kDa, together with some minor contaminants, whereas gel electrophoresis under non-denaturing conditions gave a single major band that when eluted and re-electrophoresed in the presence of sodium dodecyl sulfate showed bands at 67, 48, 32 kDa. We believe that all three bands represent oxidase components. The flavin content of the purified enzyme was 20.4 +/- 2.0 S.E. pmol of FAD/microgram of protein, whereas heme averaged 0.1 +/- 0.02 pmol/microgram and ubiquinone could not be detected. Assuming that the enzyme is composed of one 67-kDa subunit, one 48-kDa subunit, and one 32-kDa subunit (i.e. that its molecular mass is approximately 150 kDa), it can be calculated to have a turnover number of 700-1500 min-1, in agreement with a value reported previously for oxidase in a particulate O-2-forming system (Cross, A. R., Parkinson, J. F., and Jones, O. T. G. (1985) Biochem. J. 226, 881-884), and to contain the following quantities of redox carriers (mol/mol): FAD, 3.0; heme, 0.015; ubiquinone, less than 0.06. It remains to be determined whether this preparation represents the complete respiratory burst oxidase or is only the pyridine nucleotide dehydrogenating component of a more complex enzyme.     

Transient stimulation of glucose metabolism by insulin in the 1-day chick embryo

Effects of insulin upon glucose metabolism were investigated in chick embryos explanted in vitro during the first 30 h of incubation. Insulin stimulated the glucose consumption of the chick gastrula (18 h) and neurula (24 h), but had no effect on the late blastula (0 h:laying) and on the stage of six to eight somites (30 h). The increase in glucose consumption concerned both the embryonic area pellucida (AP) and extraembryonic area opaca (AO). AP responded to a greater extent (50%) and at a lower range of concentrations (0.1-1.0 ng/ml) than AO (30%; 1-100 ng/ml). Insulin had no effect on the oxygen consumption of blastoderms, whereas it stimulated the aerobic lactate production (approximately 70% of the additional glucose consumption was converted to lactate). The nanomolar range of stimulating concentrations suggests that insulin has a specific effect in the chick embryo, and that it could modulate glucose metabolism in ovo as well. The transient sensitivity of the embryo to insulin is discussed in relation to behavior of mesodermal cells.     

Distribution of male yellow dungflies around ovipasition sites: the effect of body size

Abstract.

• 1 Spatial and temporal variation in body size of yellow dungflies, Scatophaga stercoraria, gathering on and around cow droppings was studied in an Icelandic population in order to elucidate the effect of male and female size on male mating tactics.
• 2 Males copulating on droppings were on average larger than males copulating in the grass, but of similar size to males guarding ovipositing females. Males searching on droppings were smaller than males copulating or guarding females on droppings but larger than males copulating in the grass. No such differences were found in female size.
• 3 Resource-holding power of males (RHP, i.e. male: female size ratio) differed between the three mating groups and was highest for males on the droppings. Size and RHP clearly affect the tactics of copulating males. Males with low RHP tend to copulate in the grass in spite of the cost of longer copulation duration. We argue that this is caused by risk of takeovers from large searching males.
• 4 There was no change in male size with the age of individual droppings. Contrary to what might be expected, large searching males are not predominantly found at fresh droppings when the probability of catching unpaired females is highest. We suggest instead that good prospects in taking females over from other males must make the strategy to search for females on older droppings profitable.
• 5 RHP did not change with age of dropping in the three mating groups. The size of ovipositing females increased with age of dropping, probably reflecting longer copulation and egg-laying times of large females.
• 6 We found an overall positive relationship between sizes of male and female partners. This correlation was highly significant for copulating pairs in the grass. This is probably a consequence of males with low RHP copulating in the grass and fights in which larger males take over females from smaller males. A weaker, but significant, correlation was found amongst ovipositing pairs. This must be due to take-over effects. No size correlation was found for pairs copulating on droppings.

     

Cloning mammals: Current reality and future prospects

Animal husbandry would be well served by procedures that created new phenotypes by selective breeding and propagated them by cloning—that is by asexual or vegetative reproduction. Such reproductive patterns characterize some plants and some invertebrate animals. Even a few species of reptiles, amphibians, and fish exhibit parthenogenetic reproduction. Mammalian eggs can easily be provoked to develop parthenogenetically but no mammalian parthenote has ever developed to term. However the parthenote cells can be rescued by aggregating them with normal cells to make a chimera that can reach adulthood and reproduce using the parthenote cells. Replication and growth of embryos in vitro has led to twins or even quadruplets. Continued growth in vitro, as exemplified by teratocarcinomas, could lead to useful cloning. Nuclear transplantation, leading to cloning, can be carried out in mammals by using embryonic nuclei but this presents no economic advantage. Cloning with adult nuclei is not possible at present. Circumventing meiosis altogether, coupled with parthenogenesis, would lead to the most desirable mode of cloning, and this might be achieved through a series of mutations.     

Inorganic chemical investigations in the Forest Biosphere Reserve near Kalinin,USSR

To get a first impression of the degree of pollution in the Forest Biosphere Reserve 350 km northwest of Moscow, samples of Polytrichum commune and peat profiles were measured by ICP/MS after decomposition by high-pressure ashing. Comparing the results with data found in similar systems in Central Europe the Forest Biosphere Reserve can be described than medium-polluted by heavy metals. Pb shows lower values as in comparable systems in Germany, due to the fact of lower traffic density in Sowjetunion. The status of the nutritional elements Ca, Mg and K is in the same order of magnitude as found in German forest ecosystems.     

Targeted Exosomes for Drug Delivery: Biomanufacturing,Surface Tagging,and Validation

Exosomes hold great potential to deliver therapeutic reagents for cancer treatment due to its inherent low antigenicity. However, several technical barriers, such as low productivity and ineffective cancer targeting, need to be overcome before wide clinical applications. The present study aims at creating a new biomanufacturing platform of cancer‐targeted exosomes for drug delivery. Specifically, a scalable, robust, high‐yield, cell line based exosome production process is created in a stirred‐tank bioreactor, and an efficient surface tagging technique is developed to generate monoclonal antibody (mAb)‐exosomes. The in vitro characterization using transmission electron microscopy, NanoSight, and western blotting confirm the high quality of exosomes. Flow cytometry and confocal laser scanning microscopy demonstrate that mAb‐exosomes have strong surface binding to cancer cells. Furthermore, to validate the targeted drug delivery efficiency, romidepsin, a histone deacetylase inhibitor, is loaded into mAb‐exosomes. The in vitro anti‐cancer toxicity study shows high cytotoxicity of mAb‐exosome‐romidepsin to cancer cells. Finally, the in vivo study using tumor xenograft animal model validates the cancer targeting specificity, anti‐cancer efficacy, and drug delivery capability of the targeted exosomes. In summary, new techniques enabling targeted exosomes for drug delivery are developed to support large‐scale animal studies and to facilitate the translation from research to clinics.     

Effect of Repeat Dosing of Engineered Oncolytic Herpes Simplex Virus on Preclinical Models of Rhabdomyosarcoma

Rhabdomyosarcoma (RMS), a tumor of skeletal muscle origin, is the most common sarcoma of childhood. Despite multidrug chemotherapy regimens, surgical intervention, and radiation treatment, outcomes remain poor, especially in advanced disease, and novel therapies are needed for the treatment of these aggressive malignancies. Genetically engineered oncolytic viruses, such as herpes simplex virus-1 (HSV), are currently being explored as treatments for pediatric tumors. M002, an oncolytic HSV, has both copies of the γ₁34.5 gene deleted, enabling replication in tumor cells but thwarting infection of normal, postmitotic cells. We hypothesized that M002 would infect human RMS tumor cells and lead to decreased tumor cell survival in vitro and impede tumor growth in vivo. In the current study, we demonstrated that M002 could infect, replicate in, and decrease cell survival in both embryonal (ERMS) and alveolar rhabdomyosarcoma (ARMS) cells. Additionally, M002 reduced xenograft tumor growth and increased animal survival in both ARMS and ERMS. Most importantly, we showed for the first time that repeated dosing of oncolytic virus coupled with low-dose radiation provided improved tumor response in RMS. These findings provide support for the clinical investigation of oncolytic HSV in pediatric RMS.     

Acid-sensing ion channels in malignant gliomas

High grade glioma cells derived from patient biopsies express an amiloride-sensitive sodium conductance that has properties attributed to the human brain sodium channel family, also known as acid-sensing ion channels (ASICs). This amiloride-sensitive conductance was not detected in cells obtained from normal brain tissue or low grade or benign tumors. Differential gene profiling data showed that ASIC1 and ASIC2 mRNA were present in normal and low grade tumor cells. Although ASIC1 was present in all of the high grade glial cells examined, ASIC2 mRNA was detected in less than half. The main purpose of our work was to examine the molecular mechanisms that may underlie the constitutively activated sodium currents present in high grade glioma cells. Our results show that 1) gain-of-function mutations of ASIC1 were not present in a number of freshly resected and cultured high grade gliomas, 2) syntaxin 1A inhibited ASIC currents only when ASIC1 and ASIC2 were co-expressed, and 3) the inhibition of ASIC currents by syntaxin 1A had an absolute requirement for either gamma- or delta-hENaC. Transfection of cultured cells originally derived from high grade gliomas (U87-MG and SK-MG1) with ASIC2 abolished basal amiloride-sensitive sodium conductance; this inhibition was reversed by dialysis of the cell interior with Munc-18, a syntaxin-binding protein that typically blocks the interaction of syntaxin with other proteins. Thus, syntaxin 1A cannot inhibit Na(+) permeability in the absence of adequate plasma membrane ASIC2 expression, accounting for the observed functional expression of amiloride-sensitive currents in high grade glioma cells.