Covalent immobilization of the multienzyme enniatin synthetase

Summary The multienzyme enniatin synthetase was covalently immobilized to N-hydroxysuccinimide activated agarose. The stability of the immobilized enzyme at 25°C was enhanced compared to the soluble enzyme. Immobilization experiments also indicated that the enniatins are synthesized by a single molecule and thus do not require interactions of several enzyme molecules.     

Transductional mapping of nine linked chromosomal genes in Bacillus thuringiensis

Summary We have previously described a phage (63) for generalized transduction in Bacillus thuringiensis and used it for mapping of four chromosomal antibiotic resistance markers, namely nalA-rifA-strA-spcA (Landén et al. 1981). From 63 we have now isolated a host range mutant called 64 which contains 52–56 megadalton of DNA. Phage 64 was found to be a more efficient transducing vector than 63. The host range of 64 is wide, with good growth on subspecies gelechiae, kurstaki, galleriae, thuringiensis and thompsoni, restriction on some derivatives of finitimus and ostrinae and no growth on alesti, israelensis and aizawai.Using 64 and a series of new mutants of subspecies gelechiae we have no added five new genes to the antibiotic resistance group described before. The gene order found was guaB-purB-metA-novA-(purA-nalA)-rifA-strA-spcA. Linkage was also demonstrated between hisA and lysA.     

Aspects of neural plasticity in the central nervous system—III. Methodological studies on the microdialysis technique

Some methodological aspects of the intracerebral microdialysis technique have been investigated: the existence of a pressure gradient at the level of the dialyzing membrane, the substance diffusion from the microdialysis probe and the extent of tissue damage induced by the implantation of the microdialysis probe. At the level of the dialyzing membrane a rough balance between the pressure inside the probe and the one present in the extracellular fluid compartment has been observed. The pattern of substance diffusion in the tissue showed a large variability depending on the substance used and the experimental conditions. Relevant deductions can be made by the use of labeled markers. By means of this approach, the diffusion pattern of tritiated ganglioside GM1 in the tissue around the probe could be shown to follow a biexponential pattern, suggesting a two-step process of diffusion. The degree of tissue damage induced by the microdialysis probe was assessed by analyzing the glial reaction, and was measured by means of semiquantitative immunocytochemistry of glial fibrillary acidic protein immunoreactivity. Only a limited area of neuronal damage was observed in the region surrounding the microdialysis probe. The amount of glial reaction after probe implantation was shown to be comparable with that induced by the implantation of a microinjection cannula.     

WT1 mutations in patients with Denys-Drash syndrome: a novel mutation in exon 8 and paternal allele origin

Denys-Drash syndrome (DDS) is characterized by early onset nephropathy, pseudohermaphroditism in males and a high risk for developing Wilms' tumour (WT). The exact cause of DDS is unknown but germline mutations in the Wilms' tumour suppressor gene (WT1) have recently been described in the majority of DDS patients studied. These mutations occur de novo and are clustered around the zinc finger (ZF) coding exons of the WT1 gene. Analysis of exons 2–10 of the WT1 gene in constitutional DNA from five patients with DDS was carried out using the polymerase chain reaction (PCR) and direct DNA sequencing. In four out of the five patients, heterozygous germline mutations were found: a novel point mutation in exon 8 (ZF₂) at codon 377 altering the wild-type histidine to arginine, and three previously described point mutations in exon 9 (ZF₃) in the codons corresponding to amino acids ³⁹⁴Arg and ³⁹⁶Asp. In one patient, no mutations could be demonstrated. In three patients where parental DNA was available, the mutations were shown to have occurred de novo. Furthermore, since tumour DNA in two of these cases had lost the wild-type allele, polymorphic markers from the short arm of chromosome 11 were used to determine the parental origin of the mutant chromosome. In both cases, the mutant chromosome was shown to be of paternal origin. Since the majority of published WT1 mutations in DDS patients alter a RsrII restriction site in exon 9, we were able to perform PCR-based diagnosis in a female patient with early renal insufficiency and normal external genitalia.     

Rat liver foci study on coexposure with 50 Hz magnetic fields and known carcinogens

A study was performed to investigate possible interactions by magnetic fields (MF) with the processes of initiation and promotion of chemically induced preneoplastic lesions in rat liver. Male Sprague-Dawley rats were subjected to a 70% partial hepatectomy followed after 24 h by i.p. injection of diethylnitrosamine (DENA) as a tumour initiator. Starting one week after the DENA-treatment phenobarbital (PB) was given to promote growth of enzymatically altered foci of liver cells. MF was applied immediately after the partial hepatectomy and continued until sacrifice after 12 weeks of PB exposure. Homogenous horizontal AC magnetic fields with a frequency of 50 Hz and flux densities of 0.5 μT or 0.5 mT were used. The rats coexposed with MF and DENA plus PB did not gain weight as much as the rats exposed to the chemical agents only. The MF-exposure also resulted in a slight reduction in size and numbers of the focal lesions. The results suggest an interaction of MF with the processes of chemical carcinogenesis either as a result of stress or depending on effects on the proliferation of preneoplastic cells. © 1993 Wiley-Liss, Inc.     

Increased Kit/SCF receptor induced mitogenicity but abolished cell motility after inhibition of protein kinase C.

The product of the c-kit proto-oncogene, denoted Kit/SCF-R, encodes a tyrosine kinase receptor for stem cell factor (SCF). Kit/SCF-R induces proliferation, differentiation or migration of cells within the hematopoietic, gametogenic and melanogenic lineages at different developmental stages. We report here that protein kinase C (PKC) mediates phosphorylation of Kit/SCF-R on serine residues in response to SCF or PMA in intact cells. The phosphorylation inhibits SCF-induced tyrosine autophosphorylation of Kit/SCF-R. In vitro studies showed that PKC phosphorylated the Kit/SCF-R directly on serine residues and inhibited autophosphorylation of Kit/SCF-R, as well as its kinase activity towards an exogenous substrate. The PKC-induced phosphorylation did not affect Kit/SCF-R ligand binding affinity. Inhibition of PKC led to increased SCF-induced tyrosine autophosphorylation, as well as increased SCF-induced mitogenicity. In contrast, PKC was necessary for SCF-induced motility responses, including actin reorganization and chemotaxis. Our data suggest that PKC is involved in a negative feedback loop which regulates the Kit/SCF-R and that the activity of PKC determines whether the effect of SCF will be preferentially mitogenic or motogenic.     

Tight linkage between the Beckwith-Wiedemann syndrome and a microsatellite marker for the TH locus

The Beckwith-Wiedemann syndrome (BWS) is characterized by somatic overgrowth, developmental anomalies, and proneness to embryonic tumor development. The majority of cases are sporadic, but several families with an autosomal dominant mode of inheritance with variable expression and reduced penetrance have been described. In three such families, BWS has been linked to DNA markers for the insulin gene (INS) and H-ras on chromosome band 11p15. Two additional families with inherited BWS are described here. Linkage analysis has been performed with a highly informative marker for the tyrosine hydroxylase (TH) locus within the INS-IGF2 (insulin-like growth factor II)-TH gene cluser and confirms the previous observed linkage to this region (lod score 2.16 at = 0). Linkage analysis to TH provides a basis for informed genetic counselling and carrier detection in the hereditary form of the syndrome. Based on the hypothesis that IGF2 may be a candidate gene for BWS, we screened for mutations in the coding exons 7 and 9, but found no abnormalities in 5 unrelated BWS cases.     

Regional high affinity synaptosomal transport of choline in mouse brain — Influence of oxotremorine treatment

Kinetic parameters for high affinity [HA] uptake $\text{in vitro}$ in synaptosomes from different mouse brain regions were investigated. V_max was highest in the striatum [200 pmol.· mg protein^?1 · 4 min^?1], followed by the cortex [111 pmol · mg protein^?1 · 4 min^?1], hippocampus [63 pmol · mg protein^?1 · 4 min^?1], midbrain [21 pmol · mg protein^?1 · 4 min^?1] and, lowest, medulla oblongata [5 pmol · mg protein^?1 · 4 min^?1]. K_m was about the same in all brain regions [0.9–1.4 μM]. No sign of HA uptake was detected in synaptosomes from the cerebellum. A clear relationship between V_max for synaptosomal HA uptake of Ch $\text{in vitro}$ and apparent turnover of ACh $\text{in vivo}$ was found between the brain regions. Administration of oxotremorine [1 mg·kg^?1 i.p.] decreased V_max for HA uptake of Ch by 60% in the cortex and hippocampus, by 50% in the striatum and by 20% in the midbrain. This effect is in accordance with the previously observed marked decrease in turnover of ACh in these brain regions following oxotremorine treatment.     

Intracellular binding of lead in the kidney: The partial isolation and characterization of postmitochondrial lead binding components

Gel chromatography of kidney postmitochondrial fractions from control rats 2 hr after injection of ²⁰³Pb or after in vitro incubation with ²⁰³Pb disclosed the presence of two fractionated Pb-binding components plus binding in the void volume and total volume regions. The binding of Pb to the two components, with molecular weights of 11,500 and 63,000 daltons, was markedly decreased in Pb-pretreated rats. Sodium dodecyl sulfate-gel electrophoresis and autoradiography showed the presence of one major ²⁰³Pb band with an estimated molecular weight of 60,000 daltons. The 11,500-dalton peak did not incorporate ¹⁴C-leucine nor did concomitant administration of cycloheximide with the ²⁰³Pb inhibit incorporation of ²⁰³Pb activity, suggesting that the component is a preformed constituent of the kidney. In vitro incubation of brain, liver and lung postmitochondrial supernatants with ²⁰³Pb disclosed that these two binding components were also present in brain but not in liver or lung, suggesting a target tissue-specific localization for these Pb-binding macromolecules.     

Survival and growth of pea protoplasts after transformation by electroporation

Protoplasts from two different pea cultivars, Belman and Filby, were stably transformed by direct gene transfer using electroporation. Transgenic calli could be obtained after selection, when hygromycin resistance was used as the selective trait introduced into the protoplasts, while no transformants were obtained when kanamycin resistance was used as selective marker in either of the two pea cultivars tested. The effect of the field strength on survival and division rates of the protoplasts was studied. Two different culture systems and osmotica were compared for induction of sustained divisions in and regeneration of transgenic callus from the protoplasts. The choice of the culture system had a considerable effect on the initial division frequency of the treated protoplasts, as well as on the later growth of the colonies. Transformation efficiency was monitored by histochemical GUS assay, and the transgenic nature of the calli selected for resistance against antibiotics was confirmed by DNA analysis.