Small-angle X-ray scattering of pig thyroglobulin in solution (Short Communication)

Small-angle X-ray scattering measurements on native pig thyroglobulin in phosphate buffer, pH6.9, yield a radius of gyration of 6.4nm (64Å), a particle volume of approx. 1.5×10³nm³ (1.5×10⁶ų), an axial ratio of 2.2:1 (assuming an ellipsoidal shape), and a solvation of 0.63g of solvent/g of protein.     

Detection of naturally expressed receptors for gastrin-releasing peptide and tachykinins using cyanine 3-labelled neuropeptides

Summary Peptides labelled with the fluorophore cyanine 3 were used to study naturally expressed neuropeptide receptors by confocal microscopy in continuous cell lines, primary cultures, and unfixed tissue. Swiss 3T3 fibroblasts bound cyanine 3-gastrin-releasing peptide at 4°C, and internalized the peptide after 10 min at 37°C. Internalization was specific, since it was blocked by incubation with unlabelled peptide. Primary cultures of myenteric neurons of the guinea pig incubated with cyanine 3-substance P at 4°C had specific surface labelling. After 30 s at 37°C, the peptide was internalized into vesicles in both the soma and neurites. Direct observation of live neurons showed movement of fluorescent vesicles to a perinuclear region after 30 min. Endocytosis was associated with a loss of surface binding sites. Unfixed whole mounts of guinea pig and rat ileum were incubated with cyanine 3-neurokinin A at 4°C. After 5 min at 37°C, Cy3-neurokinin A was specifically internalized in neurons and smooth muscle cells. After 30 min, a perinuclear labelling occurred in some cells. Labelling in rat neurons was diminished by the NK3-R antagonist SR142801. Thus, cyanine 3-neuropeptides are valuable tools to study expression and endocytosis of naturally expressed receptors.     

Changes in Metabotropic Glutamate Receptor mRNA Levels Following Global Ischemia: Increase of a Putative Presynaptic Subtype (mGluR4) in Highly Vulnerable Rat Brain Areas

Abstract: Metabotropic glutamate receptors mediate their intracellular response by coupling to G proteins and may be divided into three subfamilies: mGluR1 and mGluR5, which stimulate phosphatidylinositol hydrolysis; mGluR2 and mGluR3, which are negatively coupled to cyclic AMP formation; and mGluR4 and mGluR6, which also inhibit forskolin-stimulated cyclic AMP formation. The mGluR4 subtypes may represent l -2-amino-4-phosphonobutyrate-sensitive presynaptic autoreceptors, and two alternatively spliced variants of the mGluR4 coding for two receptors with different C termini have been identified. Using in situ hybridization, we measured the levels of mGluR1–mGluR5 mRNA in regions of the rat brain 24 h after transient global ischemia, a time point when no neuronal damage can yet be observed morphologically. In the hippocampus, the mRNA levels for mGluR1, mGluR2, and mGluR5 were decreased, mGluR3 mRNA levels were unchanged, and the mGluR4 mRNA levels were strongly increased. The strongest increase appeared to be in the mRNA encoding mGluR4b. The mGluR4 mRNA was also increased in the parietal cortex, whereas the ventral posteromedial thalamic nucleus showed a small decrease in its mRNA content. These results suggest that vulnerable neurons react to an increased extracellular glutamate concentration by differential regulation of the mRNA for pre- and postsynaptically located metabotropic glutamate receptors.     

X chromosome linkage studies in familial Rett syndrome

Four families, each with two individuals affectecd by Rett Syndrome (RS), were analysed using restriction fragment lenght polymorphisms and microsatellite markers from the X chromosome. In two of the families, X-linked dominant inheritance of the RS defect from a germinally mosaic mother could be assumed. Therefore, maternal X chromosome markers showing discordant inheritance were used to exclude regions of the X chromosome as locations of the RS gene. Much of the short arm could be excluded, including regions containing three candidate genes, OTC, synapsin 1 and synaptophysin. Although most of the long arm was inherited in common it was possible to exclude a centromeric region. Inheritance of X chromosome markers is also presented for two families with affected aunt-niece pairs, one of which has not been previously studied at the DNA level.     

Field-scale biofiltration of gasoline vapors extracted from beneath a leaking underground storage tank

Approximately 15000 L of unleaded gasoline werereleased into the surrounding vadose zone from aleaking underground storage tank. Initialremediation was by soil vapor extraction andcombustion which soon became cost prohibitive, asadded propane was required to reach the combustionlimit of the extracted vapors. As a cost effectivealternative, a field-scale compost based biofilterwas used in conjunction with soil vapor extractionto remediate the vadose zone. The biofilter wasconstructed on site using 4:1 diatomaceousearth:composted horse manure. Results of a fivemonth study showed that the biofilter removedapproximately 90% of total petroleum hydrocarbons(TPH) and >90% of the BTEX compounds (benzene,toluene, ethylbenzene, xylene), achieving thestringent permit requirements set at either 90% TPHreduction or less than 1.36 kg per day of volatileorganic compounds (VOC's) released to theatmosphere. The biofilter showed the capacity toreadily adapt to changing environmental conditionssuch as increased contaminant loading, andvariations in temperature and moisture. Thebacterial population in the biofilter was uniformlydiverse throughout the biofilter, suggesting that aconsortium of bacteria was needed for efficientbiodegradation. The cost of biofilter set up andoperation saved 90% in the first year alone of theoperating expenses incurred by soil vapor extractionand combustion.     

Studies on the synthesis of CDP-diacylglycerol: Stimulation by GTP and inhibition by ATP and fluoride

The rat liver microsomal enzyme CTP: phosphatidate cytidylyltransferase (EC 2.7.7.41) which catalyzes the formation of CDP-diacylglycerol has been found to be markedly stimulated by GTP. The requirement for GTP is absolute, the novel GTP analogues such as guanosine 5′-[β,γ-methylene]-triphosphate, guanosine 5′-[α,β-methylene]-triphosphate, guanosine 5′-[β,γ-imido]-triphosphate and guanosine 3′-diphosphate 5′-diphosphate are without significant effect. Maximal stimulation occurs at 1 mM GTP. ATP at a concentration of 5 mM totally inhibits the formation of CDP-diacylglycerol even in the presence of optimal GTP concentration. Analogues of ATP such as adenosine 5′-[α,β-methylene]-triphosphate, adenosine 5′-[β,γ-methylene]-triphosphate and adenosine 5′-[β,γ-imido]-triphosphate are without effect on the reaction. The addition of fluoride (8 mM) likewise abolishes the stimulatory effect of GTP.     

The pharmacokinetics of tritiated ethinylestradiol in the rabbit

The disappearance of ethinylestradiol from the blood of rabbits has been studied, following the intravenous administration of this steroid. The disappearance followed two exponentials, the first having a half life ($\text{t}\text{1}\text{2}$) of 5.5 min and the second, apparently terminal exponential was also rapid ($\text{t}\text{1}\text{2}\text{= 69}\text{min}$). The plasma clearance was 150 ml/min which suggests almost total clearance of this steroid during a single passage through the liver. Bile contained a significant concentration of EE conjugates and thus this steroid could undergo enterohepatic recirculations. A large oral dose of unlabelled EE, given prior to intravenous administra tion of tritiated EE, considerably altered the pharmacokinetics of the latter by saturating both phase one metabolism (changes of the steroid nucleus) and the secretion of conjugates into bile. It was not clear whether phase two metabolism (conjugation) was also saturated.     

Genetic and developmental evidence for a repressed genital primordium in Drosophila melanogaster

Agametic, a maternal-effect mutation, causes the absence of germ cells in approximately 40% of the gonads of flies derived from homozygous females. The nature of the deficiency in the eggs produced by these flies was examined. Ultrastructural abnormalities were seen in the polar granules of some eggs shortly after fertilization. Although a normal number of pole cells form, some are abnormal with degenerating polar granules and nuclear bodies and they contain myeloid bodies. The pole cells reach the gonads and at 14 hr of development all the gonads contain germ cells. However, in 40% of the gonads the germ cells become necrotic and disappear. Thus, the source of agametic gonads in the adult is embryonic death of pole cells in some gonads. To test whether this gonadal death is an autonomous deficiency of the mutant pole cells, mosaic pole cell populations were produced by reciprocal pole cell transplantation. In both types of transplants, the mutant pole cells died autonomously. In eight instances gonads containing only donor pole cells were obtained. Since mutant pole cells die when wild-type pole cells normally begin dividing, we suggest that the lesion affects the ability of these mutant pole cells to reenter the cell cycle.