K+-evoked taurine efflux from cerebellar astrocytes: On the roles of Ca2+ and Na+

The ionic requirements for K⁺-evoked efflux of endogenous taurine from primary cerebellar astrocyte cultures were studied. The Ca²⁺ ionophore A23187 evoked taurine efflux in a dose-dependent fashion with a time-course identical to that of K⁺-induced efflux. The Ca²⁺-channel antagonist nifedipine had no effect upon efflux induced by 10 or 50 mM K⁺. In addition, verapamil did not antagonize 50 mM K⁺-evoked efflux except at high, non-pharmacological concentrations (>100 M), and preincubation with 2 M -conotoxin had no effect on 50 mM K⁺-evoked efflux. Similarly, preincubation with 1 mM ouabain had no effect on the amount of taurine released by K⁺ stimulation, but did accelerate the onset of efflux by 2–4 min. Although 2 M tetrodotoxin had no effect on K⁺-evoked release, replacing Na⁺ with choline abolished the taurine efflux seen in response to K⁺ stimulation. Together, these findings suggest that neuronal N- and L-type Ca²⁺- and voltage-dependent Na⁺-channels are not involved in the influx of Ca²⁺ which appears to be necessary for K⁺-evoked taurine efflux, and that in addition to Ca²⁺, extracellular Na⁺ is also required.     

Effects of pollen donors on seed formation inEspeletia schultzii (Compositae) populations at different altitudes

The influence of different pollen donors on seed formation was investigated in three populations ofEspeletia schultzii that differ in environmental conditions and life history characteristics. Self pollen and pollen from different donors (< 15m apart) within each population was used in a diallel design in order to test the genetic base of seed set variation. Three measures of seed formation were used: (1) achene number; (2) proportion of filled achenes (fruits) that distinguishes between achenes with seeds and empty achenes; (3) proportion of aborted seeds that distinguishes between viable and aborted seeds. Self-pollinations resulted in empty achenes. Achene number did not vary between the different pollen donors. A bimodal pattern of filled achenes was found in two populations in two consecutive years. On the other hand, a unimodal pattern was found in crosses between more distant donors (> 30m). These patterns seems to be the results of a sporophytic incompatibility system. Seed abortion was highest at the higher elevations and seems to be correlated with elevation rather than with any genetic effect.     

An oxygen dependence in chromium mutagenesis

Evidence is provided that mutagenicity in Salmonella by a chromium(VI) salt and a chromium(III) compound has a differential dependence on the presence of oxygen. The mutagenic chromium(III) compound, cis-dichlorobis(2,2'-bipyridyl)chromium(III), reverted Salmonella strains, TA102 and TA2638, only under aerobic conditions. Potassium dichromate (chromium VI) required the presence of oxygen to revert the Salmonella strain TA102 but induced a moderate reversion frequency in TA2638 under anaerobic conditions. The data also support a role for oxygen radicals in chromium-mediated mutagenesis and suggests at least two pathways by which chromium compounds can induce mutations.     

Hypervariability of intronic simple (gt)n(ga)m repeats in HLA-DRB genes

We have investigated the extent of DNA variability in intronic simple (gt)_n(ga)_m repeat sequences and correlated this to sequence polymorphisms in the flanking exon 2 of HLA-DRB genes. The polymerase chain reaction (PCR) was used to amplify a DNA fragment containing exon 2 and the repeat region of intron 2. The PCR products were separated on sequencing gels in order to demonstrate length hypervariability of the (gt)_n(ga)_m repeats. In a parallel experiment, the PCR products were cloned and sequenced (each exon 2 plus adjacent simple repeats) to characterize the simple repeats in relation to the HLA-DRB sequences. In a panel of 25 DRB1, DRB4, and DRB5 alleles new sequences were not detected. Restriction fragment length polymorphism (RFLP) subtyping of serologically defined haplotypes corresponds to translated DNA sequences in 85% of the cases, the exceptions involving unusual DR/DQ combinations. Many identical DRB1 alleles can be distinguished on the basis of their adjacent simple repeats. We found group-specific organization of the repeats: the DRw52 supergroup repeats differ from those of DRB1*0101, DRB4*0101, and DRB5*0101 alleles and from those of pseudogenes. Finally, we amplified baboon DNA and found a DRB allele with extensive similarity to DRB1 sequences of the DRw52 supergroup. The simple repeat of the baboon gene, however, resembles that of human pseudogenes. In addition to further subtyping, the parallel study of polymorphic protein and hypervariable DNA alleles may allow conclusions to be drawn on the relationships between the DRB genes and perhaps also on the theory of trans-species evolution.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M 34258.     

Reinfection of virus free mice with mouse mammary tumour virus

BR6/Icrf mice carrying a milk-transmitted mammary tumour virus (MMTV) develop tumours after several pregnancies. If the mice are freed from MMTV, no tumours develop. In the experiments described in this paper, MMTV was reintroduced into MMTV-free mice by foster nursing, which was least effective if the pups were exposed to the virus only during the first week of life. Exposure for even a short time after that age led to a tumour incidence similar to that found in normally infected mice. Reinfection was also achieved by injection of MMTV-containing milk into weanling or pregnant mice, and was then transmitted naturally to the next generation.     

Paleohistology of Paget's disease in two medieval skeletons.

Paget's disease has been ascribed several times to specimens of archeological bone but, in the absence of microscopic examination, the evidence remains insubstantial. Suspected metabolic bone disease is described here in the archeological remains of a skeleton from a 16th century burial ground at Wells Cathedral, England and from a single medieval sacrum recovered from a large deposit of disarticulated bones from a churchyard at Barton-on-Humber, England. Radiographs showed apparent structural abnormality in one femoral shaft and calcaneus and in the isolated sacrum. Histomorphometry on undecalcified bone cores confirmed the regions of abnormality and showed not only increased trabecular width but also areas of "mosaic" woven bone together with extensive resorption cavities; these features contrasted with the normal structure and organized lamellar bone from sites elsewhere. Despite post-interment changes in surrounding tissues, the morphological stability of some of the osteocytes was remarkable. Preservation of the histology was sufficient to permit the assignment of a metabolic bone disorder and the nature of the sclerosis was consistent with Paget's disease.     

Metabolism of halogenated bisphosphonates by the cellular slime mould Dictyostelium discoideum.

Methylenebisphosphonate and its monofluoro-, difluoro- and dichloro- derivatives inhibited growth of amoebae of Dictyostelium discoideum. Dichloromethylenebisphosphonate was the most potent inhibitor of amoebal growth whereas difluoromethylenebisphosphonate was the least potent inhibitor. Each of the bisphosphonates was taken up by the amoebae and incorporated into the corresponding beta, gamma-methylene analogue of adenosine triphosphate. Two of the bisphosphonates were also incorporated into the corresponding analogues of diadenosyl tetraphosphate. No correlation was found between the ability of the bisphosphonates to inhibit amoebal growth and the extent to which they were metabolised.     

Linkage of the acetylcholine transporter-vesamicol receptor to proteoglycan in synaptic vesicles.

The relationship of the acetylcholine transporter-vesamicol receptor (AcChT-VR) to proteoglycan in Torpedo electric organ synaptic vesicles was investigated. The cholate-solubilized VR was immunoprecipitated by a monoclonal antibody directed against the SV1 epitope located in the glycosaminoglycan portion of the proteoglycan. AcChT that was photoaffinity-labeled with a tritiated high-affinity analogue of AcCh [cyclohexylmethyl cis-N-(4-azidophenacyl)-N-methylisonipecotate] and then denatured in sodium dodecyl sulfate also immunoprecipitated. The labeled AcChT exhibited a M(r) range of 100,000-200,000. Proteoglycan did not engage in detectable nonspecific reversible aggregation that might mask the presence of another subunit during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In vesicles permeabilized with cholate, the enzymes keratanase and testicular hyaluronidase inactivated binding of vesamicol and destroyed the SV1 epitope without detectable proteolysis. Other glycosaminoglycan-degrading enzymes were without effect. The results demonstrate that the AcChT-VR and proteoglycan are very strongly linked and that glycosaminoglycan-like polysaccharide controls the conformation of the VR. The unexpected linkage to proteoglycan suggests that AcChT-VR in intact terminals might communicate with extracellular matrix and participate in stabilization and operation of the synapse.     

High-resolution X-ray study of deoxyhemoglobin Rothschild 37 beta Trp----Arg: a mutation that creates an intersubunit chloride-binding site.

The mutation site in hemoglobin Rothschild (37 beta Trp----Arg) is located in the "hinge region" of the alpha 1 beta 2 interface, a region that is critical for normal hemoglobin function. The mutation results in greatly reduced cooperativity and an oxygen affinity similar to that of hemoglobin A [Gacon, G., Belkhodja, O., Wajcman, H., & Labie, D. (1977) FEBS Lett. 82, 243-246]. Crystal were grown under "low-salt" conditions [100 mM Cl- in 10 mM phosphate buffer at pH 7.0 with poly(ethylene glycol) as a precipitating agent]. The crystal structure of deoxyhemoglobin Rothschild and the isomorphous crystal structure of deoxyhemoglobin A were refined at resolutions of 2.0 and 1.9 A, respectively. The mutation-induced structural changes were partitioned into components of (1) tetramer rotation, (2) quaternary structure rearrangement, and (3) deformations of tertiary structure. The quaternary change involves a 1 degree rotation of the alpha subunit about the "switch region" of the alpha 1 beta 2 interface. The tertiary changes are confined to residues at the alpha 1 beta 2 interface, with the largest shifts (approximately 0.4 A) located across the interface from the mutation site at the alpha subunit FG corner-G helix boundary. Most surprising was the identification of a mutation-generated anion-binding site in the alpha 1 beta 2 interface. Chloride binds at this site as a counterion for Arg 37 beta. The requirement of a counterion implies that the solution properties of hemoglobin Rothschild, in particular the dimer-tetramer equilibrium, should be very dependent upon the concentration and type of anions present.